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Optimization of chemical reporters of O-GlcNAc for improved specificity and metabolic mapping

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Optimization of chemical reporters of O-GlcNAc for improved specificity and metabolic mapping

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Content OPTIMIZATION OF CHEMICAL REPORTERS OF O-GlcNAc FOR IM-
PROVED SPECIFICITY AND METABOLIC MAPPING
by
Balyn Wood Zaro
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
December 2014
Copyright 2014 Balyn Wood Zaro
Acknowledgements
Thank you to my parents for encouraging me to pursue my interests and to in-
dulge my curiosities no matter how far away, obscure or improbable. To Jeff for
being patient and embracing all the ‘nerd stuff’. I love you. To Hubert for con-
vincing me to join the Pratt Lab and helping me to become a careful scientist in
the early days. To Tharindumala for her thoughtfulness and warmth, in addition
to her scientific talents. To Kelly for being a great labmate and friend. I am going
to miss you everyday and know that whoever I have lunch with in my post-doc
will be a poor substitute. As for lab leadership, I am passing the torch to you. To
Maya for her many home-cooked meals, long conversations and advice. You filled
the role of wise female mentor perfectly. And most importantly, to Matt for put-
ting your utmost faith in me as your first student, for teaching me to read litera-
ture voraciously, think critically and write clearly, for showing me how to make
figures and give a decent talk, for practicing restraint when I clicked az-with-az
and alk-with-alk, for being my carpool buddy, for letting me fly to New York once
a month for a year and a half and only getting a little angry, for taking me out for
a beer when I’ve had a bad (or even good) day, for everything, really.
ii
Table of Contents
Acknowledgements
List of Figures
List of Schemes
Abstract
Chapter One. The Chemistry and Biology of O-GlcNAc Modifica-
tion
Introduction
The Biochemical Consequences of O-GlcNAc Modification
Methods of Studying O-GlcNAc Modification
Chapter One References
Chapter Two. A new metabolic chemical reporter reveals novel
O-GlcNAc modified proteins including the ubiq-
uitin ligase NEDD4-1
Introduction
Results
Discussion
Conclusion
Materials and Methods
Chapter Two References
Chapter Three. Robust in-gel fluorescence detection of mucin-type
O-linked glycosylation
Introduction
Results
Discussion
Conclusion
Materials and Methods
Chapter Three References
Chapter Four. N-Propargyloxycarbamate monosaccharides as
metabolic chemical reporters of carbohydrate sal-
vage pathways and protein glycosylation
Introduction
Results
Discussion and Conclusion
Materials and Methods
Chapter Four References
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Chapter Five. An alkyne-aspirin chemical reporters for the detec-
tion of aspirin-dependent protein modification in
living cells
Introduction
Results
Discussion
Conclusion
Materials and Methods
Chapter Five References
Chapter Six. Changes in metabolic chemical reporter structure
yield a selective probe of O-GlcNAc modification
Introduction
Results
Discussion and Conclusion
Materials and Methods
Chapter Six References
Chapter Seven. A chemical reporter for visualizing metabolic
cross-talk between carbohydrate metabolism and
protein acetylation
Introduction
Results
Discussion
Conclusion
Materials and Methods
Chapter Seven References
Chapter Eight. Investigation of O-GlcNAc glycosylation on the E3
ubiquitin ligase NEDD4-1
Introduction
Results
Discussion and Conclusion
Materials and Methods
Chapter Eight References
Chapter Nine. Synthesis of O-GlcNAc-related compounds and
additional small molecules
Introduction
Materials and Methods
Chapter Nine References
References
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Appendices
Appendix A: Mass Spectroscopy Tables
Appendix B: NMR Spectra
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List of Figures
Figure 1-1: The hexosamine biosynthetic pathway (HBP).
Figure 1-2: Known biochemical roles of O-GlcNAc.
Figure 1-3: Chemical reporting strategies employed to study
O-GlcNAc modification.
Figure 1-4: Methods for enrichment, identification and inter-
rogation of O-GlcNAc modification on proteins.
Figure 1-5: A selection of small molecules to study O-GlcNAc
modification.
Figure 2-1: The HBP and chemical reporters of O-GlcNAc
modification.
Figure 2-2: Fluorescence detection of O-GlcNAc-modified pro-
teins by GlcNAz and GlcNAlk.
Figure 2-3: Characterzing GlcNAz and GlcNAlk protein label-
ing.
Figure 2-4: The incorporation efficiency of GlcNAlk in a vari-
ety of cell lines
Figure 2-5: Selective enrichment and identification of O-
GlcNAc-modified proteins.
Figure 2-6: Identification of O-GlcNAlk-modified proteins.
Figure 3-1: The GalNAc salvage pathway.
Figure 3-2: Fluorescent detection of mucin-type O-linked gly-
coproteins.
Figure 3-3: Characterization of Ac4GalNAz labeling.
Figure 3-4: Characterizing the metabolic fate of Ac4GalNAz.
Figure 3-5: GalNAz labels proteins in a variety of cell lines.
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Figure 4-1: N-Propargylcarbamate-containing metabolic
chemical reporters incorporated onto proteins.
Figure 4-2: Characterization of N-propargyloxycarbamate
(Poc) bearing metabolic chemical reporters.
Figure 4-3: Flow cytometry analysis of metabolic chemical re-
porter incorporation.
Figure 4-4: Incorporation of metabolic chemical reporters into
the O-GlcNAcylation pathway.
Figure 5-1: AspAlk is a chemical reporter of aspirin-dependent
protein modification.
Figure 5-2: Characterization of AspAlk labeling.
Figure 5-3: Kinetic analysis of AspAlk labeling.
Figure 5-4: Identification of potential aspirin-dependent ace-
tylation substrates.
Figure 6-1: Metabolic chemical reporters (MCRs).
Figure 6-2: Ac36AzGlcNAc labels proteins in living cells.
Figure 6-3: The GlcNAc salvage pathway.
Figure 6-4: Investigation of 6AzGlcNAc metabolism.
Figure 6-5: LC-MS analysis of 6AzGlcNAc-1-phosphate pro-
duction by AGM1.
Figure 6-6: Fluorescence incorporation of MCRs in a variety of
cell lines.
Figure 6-7: Characterization of Ac36AzGlcNAc.
Figure 6-8: Glycoprotein specificity of 6AzGlcNAc.
Figure 6-9: Identification of O-GlcNAcylated proteins using
6AzGlcNAc.
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Figure 7-1: Using metabolic chemical reporters (MCRs) to de-
tect cellular metabolism.
Figure 7-2: Characterization of proteins that are labeled by the
MCR 1-deoxy-GlcNAlk.
Figure 7-3: Characterization of 1-deoxy-GlcNAlk labeling.
Figure 7-4: Dose-dependence and dynamics of 1-deoxy-
GlcNAlk protein labeling.
Figure 7-5: Generality of 1-deoxy-GlcNAlk labeling.
Figure 7-6: Toxicity of 1-deoxy-Ac3GlcNAlk.
Figure 7-7: Identification of posttranslationally modified pro-
teins using 1-deoxy-GlcNAlk.
Figure 7-8: Identification of proteins labelled by GlcNAlk
Figure 8-1: NEDD4 and its substrates.
Figure 8-2: The C2 domain of NEDD4 is O-GlcNAcylated.
Figure 8-3: NEDD4 stability it response to O-GlcNAcylation
and cell stress.
Figure 8-4: O-GlcNAcylation of the C2 domain in vitro.
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List of Schemes
Scheme 2-1: Synthesis of azido-azo-biotin.
Scheme 6-1: Synthesis of Ac46AzGlcNAc
Scheme 6-2: Synthesis of alkyne-azo-biotin.
Scheme 6-3: Synthesis of 6AzGlcNAc-1-phosphate
Scheme 7-1: Synthesis of Ac3-1-DeoxyGlcNAc, Ac3-1-
DeoxyGlcNAlk, and 1-DeoxyGlcNAlk.
Scheme 8-1: Synthesis of Thiamet-G.
Scheme 9-1: Synthesis of Ac5SGlcNAc.
Scheme 9-2: Synthesis of 1,3-Dibromoacetone.
Scheme 9-3: Synthesis of 3-(2-(bromomethyl)-1,3-dioxolan-2-
yl)prop-2-en-1-amine.
Scheme 9-4: Synthesis of UDP-GalNAlk.
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Abstract
Post-translational modifications (PTMs) are ancillary decorations that are trans-
ferred onto fully-synthesized proteins. These modifications have been shown to
significantly alter the fate and function of their substrates, and they include pro-
tein acetylation, phosphorylation, ubquitination, lipidation and glycosylation,
among others. PTMs have been shown to be vital for development and are often
misregulated in human disease. Throughout my graduate research, I have been
particularly focused on the nutrient-senstitive, intracellular-glycosylation PTM
N-acetylglucosamine (O-GlcNAc) and, to lesser extents, acetylation and mucin-
type O-linked glycosylation. O-GlcNAc modification is a dynamic, signaling PTM
that has been implicated in diabetes, neurodegeneration and cancer and is known
to be upregulated in response to cell stress as a cytoprotective mechanism. In or-
der to study O-GlcNAc’s role in disease progression, I have developed and opti-
mized chemical reporters of O-GlcNAc that behave similarly to endogenous O-
GlcNAc but are equipped with bioorthogonal functionality (azides or alkynes)
that can be reacted with corresponding alkyne- or azide-containing tags through
Cu(I)-catalyzed Azide-Alkyne Cycloadditon (CuAAC) to visualize the modification
and/or identify protein substrates. Importantly, these new chemical reporters of
O-GlcNAc are significantly more selective than what was previously available. I
employed these probes to identify novel O-GlcNAc substrates as well as charac-
terize the specific biochemical role of O-GlcNAcylation on a particular protein of
interest, NEDD4-1. I have also utilized this strategy in developing chemical re-
x
porters of protein acetylation and cell surface glycosylation. In addition, I have
exploited chemical reporters in order to read-out on metabolic cross-talk between
biosynthetic pathways as a way to further understand how cells amend their
metabolic machinery in order to adapt to changes in nutrient availability and cel-
lular demand.
xi
Chapter One. The Chemistry and Biology of O-GlcNAc
Modification
Introduction
Protein stability, localization and function are often regulated by post-
translational modifications (PTMs) including ubiquitination, lipidation, acetyla-
tion, nitrosylation, phosphorylation and glycosylation. There is compounding
evidence that these PTMs are critical for cellular development, homeostasis and
survival. Dynamic PTMs such as phosphorylation, ubiquitination and acetylation,
in particular, are important in cell-signaling events. N-acetylglucosamine, or O-
GlcNAc, modification is another lesser-known, dynamic PTM that is abundant
but poorly understood (1,2). O-GlcNAcylation, in contrast to large, cell-surface
glycosylation events such as mucin-type O-linked or N-linked glycosylation, oc-
curs in the nucleus, cytosol and mitochondria of the cell and consists of a single
monosaccharide installed onto the side-chain of serine or threonine residues. O-
GlcNAc glycosylation has been found in higher eukaryotes and some bacteria.
The modification is unique in that there is a single enzyme, O-GlcNAc Transfe-
rase (OGT), that transfers the sugar onto the protein and one that removes the
modification, termed O-GlcNAcase (OGA) (Figure 1-1) (3). Both OGT and OGA
are required for stem- and differentiated-cell survival and embryo development
in mammals and insects (4,5). Tissue-specific knockouts of OGT in mice have
been performed and have exhibited pronounced phenotypes, including
1
apoptosis-mediated cell death in T-cells, hyperphosphorylation of the neurofibril-
lary protein tau neurons, and growth arrest in fibroblasts (4). However, OGT is
not required for Caenorhabditis elegans (C. elegans) survival, despite knockout
of the gene resulting in metabolic misregulation and nutrient-storage abnormali-
ties (6). Therefore, OGT-deficient C. elegans provide a unique opportunity to in-
terrogate the role of OGT and O-GlcNAc glycosylation in biological processes in a
model organism.
Figure 1-1. The hexosamine biosynthetic pathway (HBP). Nutrients and metabolites are
shuttled into the HBP, resulting in the generation of a UDP-GlcNAc donor sugar.
The hexosamine biosynthetic pathway (HBP) generates the high-energy uridine
diphosphate O-GlcNAc donor sugar (UDP-GlcNAc) that is transferred onto target
proteins by OGT. The HBP lies at the confluence of 4 metabolic pathways (glu-
cose, amino acid, fatty acid and nucleic acid), setting it up as cumulative readout
of cellular metabolic activity (Figure 1-1). Approximately 2 - 5% of available glu-
2
cose in a cell is shunted through the HBP. First, glucose is taken into the cell by
glucose transporter, phosphorylated at C6 by hexokinase to generate glucose-6-
phosphate. Glucose-6-phosphate is then converted to fructose-6-phosphate at
which time Glutamine/Fructose-6-Phosphate Amidotransferase (GFAT) gener-
ates glucosamine-6-phosphate. This step is known to be the rate-limiting step in
O-GlcNAc biosynthesis (7). Glucosamine-6-phosphate is then acetylated by the
enzyme Glucosamine-6-phosphate acetyl transferase (GAT) with one equivalent
acetyl-CoA to generate N-acetylglucosamine-6-phosphate. N-acetylglucosamine
mutase (AGM) mutates the sugar to form N-acetylglucosamine-1-phosphate, the
substrate for N-acetylglucosamine pyrophosphorylase (AGX1). AGX1 transfers
uridine monophosphate onto the sugar to form the product, UDP-GlcNAc. The
HBP is known to be a nutrient-sensing pathway, experiencing changes in flux due
to nutrient levels. Notably, OGT alters its KM for target substrates in response to
changes in levels of the donor sugar, thereby sensing levels of contributing me-
tabolites (8).
Due to the dynamic nature of the PTM, O-GlcNAc is implicated as regulator of
vital biochemical processes. It has been shown that O-GlcNAc modification can
alter a protein’s localization (9), stability (10), activity (10-12) and protein-protein
interactions (Figure 1-2B) (13-15). Additionally, O-GlcNAcylation plays a regula-
tory role in cell-cycle progression and cell fate and function (16). O-GlcNAc is also
sensitive to cell stress events, including heat, osmotic pressure, hypoxia, trauma
3
hemorrhage and others (17,18) (Figure 1-2A). Increasing O-GlcNAcylation is
thought to be protective, as upregulation of O-GlcNAc results in improved sur-
vival during heart attack and ischemia/reperfusion (7,19,20). O-GlcNAc modifi-
cation is increased in some cancers (21,22) and is implicated in insulin resistance
in diabetes, although this is still somewhat debated (see below) (2,23) (Figures 1-
2A and 1-2B). Taken together, O-GlcNAc modification is a sensitive metabolic
output that modifies proteins in response to changes in nutrient flux and de-
mand. Further details as to O-GlcNAc’s specific biochemical role in cellular ho-
meostasis and disease are outlined below.
The Biochemical Consequences of O-GlcNAc Modification
O-GlcNAc and Gene Transcription
O-GlcNAc modification as well as OGT have been shown to regulate transcription
of genes associated with human disease, metabolism and development. The tran-
scription factor Stat5 is O-GlcNAcylated at Thr92, and glycosylation is required
for binding to its coactivator for subsequent gene transcription (14). Stat5 regu-
lates expression of genes associated with cell-cycle progession, inflammation and
apoptosis. Sp1, the transcriptional modulator of genes associated with a variety of
cellular processes including cell-cycle progression, immune response and DNA
damage is, too, O-GlcNAcylated, and glycosylation has also been shown to inhibit
its protein-protein interactions (24). Ten eleventranslocation-2 and Ten
eleventranslocation-3 (TET-2 and TET-3) enzymes, two key regulators of gene
4
transcription, have been shown to recruit OGT to chromatin and O-GlcNAcylate
histones, particularly H2B Ser112 (25). Histone H2B glycosylation at Ser112 pro-
motes ubiquitination at K120 to drive gene transcription (13). Additionally, OGT
bound to TET-2 is found at transcriptional start sites, demonstrating its non-
enzymatic function as a bonafide transcriptional coregulator (25). Akt1 is a also a
regulator of gene transcription and promotes cell-survival during stress. Phos-
phorylation at Thr308 and Ser473 are required for activation, but Akt1 is also a
substrate for OGT. Glycosylation does not affect basal Akt1 activity/
phophorylation, but treatment with IGF-1 independently activates and glycosy-
lates Akt1 (9). Proposed mechanisms include O-GlcNAc functioning as a signal-
ing molecule for nuclear transport or that O-GlcNAc may help Akt1 associate with
the nuclear pore complex and/or transport proteins (9).
Figure 1-2. Known biochemical roles of O-GlcNAc. (A) O-GlcNAc modification is
upregulated under cell stress and in cancer and diabetes (green arrows) but is downregu-
lated in neurodegenerative disease states (red arrow). (B) Known substrates of O-
GlcNAcylation and the affect on the function/activity of the protein (positive regulator -
green arrow, negative regulator - red arrow).
5
OGT has also been shown to be regulated by phosphorylation via AMP-activated
protein kinase (AMPK). Activation of AMPK, and thus phosphorylation of OGT at
Thr444, targets OGT to the nucleus and affects substrate binding (16). AMPK-
mediated phosphorylation has specifically been shown to preclude OGT from
binding with chromatin, preventing histone O-GlcNAcylation (26). Histones
H2A, H2B, H3 and H4 are all O-GlcNAcylated, and it has been demonstrated that
occupancy of these sites are important for gene transcription during cell stress
and proliferation (27,28).
O-GlcNAc and Metabolism/Signal Transduction
Cellular O-GlcNAc levels are extremely sensitive to nutrient availability, and O-
GlcNAcylation events can further modulate downstream signaling and metabolic
pathways in response to the cellular environment. Under glucose depravation,
UDP-GlcNAc equivalents can be generated through degradation of glycogen in
order to meet cellular demands (29). This is attributed to an upregulation of the
enzymes glycogen phosphorylase and GFAT (29). O-GlcNAcylation itself has
been shown to be a regulator of cellular metabolism, with the modification be-
having as a barometer for nutrient availability and demand. For example, O-
GlcNAcylation is implicated as a regulator of gluconeogenesis. The O-
GlcNAcylated protein Host Cell Factor C1 (HCF-1) serves as the adaptor protein
to recruit PGC-1α, a transcriptional coactivator of gluconeogenetic genes, to OGT
for glycosylation. O-GlcNAcylation stabilizes PGC-1α by promoting association
6
with the deubiquitinase BAP1, thus driving transcription of genes required for
glucose biosynthesis (30). O-GlcNAc modification also inhibits the 26S protea-
some, allowing cells to respond to changes in cellular demand for amino acid
building blocks and vital regulatory proteins (12). In addition, O-GlcNAc levels
have also been shown to regulate cardiac and lipid metabolism (31,32).
O-GlcNAc and Development/Cell-Cycle Progression
As stated previously, O-GlcNAc modification is indispensable for development
and survival in mammals and Drosophila and is a vital link between cellular
development/cell-cycle progression and nutrient availability. The developmental
protein GAT, an enzyme in the HBP (Figure 1-1), is required for survival in mice
and is sensitive to UDP-GlcNAc levels for activity. A decrease in cellular UDP-
GlcNAc levels has been shown to result in developmental defects in a mouse
model (33). HCF-1, the important cell-cycle regulator and transcriptional co-
activator, is cleaved and O-GlcNAcylated in the active site of OGT (34,35). O-
GlcNAcylation and cleavage of HCF-1 affects the protein’s subcellular localization
as well as cellular development (36). O-GlcNAc has also been shown to be en-
riched at the spindle and mid-body of mammalian cells and competes with phos-
phorylation for occupancy on proteins associated with mitosis (37). Increased O-
GlcNAcylation of developmental proteins allows cells to adjust their cell-cycle in
response to changes in nutrient availability. The developmental protein YY1, a
zinc-finger DNA-binding transcriptional regulator, is typically bound to the pro-
7
tein Rb (retinoblastoma protein) in order to regulate its activity during cell devel-
opment. Glycosylation of YY1 has been shown to prevent heterodimerization of
YY1 and Rb, altering gene transcription during cellular development (38). O-
GlcNAc modification also regulates activity of Oct4 and Sox2, transcription fac-
tors responsible for expression of genes vital to pluripotency in embryonic stem
cells (39) as well as PPAR!, a transcriptional modulator of genes associated with
adipocyte differentiation (32,40). Additonally, O-GlcNAcylation is indispensable
for fibroblast growth factor (FGF) signaling in Drosophila. Glycosylation of the
adaptor protein Dof (Downstream of FGF receptor) is crucial for signal transduc-
tion (41).
O-GlcNAc and Cell Stress/Inflammation
O-GlcNAcylation is upregulated in response to a variety of cell stresses including
oxidative stress, heat stress and osmotic stress, among others (18,42,43). In-
creased O-GlcNAcylation during cell stress has been demonstrated to be pro-
survival, allowing cells to become more robust. Additionally, stress-induced ex-
pression of heat shock proteins has been shown to be tightly regulated by O-
GlcNAcylation (18). O-GlcNAc modification has also been shown to be upregu-
lated following injury, and this hyperglycosylation inhibits the inflammatory re-
sponse (44,45). Treatment of rats with glucosamine during a trauma-hemorrhage
and resuscitation event shows improved survival and organ function directly due
to increased O-GlcNAcylation (19). O-GlcNAc is also elevated in mouse cardiac
8
myocytes following ischemic preconditioning, and this has been shown to be a
mechanism by which these mice are protected against a subsequent cardiac in-
jury event. Specifically, increased O-GlcNAcylation rendered the mice more resis-
tant to the decrease in mitochondrial membrane potential associated with ische-
mia (20,46). Teo et al. reported the glycosylation of new proteins following
trauma-hemorrhage and resuscitation, suggesting that new O-GlcNAcylation
events may be important for survival (17).
Recently, O-GlcNAcylation and the HBP have been linked to the unfolded protein
response (UPR) (47,48). During cell stress events, misfolded proteins accumulate
in the endoplasmic reticulum, eliciting the UPR. The UPR consists of three signal
transduction pathways that respond in order to determine cell survival under du-
ress: IRE1, PERK and ATF6. IRE1, in particular, is an RNAase that splices mRNA
of Xbp-1 to generate the pro-survival transcription factor Xbp-1s. Much like O-
GlcNAcylation, Xbp-1 is required for survival during hypoxia and tumorigenesis
(49). Hill and co-workers and Antebi and co-workers independently reported that
transcription of Glutamine/Fructose-6-Phosphate Amidotransferase Isoform 1
(GFAT-1), the enzyme responsible for rate limiting step in the HBP, is transcrip-
tionally regulated by Xbp-1s (47,48). Therefore, activation of the UPR during cell
stress promotes transcription of GFAT-1, thereby increasing flux through the
HBP, generating more UDP-GlcNAc and increasing O-GlcNAcylation.
9
O-GlcNAc has also been implicated in the NF-κB signaling pathway. Ra-
makrishnan et al. reported that O-GlcNAcylation at Ser350 of NF-κB c-Rel
subunit increases expression of T-cell receptor signaling genes, which combat
autoimmune response in hyperglycemic conditions (50). Glucose metabolism,
the hexosamine biosynthetic pathway and O-GlcNAc modification affect activa-
tion of NF-κB-driven gene transcription (51). Specifically, glycosylation of the p65
subunit has been shown to be required for dissociation from IκB and nuclear
translocation and NF-κB transcriptional activity (52). Additionally, OGT itself lo-
calizes to NF-κB promoters, targeting p65 for acetylation and driving transcrip-
tion of downstream NF-κB targets (53).
Apoptosis-related gene transcription has been shown to be affected by O-GlcNAc
modification. Glycosylation stabilizes Δ-Lactoferrin, a transcription factor that
drives transcription of several apoptosis-related genes including Skp1 and Bax by
blocking polyubiquitination and proteasomal turnover (10). O-GlcNAcylation of
E-cadherin has also been shown to promote apoptosis. In order for E-cadherin to
localize to the cell membrane, it must first bind PIPKIɣ, and O-GlcNAcylation
prevents this interaction, inhibiting its transport to the cell surface (54). Addi-
tionally, Snail1, a repressor of E-cadherin transcription, is O-GlcNAcylated at
Ser112. This stabilizes Snail1 and represses expression of E-cadherin (55).
10
O-GlcNAc and Diabetes
Given glycosylation’s intimate connection with glucose metabolism, it is not sur-
prising that O-GlcNAcylation has been shown to be upregulated in diabetes. No-
tably, FOXO1, a key transcriptional regulator of gluconeogenesis and glucose
homeostasis as well as enzymes that reduce reactive oxygen species, is O-
GlcNAcylated, and this modification is increased in diabetic hepatocytes (56). O-
GlcNAc modification has also been shown to directly contribute to insulin resis-
tance. In 3T3-L1 adipocytes Vosseller and co-workers reported the presentation
of insulin resistance due to decreased Akt signaling in response to decreased O-
GlcNAcylation upon treatment with the OGA inhibitor PUGNAc (57). Addition-
ally, treatment with PUGNAc also resulted in the increased glycosylation of insu-
lin receptor substrate 1 (IRS1) and β-catenin, two important effector proteins in
the insulin signaling pathway (57). In Caenorhabditis elegans (C. elegans), dele-
tion of Oga-1 and Ogt-1 results in decreased lipid storage and increased cellular
levels of glycogen and trehalose, consistent with insulin-resistance (6). Oga-1
knockout C. elegans display effects of insulin resistance analogous to that of type-
2 diabetes, revealing a mechanism by which nutrient availability can directly af-
fect insulin signaling in human disease (58). In Drosophila O-GlcNAc has also
been shown to disrupt glucose-insulin homeostasis and promote insulin resis-
tance (59). However, the role of O-GlcNAc in insulin resistance has been refuted.
Treatment with a highly-specific OGA inhibitor, NButG, does not result in insulin
resistance or disruption of glucohomeostasis in cells or in vivo (23,60,61). This
11
suggests that the affects of PUGNAc could be due to off-target inhibition of lyso-
somal Hexosaminidase B and not OGA. Further exploration is required in order
to fully elucidate the role of O-GlcNAc glycosylation in diabetic metabolism.
O-GlcNAc modification has also been implicated in cardiac disease, including
heart damage associated with diabetes (62). In diabetic rat and mice models, O-
GlcNAcylation of Ca
2+
/Calmodulin-dependent protein kinase II (CaMKII) at
Ser279 activates the enzyme, resulting in misregulation of Ca
2+
concentrations
and triggering cardiac mechanical disfunction and arrhythmia (63). It has also
been shown that insulin itself can promote O-GlcNAcylation. Insulin has been
shown to directly promote tyrosine phosphorylation of OGT, thus further activat-
ing the glycosyltransferase (64). Additionally, O-GlcNAcylation of signaling pro-
teins has been associated with the pathogenesis of diabetic retinopathy which
leads to blindness in diabetic patients (65).
O-GlcNAc in Neurodegeneration and Aging
Whereas O-GlcNAc levels are markedly increased in diabetes, the opposite is true
in neurodegenerative disease and aging. Typically O-GlcNAc heavily decorates
proteins at neuronal synapses and is vital for synaptic plasticity, but glucose up-
take and O-GlcNAcylation are lowered in Alzheimer’s disease (66). In a starved
mouse model, decreased O-GlcNAc results in hyperphosphorylation of the neuro-
fibrillary protein Tau, and it has been shown in rat brains that O-GlcNAcylation
directly affects Tau phosphorylation (67). More specifically, Vocadlo and cowork-
12
ers determined that glycosylation of Tau blocks phosphorylation at Thr231 and
Ser396 in PC-12 cells and at Ser422 in rat cortex and hippocampus tissues (68).
However, further study revealed that increased glycosylation of Tau in hemizy-
gous JNPL3 tau transgenic mice using the OGA inhibitor Thiamet-G blocked ag-
gregation and decreased neuronal cell death but did not alter tau phosphoryla-
tion. This suggests a mechanism by which O-GlcNAc can directly stabilize pro-
teins, preventing them from aberrant aggregation. Indeed, TAK-1 is another pro-
tein whose aggregation can be blocked by O-GlcNAcylation (69). Finally, it has
been shown that increased GFAT-1 expression is linked to improved longevity
and inhibition of protein aggregation, further supporting the importance of O-
GlcNAcylation in protein aggregation/misfolding disease (47). In addition to pro-
tein glycosylation being implicated in Alzheimer’s disease, O-GlcNAc modifica-
tion has been identified on the Parkinson’s disease-related protein α-synuclein
(α-syn) and is also implicated in amyotrophic lateral sclerosis (ALS) (70-72). A
hallmark of ALS in a mouse model of the disease is a marked decrease in O-
GlcNAcylation in motor neurons of the spinal cord (72). Our laboratory recently
reported that a glycosylated peptide of α-syn does not participate in the α-syn ag-
gregation associated with Parkinson’s pathology, suggesting a mechanism by
which O-GlcNAcylation of α-syn plays an inhibitory role in protein aggregation
(70).
13
O-GlcNAcylation has also been implicated in longevity and memory formation.
Promoters of genes vital for longevity including daf-16 are O-GlcNAcylated and
site occupancy has been shown to affect activity (73). Additionally, the Hsieh-
Wilson laboratory has demonstrated that cyclic AMP-responsive element binding
protein (CREB), a transcription factor that is important for the development of
long-term memory, is O-GlcNAcylated. This gycosylation event prevents CREB’s
association with the cofactor TAFII-130. Importantly, this effect is independent
of CREB phosphorylation, demonstrating O-GlcNAc’s role as a direct regulator of
memory-related gene transcription (74,75).
O-GlcNAc and Cancer
O-GlcNAc modification has been shown to be elevated in a variety of cancers in-
cluding chronic lymphocytic leukemia (CLL), breast cancer, colon cancer, pan-
creatic cancer and lung cancer (21,22,76,77). O-GlcNAc levels are associated with
disease progression and prognosis, and globally elevating O-GlcNAc levels in
cancer cells increases anchorage-dependent growth and promotes cancer cell in-
vasion (21,77). OGT is also often upregulated in cancer, and knockdown of OGT
leads to a decrease in tumorigenesis and cell invasion (22). Ma et al. reported that
increased O-GlcNAcylation in pancreatic cancer promotes cancer-cell growth and
proliferation through maintenance of NF-κB signaling and protecting cells from
apoptosis (76). Glycosylation of specific proteins has also shown to be important
in cancer. O-GlcNAcylation of the structural protein Cofilin at Ser108 promotes
14
breast cancer cell invasion by targeting the protein for invadopodia localization.
O-GlcNAcylated Cofilin stabilizes the invadopodia, promoting cell invasion (78).
The transcription factor Snail1 is also O-GlcNAcylated at Ser112 under hypergly-
cemic conditions. This occupancy blocks phosphorylation, repressing transcrip-
tion of the epithelial-mesenchymal transition program and allowing cancer cells
to become invasive (55). Cancer has been shown to exhibit an altered metabolism
of nutrients in order to optimize cancer cell growth and proliferation.
As a nutrient-sensing modification, O-GlcNAc is intimately tied to glucose me-
tabolism. Phosphofructokinase, a key enzyme in glucose metabolism, is O-
GlcNAcylated at S529, and the modification inhibits activity, resulting in in-
creased metabolic flux through the pentose phosphate pathway in order to gener-
ate more building blocks for tumor cell proliferation. Mutation of S529 results in
a decrease in tumorigenesis (79).
Methods of Studying O-GlcNAc Modification
In order to further elucidate the function of O-GlcNAcylation, several strategies
have been employed to identify target substrates, visualize glycosylation dynam-
ics and manipulate modification levels. Due the the small size and charge-neutral
nature of this glycosylation, O-GlcNAc modification poses a challenge for tradi-
tional biochemical strategies. Additionally, it has been shown that OGT and OGA
have non-enzymatic functions within the cell, including participating in gene
15
transcription and regulation (30). To circumvent these challenges, chemical re-
porters have emerged as invaluable tools in understanding O-GlcNAc modifica-
tion.
Metabolic chemical reporting
Metabolic chemical reporters allow for the incorporation of modified analogs of
O-GlcNAc onto target O-GlcNAcylated proteins over a given time period. These
analogs are equipped with a biologically inert functionality, typically an azide or
alkyne, that allow them to be selectively reacted with a corresponding fluores-
cence or affinity tag (Figure 1-3). The three most common bioorthogonal chemis-
tries that are currently exploited for metabolic reporters are Staudinger ligation,
CuAAC and strain promoted azide-alkyne cycloaddition (SPAAC). Staudinger
ligation, which utilizes the unique reactivity of azides with triarylphosphines, al-
lows for the generation of an amide bond that covalently binds the chemical re-
porter to a tag for visualization and/or enrichment. The azide functionality can be
also reacted with a complementary alkyne-bearing tag under Cu(I)-catalyzed
[3+2] azide-alkyne cycloaddition (CuAAC). CuAAC can be performed at physio-
logical pH within 1 hour. It has also been shown that the orientation can be re-
versed with an alkyne chemical reporter reacted with an azide tag for improved
signal-to-noise (80,81). More recently, SPAAC has been utilized, which allows for
Cu-free conditions, ideal for in vivo labeling and improved reaction kinetics
(82,83).
16
GlcNAz/GalNAz
The Bertozzi laboratory first pioneered the metabolic installation of biologically-
inert functionality onto target glycoproteins through the development of the
ketone-bearning chemical reporter N-levulinoylmannosamine (ManLev) (84).
ManLev can be transformed to the corresponding sialic acid and efficiently in-
corporated on cell-surface glyans. However, ketone- and aldehydecontaining me-
tabolites exist in cells, and thus a N-levulinoylglucosamine analog would not be a
truly bioorthogonal chemical reporter of intracellular glycosylation. To circum-
vent this issue, Bertozzi and coworkers employed a bioorthogonal azide moiety to
generate a fully acetylated N-azidoacetylmannosamine, which demonstrates not
only improved cellular uptake due to the acetylated hydroxyl groups but also al-
lows for more efficient incorporation of the chemical reporter onto the cell sur-
face (85). In order to investigate O-GlcNAc modification using the same chemical
reporter strategy, a new chemical reporter was developed, Ac 4-N-
azidoacetylglucosamine (GlcNAz), that enters the HBP by way of the GlcNAc sal-
vage pathway to generate the corresponding UDP donor sugar, UDP-GlcNAz and
can be efficiently incorporated on to target proteins (Figure 1-5A) (86). Impor-
tantly, GlcNAz is also tolerated by OGA, which allows for the chemical reporter to
be utilized as a tool for understanding the dynamics of O-GlcNAc modification
(87). GlcNAz can be reacted with a corresponding phosphine-biotin or
17
phosphine-FLAG tag for Western blotting visualization of the GlcNAz-labelled
proteome (87,88).
More recently, it has been reported that Ac4-N-azidoacetylgalactosamine (Gal-
NAz) is also an O-GlcNAc chemical reporter (Figure 1-5A) (89,90). The epime-
rase GALE, which interconverts UDP-GalNAc to UDP-GlcNAc and vice versa,
also accepts UDP-GalNAz and UDP-GlcNAz as substrates. Utilizing GalNAz in
conjunction with cellular fractionation strategies, Boyce et al reported the modifi-
cation of 18 glycoproteins using GalNAz labeling followed by Staudinger ligation
with phosphine-biotin, enrichment and MS identification. However, we and oth-
ers have shown that under high-glucose labeling conditions, GalNAz does not la-
bel O-GlcNAc modified proteins and does label mucin-type O-linked and N-
linked glycoproteins. These opposing experiments were conducted in different
cell lines, and therefore it is believed that the incorporation/interconversion of
chemical reporters may be cell-type specific.
GlcNAlk
As mentioned earlier, alkyne chemical reporters in combination with azide-
bearing tags have improved signal-to-noise ratios. We reported the development
of an alkyne-containing O-GlcNAc analog, Ac4-N-alkyneacetylglucosamine
(GlcNAlk), that is more selective for O-GlcNAc modification than the azide ana-
log GlcNAz (Figures 1-3 and 1-5A, Chapter 2) (91). Importantly, we characterized
18
the metabolic fate of 4 chemical reporters of glycosylation, including the 2 new
alkyne reporters: GlcNAz, GlcNAlk, GalNAz and GalNAlk. Whereas the azide-
bearing GlcNAz and GalNAz were accepted by GALE as previously reported, it
appears that alkyne-bearing reporters, GlcNAlk and GalNAlk, are not well toler-
ated by the enzyme. Therefore, GlcNAlk is not incorporated onto mucin-type O-
linked cell surface glycans. This allowed us to more-selectively visualize/identify
O-GlcNAcylated proteins without the use of cellular fractionation. In order to
visualize labeling, we utilized the corresponding azido- or alkyne-rhodamine tags
previously developed in the Francis lab (Chapter 2) (92).
6AzGlcNAc
While the development of GlcNAlk represents an improvement in the selectivity
of chemical reporters of O-GlcNAc modification, our laboratory wanted to chemi-
cally refine the reporter to only read-out on O-GlcNAc modification. To this end,
we synthesized analogs of GlcNAz where we changed the location of the azide
moiety. More specifically, we synthesized the C6 azide-equipped GlcNAc analog
6AzGlcNAc (Figure 1-5A, Chapter 6). To compare the labeling ability of 6AzGlc-
NAc, we treated cells with 6AzGlcNAc or GlcNAz lysed and subjected to the solu-
ble lysate to alk-rho under CuAAC conditions. In-gel fluorescence scanning re-
vealed 6AzGlcNAc labeling had a similar band pattern that was slightly lower in
intensity to that of GlcNAz. We also demonstrated that 6AzGlcNAc modification
is dynamic, thus behaving similarly to endogenous GlcNAc residues. The meta-
19
bolic fate of 6AzGlcNAc was also characterized, revealing that while the reporter
does modify O-GlcNAcylated proteins it is not incorporated into any extracellular
modifications. Additionally, we performed a large-scale proteomics experiment to
compare the proteins modified by GlcNAz, GalNAz and 6AzGlcNAc. Indeed,
6AzGlcNAc was only found on nuclear and cytosolic proteins. The small chemical
alteration of moving the azide to a different location on the sugar significantly al-
tered the fate of our chemical reporter, thus demonstrating the power of having
complete chemical control in metabolic reporter development.
GlcNDAz
While identifying O-GlcNAcylated proteins has proven invaluable to understand
ing the biochemical role of the PTM, it is also vital that we understand how O-
GlcNAcylation affects protein-protein interactions and if O-GlcNAc directly par-
ticipates in protein binding-partner recognition either as a recruiting or inhibi-
tory residue. To tackle this, Kohler and co-workers have developed a chemical-
and genetic-engineering strategy for identifying binding partners of O-GlcNAc-
modified proteins (93). The chemical reporter, a photocrosslinking analog of
GlcNAc equipped with a diazirine-modified N-acyl group, is termed GlcNDAz.
The alkyl diazirine functional group has been previously used to identify binding
partners of other PTMS including cell-surface glycosylation and lipidation (94-
97). Photocrosslinking using the relatively-small diazirine moiety is initiated by
ultraviolet light which generates the activated carbene intermediate that readily
20
inserts into heteroatom-H bonds. Unfortunately, its downstream products are
not well tolerated by O-GlcNAc salvage pathway enzymes NAGK and AGM1(93).
To circumvent these challenges and generate the UDP-GlcNDAz sugar donor, a
fully-protected precursor Ac3GlcNDAz-1-P(Ac-SATE)2 was synthesized (Figure 1-
5A). Ac3GlcNDAz-1-P(Ac-SATE) 2 can easily diffuse through the plasma mem-
brane and intracellular esterases hydrolyze the acetyl groups on the sugar gener-
ating GlcNDAz-1-P. Due to the increased size of the diazirine-modified N-acyl
group, GlcNDAz-1-P is not accepted by endogenous AGX1 (Figure 1-1). Expres-
sion of a mutant AGX1 that has an increased hydrophobic pocket, AGX1 F383G,
allows for generation of UDP-GlcNDAz and the subsequent transfer of GlcNDAz
onto target O-GlcNAcylated proteins. Photocrosslinking can then ligate
GlcNDAz-modified proteins to their binding partners. GlcNDAz proteins, and
now chemically-bound binding partners, can then be enriched using an O-
GlcNAc antibody or specific proteins of interest can be immunoprecipitated for
Western blotting or proteomics experiments.
While GlcNDAz does read out on O-GlcNAc modification, it not accepted by the
enzyme O-GlcNAcase, and therefore the modification is permanent. Despite this
limitation, GlcNDAz is a valuable reporter of O-GlcNAc modification due to its
ability to exclusively modify sites of O-GlcNAcylation. Importantly, UDP-
GlcNDAz is not accepted by the enzyme GALE, which interconverts UDP-GlcNAc
21
to UDP-GalNAc, allowing for the specific study of the affects of O-GlcNAcylation
on protein-protein interactions.
Figure 1-3. Chemical reporting strategies employed to study O-GlcNAc modification.
Metabolic chemical reporters, equipped with biologically inert functionality (alkyne seen
here) and behave similarly to native GlcNAc, are fed to live cells, and the treated cells are
subsequently lysed and subjected to Cu(I)-catalyzed Azide-Alkyne Cycloaddition
(CuAAC) with a corresponding visualization or affinity azide tag. Chemoenzymatic
chemical reporting involves the post-lysis transfer of an azide-equipped monosaccharide
GalNAz onto O-GlcNAc residues for reaction with visualization or affinity tags.
Chemoenzymatic chemical reporting
An alternative to metabolic chemical reporting involves the post-lysis enzymatic
transfer of a reporter probe onto proteins of interest. Chemoenzymatic chemical
reporting has been used to identify novel O-GlcNAcylated proteins, sites of modi-
fication, as well as elucidate the role of O-GlcNAc modification on the fate and
function of target proteins (Figure 1-3) (74,75,98-101).
22
Radiolabelled UDP-[
3
H]Galactose
The first O-GlcNAc chemical reporting strategy was reported by Holt and co-
workers. It was previously known that UDP-[
3
H]galactose can be transferred
onto terminal N-acetylglucosamine residues of cell surface glycosylation using
recombinant β-1,4-galactosyltransferase (GalT). This strategy has been employed
in visualizing mucin-type O-linked and N-linked glycosylation events. However,
it was discovered that the known O-GlcNAc modified protein Band 4.1 was also
labelled by [
3
H]galactose during incubation with GalT (102). Unfortunately, this
method does not allow for protein enrichment and identification.
Ketone
In order development a reporting strategy that would allow for eventual protein
visualization and identification, the Hsieh-Wilson laboratory employed a geneti-
cally engineered galactosyltransferase to selectively deliver orthogonal function-
ality onto O-GlcNAcylated proteins following lysis. A uridine disphosphate keto-
galactose can be transferred to O-GlcNAc residues using the mutant β-1,4-
galactosyltransferase (GalT Y289L), which has an enlarged binding pocket to ac-
commodate the ketone moiety at the C2 position of the UDP donor sugar (103).
The newly-formed disaccharide can then be selectively reacted with an aminooxy-
biotin tag for further enrichment and/or visualization using streptavidin-HRP.
Utilizing this strategy, Khidekel et al were able to visualize substoichiometric lev-
els of O-GlcNAcylation on α-A-crystallin, demonstrating an improved sensitivity
23
when compared to traditional chemoenzymatic radiolabeling (103). Importantly,
the use of the chemoenzymatic chemical reporter allows for a ‘snapshot’ of O-
GlcNAc modification at a given time without disrupting metabolic and signaling
pathways. Hsieh-Wilson and coworkers also utilized this chemoenzymatic re-
porter in conjunction with a isotopic labeling strategy for quantitative proteomics
in order to elucidate the dynamics of O-GlcNAc modification in the brain (101).
Azide
Further optimization of the chemoenzymatic O-GlcNAc chemical reporter came
in the development of an azide-modified UDP chemical reporter, N-
Azidoacetylgalactosamine (UDP-GalNAz) that is also a substrate for GalT Y289L
but allows for more selective and robust reactivity in comparison to previous
strategies (Figure 1-3) (98). Utilizing UDP-GalNAz and a corresponding alkyne-
containing tetramethyl-6-carboxyrhodamine (alkyne-TAMRA), Clark et al re-
ported the first fluorescence visualization of O-GlcNAc modified proteins and,
following enrichment with an anti-TAMRA antibody, subjected the labelled pro-
teins, which were isolated from rat forebrains, to mass-spectroscopy analysis.
Over 200 proteins were identified, most of which were novel substrates. Not sur-
prisingly, the O-GlcNAc-modified proteins identified are implicated in a variety
of cellular processes including, but not limited to, metabolism and signal trans-
duction. The UDP-GalNAz chemical reporter has also been utilized with an
alkyne-biotin affinity reagent for O-GlcNAc enrichment and subsequent mass-
24
spectroscopy analysis (Figure 1-4A) (37,104). First, proteins were trypsin di-
gested, and the ensuing peptides subjected to chemoenzymatic transfer of Gal-
NAz onto all GlcNAc-residues, including any potential terminal GlcNAc residues
on cell-surface N-linked glycans. The labeled peptides were then treated with
PNGase-F in order to remove any unwanted glycans, and the resulting peptides
reacted with the photo-cleavable biotin-PEG-alkyne tag (biotin-PEG-PC-alk).
Following streptavidin enrichment, the samples were subjected to β-elimination
Michael addition (BEMAD) which replaces the GlcNAc-GalNAz-Alkyne-Biotin
tag with a dithiothrietol residue and allows for site identification. Smith and co-
workers also employed the UDP-GalNAz chemical reporting strategy in conjunc-
tion with the biotin-PEG-PC-alk tag but did not subject proteins to BEMAD prior
to alternating electron-transfer dissociation (ETD) and collision-induced disso-
ciation (CID) mass spectroscopy analysis (Figure 1-4A) (105). Identification was
determined from the photo-cleaved peptides for 274 O-GlcNAc substrates and
458 sites of modification (105).
Visualization/Affinity Tags
In addition to reporter development, advances in fluorescence and affinity tag
chemistries have enabled researchers to more readily and robustly visualize and
identify O-GlcNAc modified proteins. These tags are also amenable to investigat-
ing the dynamics and function of O-GlcNAcylation.
25
Figure 1-4. Methods for enrichment, identification and interrogation of O-GlcNAc
modification on proteins. (A) Enrichment of GalNAz-labelled proteins with biotin-PEG-
PC-alkyne. GalNAz-labelled cell lysate is subjected to treatment with PNGase-F and CIP
followed by CuAAC with biotin-PEG-PC-alkyne. Biotinylated lysate is enriched using
streptavidin beads and modified proteins subjected to UV cleavage prior to mass spec-
troscopy. (B) Investigation of O-GlcNAc modification of proteins using the mass-shift
tag. Ketogalactosamine-labelled cell lysate was reacted with an aminooxy-PEG mass-tag,
thus shifting the molecular weight of the protein incrementally with respect to number of
sites of modification. SDS-PAGE and Western blotting of labelled lysates reveals number
of residues modified by O-GlcNAc. (C) Enrichment and identification of O-GlcNAcylated
substrates using a metabolic chemical reporter. GlcNAlk-modified lysate is subjected to
CuAAC with azido-azo-biotin and the biotinylated fraction enriched with streptavidin
beads. Chemical cleavage of the enriched lysate using sodium dithionite liberated
GlcNAlk-modified proteins for proteomics experiments.
Mass-shift tag
While a myriad of proteins are known to be O-GlcNAcylated, little is known about
the dynamics and in vivo levels of O-GlcNAc on proteins. Mass spectroscopy can
identify sites of O-GlcNAc modification, but it does not determine the number of
O-GlcNAcylation events on a protein at a given time. In order to address this, the
Hsieh-Wilson laboratory developed a mass-tag approach in which ketogalactose-
26
labeled lysate can be selectively reacted with an aminooxy-bearing PEG mass-tag
(Figure 1-4B) (75). Proteins can then be separated by SDS-PAGE, and Western
blotting for a protein of interest reveals distinctive mass shifts corresponding to
each site of glycosylation. This approach has been utilized to investigate the gly-
cosylation for several known O-GlcNAc modified proteins including the tran-
scription factor CREB, which is implicated in long-term memory formation and
neronal plasitcity, and the metabolic enzyme PFK1, which contributes to cell pro-
liferation and tumorigenesis (75,79).
Cleavable Linkers
To improve retrieval of GalNAz-GlcNAc-modified peptides after enrichment,
Hart and coworkers optimized the biotin affinity tag (37,104). A new linker
briefly described above, termed PC-PEG-biotin-alkyne, is equipped with a termi-
nal biotin tag, a photocleavable 1,2-(nitrophenyl)ethyl moiety and the requisite
alkyne functionality (Figure 1-4A). Following enrichment and BEMAD, the pep-
tides are eluted and are now tagged with an aminomethyltriazole, a basic residue
that allows for improved fragmentation of the tryptic peptide and thus improved
MS signal (Figure 1-4A). Employing this technology, glycosylation sites on several
proteins implicated in neurodegenerative disease were identified from target-
protein-enriched rat brains, including eight O-GlcNAc sites on the protein tau
(37,104). In separate experiment using a tandem CTD/ESI mass spectroscopy
strategy that circumvented the use of BEMAD, which cleaves potential PTMs of
27
interest in addition to the GalNAz-GlcNAc disaccharide, hundreds of other O-
GlcNAcylated proteins and their sites were also identified using PC-PEG-biotin-
alkyne (Figure 1-4A) (105).
A chemical alternative to the photocleavable linker has also been utilized as an
enrichment tool in identifying O-GlcNAc modified proteins labelled with chemi-
cal reporters (Chapter 2) (91). Equipped with an reducible azo bond, azido- or
alkyne-azo-biotin can be selectively reacted with the corresponding alkyne- or
azide-equipped O-GlcNAc chemical reporter for subsequent enrichment and
Western blotting and/or proteomics (Figure 1-4C) (106,107). We reported the use
of these cleavable biotin tags for the identification of GlcNAlk-modified proteins
(Chapter 2) (91,108). Due to the improved specificity of our chemical reporters
under optimized labeling conditions, we employed GlcNAlk in conjunction with a
chemically cleavable azido-azo-biotin tag to enrich for GlcNAlk-modified proteins
and identify them by mass spectroscopy. Over 350 putatively O-GlcNAc-modified
proteins were identified, 279 of which were previously unknown.
Small molecule modulators of O-GlcNAc modification
Small-molecule regulation of O-GlcNAc dynamics is a desirable therapeutic tar-
get in human disease, and, as a result, efforts have been made to develop inhibi-
tors of OGT and OGA.
28
OGT inhibitors
The first OGT inhibitor identified was the uracil analog alloxan (Figure 1-5B)
(109) .The proposed mechanism of action is such that alloxan binds to the uracil
pocket of OGT, rendering the enzyme inactive (110). Unfortunately, the com-
pound is cytotoxic, unstable and has off-target effects (109). The Walker labora-
tory developed a high-throughput screening assay for OGT inhibition and identi-
fied 3 candidate small-molecules from the Institute of Chemistry and Cell Biology
library at Harvard Medical School (111). One of these compounds, an irreversible
inhibitor containing a benzoxazolinone (BZX) scaffold, was demonstrated to de-
crease global O-GlcNAcylation in breast cancer cells upon treatment at 500 µM
for 48 hr (BZX1, Figure 1-5B) (22). As a testament to the importance of O-GlcNAc
modification in cancer cell growth and proliferation, treatment with this inhibitor
revealed a decrease in MCF-10A-ErbB2 cell growth and invasion in both soft agar
assays as well as 3D cell culture experiments. The Walker lab further improved
the potency of this drug by building a small suite of molecules and identified an
inhibitor equipped with a ketone on the BZX moiety as well as a p-methoxy group
on the adjoining phenyl ring (BZX2, Figure 1-5B) (112).A crystal structure of this
diphosphate-mimetic inhibitor reveals that the molecule irreversibly crosslinks
the active-site lysine and a neighboring cysteine, thus rendering the enzyme inac-
tive (112).
29
Two sub-millimolar OGT inhibitors were developed in the van Aalten lab: a gly-
cosylthiophosphate analog of UDP-GlcNAc, termed UDP-S-GlcNAc and C-UDP,
an α,β-methylene bisphosphonate of UDP (Figure 1-5B) (110). These small mole-
cules were shown to successfully bind to the active site of OGT in a manner simi-
lar to that of native UDP-GlcNAc, as determine by crystal structure, but failed to
effectively decrease O-GlcNAc levels in living cells. Recently, the Vocadlo lab re-
ported the development of a low-micromolar and highly-selective inhibitor of
OGT (113). 1,3,4,5-tetraacetyl-2-acetamido-2-deoxy-5-thio-D-glucopyranose
(Ac5SGlcNAc, Figure 1-5B, 9.8) effectively enters the HBP by way the of GlcNAc
salvage pathway in a manner similar to the metabolic chemical reporters and re-
sults in the generation of UDP-5SGlcNAc, an inhibitor of OGT. For synthesis of
Ac5SGlcNAc see Chapter 9.
30
Figure 1-5. A selection of small molecules to study O-GlcNAc modification. (A) Meta-
bolic chemical reporters of O-GlcNAc. (B) Small molecules for O-GlcNAc modulation
have been developed to chemically raise (OGA inhibitors) or lower (OGT inhibitors) O-
GlcNAc levels.
OGA inhibitors
Horsch et al first reported the development of a (phenylcarbamoyl)oxime analog
of GlcNAc, termed PUGNAc, as a reversible, nanomolar inhibitor of O-GlcNAcase
(Figure 1-5B) (114). Unfortunately, in the same report, the small molecule was
also shown to inhibit lysosomal Hexosaminidases A and B (HEX-A and HEX-B).
Hart and coworkers showed in 3T3-L1 adipocytes that increasing O-GlcNAc lev-
els by treatment with PUGNAc prevents Akt phosphorylation, resulting in the in-
creased activation of Akt and thus insulin resistance (57). However, use of an-
31
other, more specific OGA inhibitor, NButGT (Figure 1-5B, see below) has not
been able to recapitulate these results, suggesting that Akt activation is an off-
target effect of PUGNAc treatment (61). Recently it has been shown that PUGNAc
treatment increases levels of membrane-localized free oligosaccharides including
gangliosides due to HEX-A and HEX-B inhibition, and this incomplete turnover
of N-linked glycan structures mimics conditions of lysosomal storage disorders
(115). Importantly, accumulation of gangliosides has been shown to induce insu-
lin resistance, suggesting a possible mechanism by which PUGNAc treatment re-
sults in decreased insulin sensitivity (116).
The antibiotoic streptozotocin (STZ) is a GlcNAc analog containing a nitrosamino
substitution that has been shown to inhibit OGA at micromolar concentrations
(Figure 1-5B) (117). Toleman et al reported the mechanism of OGA inhibition
through the generation of a transition state analog that is not amenable to hydro-
lytic release (118). Importantly, STZ does not inhibit HEX-A and HEX-B. How-
ever, STZ is known to induce diabetes through the release of its nitric oxide moi-
ety (119). Additionally, internucleosomal DNA damage is also a side effect of STZ
treatment.
Employing a variety of fluorinated GlcNAc analogs, Macauley et al determined
that the catalytic mechanism of OGA involves anchimeric assistance, in which the
2-acetamido group is indispensable in the formation and subsequent hydrolysis
32
of a bicyclic oxazoline intermediate (120). With a greater understanding of the
biochemical role of the N-acetate functional group, a more specific, sub-
micromolar OGA inhibitor was developed by Vocadlo and coworkers, 1,2-
dideoxy-2’-methyl-α-D-glucopyranoso-[2,1-d]-Δ2’-thaizoline or NAG-thiazoline,
which mimics the bicyclic intermediate but cannot be broken down (120). Fur-
ther optimization of the bicyclic structure resulted in an even more specific small-
molecule termed NButGT that is 1200-fold more selective for OGA than PUGNAc
(Figure 1-5B) (61). Unfortunately, these thiazoline analogs, while specific for
OGA, are not stable in media for prolonged periods of time (23,60). Nevertheless,
NButGT was successfully employed as an OGA inhibitor in rodents, and, impor-
tantly, while O-GlcNAc levels were increased, insulin resistance as well as pertur-
bations to glucose homeostasis were not reported even at 8 months of treatment
(23).
GlcNAcstatin is a picomolar inhibitor of bacterial OGA that is 10
6
-fold more se-
lective for OGA than hexosaminidases (121). However, this bicyclic carbohydrate
analog is synthetically rigorous to make and has not yet been tested in mammal-
ian cells (68). Another small-molecule OGA inhibitor 6-acetamido-6-deoxy-
castanospermine (6-Ac-Cas) has been shown to elevate O-GlcNAc levels (Ki value
300 nM) and does not induce insulin resistance in cell culture (60). However, 6-
Ac-Cas is also an inhibitor of HEX-B with similar efficacy (Ki value 250 nM) (60).
Importantly, however, Vocadlo and co-workers employed 6-Ac-Cas, PugNAc and
33
NButGT to show that insulin resistance that is exhibited upon PUGNAc treat-
ment can be attributed to HEX-B inhibition and not to OGA inhibition (60).
Thiamet-G, the most potent (Ki value = 21 nM) and selective small molecule in-
hibitor of OGA, was initially reported by Yuzwa et al and the binding profile is
similar to that of NButG (Figure 1-5B) (68). Thiamet-G was initially employed for
both in vitro and in vivo manipulation of O-GlcNAc levels on protein Tau, a
microtubule-associated protein that is oligomerized in neurodegerative diseases
including Alzheimer’s (68). Tau is both O-GlcNAc modified as well as phosphory-
lated, and it has been shown that treatment with Thiamet-G decreases phospho-
rylation of the protein and also that O-GlcNAc modification on its own, inde-
pendent on Tau’s phosphorylation state, can stabilize the protein as well as de-
crease the formation of Tau-containing aggregates (68,69). Importantly,
Thiamet-G can cross the blood-brain barrier and subsequently inhibit OGA activ-
ity as seen by a dose-dependent increase in O-GlcNAc levels in mouse brains fol-
lowing intravenous delivery of the small molecule (69). Therefore, Thiamet-G is
the first known OGA inhibitor that is also a candidate for use as a therapeutic
treatment.
Conclusion
While the biochemical role of O-GlcNAc modification continues to be defined,
there are still large gaps in our understanding. For instance, the precise role(s) of
34
O-GlcNAc in diabetic insulin signaling remain to seen. Also, the mechanism by
which glycosylation protects cells during stress is still poorly understood. In addi-
tion to understanding how O-GlcNAc affects its substrates, there is need to fur-
ther investigate the enzymes that mediate the modification, OGT and OGA. There
are 3 isoforms of OGT, short, nuclear/cytosolic and mitochondrial, but their sub-
strate specificity and biological function are not well characterized.
In order to further understand the the consequences of O-GlcNAcylation, several
chemical tools and techniques have been developed. However, these technologies
are still lacking in their ability to quantitatively modify and identify O-GlcNAc
substrates. The chemoenzymatic chemical reporting strategy requires a cumber-
some protocol and commercially available kits are not quantitative as sold. The
promiscuity of metabolic chemical reporters poses a challenge, and reporters that
label specific types of glycosylation must be developed. Additionally, mass spec-
troscopy techniques must be optimized in order to preserve native GlcNAc link-
ages during proteomics experiments. Concerning the development of small-
molecule modulators of O-GlcNAc, there have been some impressive advances
but molecules amenable to therapeutic treatment have yet to be developed. The
crystal structure of human OGT has been solved, which should lend to the devel-
opment of more-potent, drug-like inhibitors (122). While the structure of the
human isoform of OGA has not been solved, bacterial analogs have been crystal-
lized and should also contribute to drug discovery (123,124). With continued de-
35
velopment in chemical approaches and mass spectroscopy, the role of O-
GlcNAcylation in cell biology and human disease will be revealed.
36
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Chapter Two. A New Metabolic Chemical Reporter Reveals
Novel O-GlcNAc Modified Proteins Including the Ubiquitin
Ligase NEDD4-1
*
Introduction
The glycosylation of serine and threonine residues by the monosaccharide
GlcNAc (O-GlcNAc) is an important and abundant post-translational modifica-
tion (PTM) found in a wide range of organisms from Arabidopsis thaliana to
humans (1,2). This nuclear and cytoplasmic modification is dynamic (3), through
addition and removal by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA),
respectively, and can compete with phosphorylation, setting it up as key regulator
of signaling pathways (4,5). A variety of proteins have been shown to be O-
GlcNAc modified including regulators of transcription and translation, cytoskele-
tal proteins, signaling proteins, and metabolic enzymes. O-GlcNAc modification
has been shown to affect protein function through changes in protein localization
(6), stability (7), molecular interactions (8-10), and enzymatic activity (11); how-
ever, the biochemical consequences of many O-GlcNAc modification events are
unknown.
59
*
Yu-Ying Yang and Howard C. Hang (The Rockefeller University) contributed to the work pre-
sented in this chapter.
Despite an incomplete molecular understanding of O-GlcNAc modification of
many proteins, this PTM has been implicated in many biological processes. O-
GlcNAc is indispensable for development in both mice and Drosophila because
knockouts of OGT are lethal (12,13). In addition, Caenorhabditis elegans mutants
lacking OGT or OGA display defects in nutrient storage and Dauer formation
(14,15), and O-GlcNAc is also involved in metabolism and related diseases such as
type II diabetes (16). Global O-GlcNAc modification of proteins has also been
shown to be elevated when cells are subjected to a variety of cellular injuries in-
cluding heat stress, oxidative stress, hypoxia, ischemia reperfusion, and trauma
hemorrhage (17,18) and additionally in several different types of cancers (19-21).
Finally, several lines of evidence point toward O-GlcNAc playing a key role in
neuronal function and neurodegeneration (22). For example, O-GlcNAc is en-
riched at synapses (23,24), and mice with neuron-specific deletion of OGT dis-
play locomotor defects before neonatal death (25). In addition, components of
proteinaceous aggregates found in neurodegenerative diseases, including the mi-
crotubuleassociated protein tau and α-synuclein (26), are modified with O-
GlcNAc, and reduced levels of O-GlcNAc have been observed in Alzheimer’s dis-
ease (16,27).
Because O-GlcNAc is clearly important in a wide range of cellular processes, a
handful of complementary methods have been developed for the identification
and visualization of O-GlcNAcylation. Teo et al. recently generated a small panel
60
of O-GlcNAc-specific IgG monoclonal antibodies (28). When used together, they
can overcome any individual antibody’s requirement for different but overlapping
peptide determinants resulting in the identification of over 200 proteins. As a
complement to antibodies, two bioorthogonal reactions, Staudinger ligation and
Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC) (29,30), have been
used for the analysis of PTMs and small molecule–protein interactions. This ap-
proach has been applied to analyze protein glycosylation (31), acetylation (32),
and lipidation (33-35), as well as covalent small molecule inhibitors (36,37). One
method that takes advantage of CuAAC is a version of the chemoenzymatic tech-
nology developed by the Hsieh-Wilson laboratory (38-42) and subsequently
commercialized by Invitrogen. Specifically, an engineered β-1,4-
galactosyltransferase (Y298L GalT) is used to transfer an azide-containing mono-
saccharide donor, UDP-GalNAz, to O-GlcNAc-modified proteins. The azide can
then be reacted under CuAAC conditions with fluorescent and biotin tags for
visualization and identification, respectively. Because the enzymatic addition of
GalNAz is quantitative, this approach is exquisitely sensitive and has been used to
identify over 200 O-GlcNAc-modified proteins, including many that overlap with
those identified by antibodies.
Another approach, originally developed by Bertozzi and coworkers, relies on the
promiscuity of the hexosamine biosynthetic pathway (HBP) to metabolically de-
liver azides into O-GlcNAc modified proteins (43-47). Cells can be treated with
61
the fully protected azide-bearing monosaccharide Ac4GlcNAz or Ac4GalNAz. After
deprotection by cellular enzymes, GlcNAz can enter the HBP by action of GlcNAc
kinase, and GalNAz can be metabolically transformed to GlcNAz, resulting in
modification of proteins by GlcNAz (Figure 2-1A). This approach was first used to
react O-GlcNAc-modified proteins with biotin and peptide tags using the Staud-
inger ligation (Figure 2-1B) for visualization and identification by Western blot-
ting and mass spectrometry (43-46), resulting in the discovery of approximately
200 proteins when using GlcNAz and 18 proteins when using GalNAz (47). How-
ever, these immunoblotting methods are not ideal for analyzing quantitative
changes in O-GlcNAc modification required for investigations of dynamics and
regulation. In addition, several experiments have shown that CuAAC outperforms
the Staudinger ligation for the analysis of cell lysates (33,37,48). However, the
enrichment of GlcNAz-labeled proteins using CuAAC only resulted in the identifi-
cation of 32 proteins (46).
We describe here an improvement in the use of metabolic chemical reporters of
O-GlcNAc in combination with CuAAC conditions that resulted in the identifica-
tion of 374 proteins, including many previously unidentified potential O-GlcNAc
substrates. In addition, we observed robust fluorescent visualization of O-GlcNAz
and successful metabolic incorporation and visualization of an alkyne functional-
ized GlcNAc analogue, termed GlcNAlk. We performed a cellular analysis of the
metabolic fates of both GlcNAz and GlcNAlk, along with their corresponding ga-
62
lactose isomers, GalNAz and GalNAlk, demonstrating that GlcNAlk may be a
more specific reporter of O-GlcNAc modification than GlcNAz. Finally, we used
GlcNAlk to discover O-GlcNAcylation of the ubiquitin ligase NEDD4-1. NEDD4-1
has been shown to regulate the cell surface turnover of a handful of transmem-
brane receptors, participate in proliferative signaling in cancer, and facilitate the
budding of viral particles including HIV (49,50). Despite its importance in a vari-
ety of biological processes, the regulation of NEDD4-1 is not well understood.
Our discovery of the O-GlcNAcylation of NEDD4-1 potentially uncovers an im-
portant regulatory mechanism.
63
Figure 2-1. The HBP and chemical reporters of O-GlcNAc modification. (A) Glucose,
glutamine, and acetyl-CoA are transformed by the metabolic enzymes of the HBP to
UDP-GlcNAc, which can be used by OGT to modify protein substrates and reversed by
OGA. Per-O-acetylated analogues are deacetylated by intracellular esterases and then
enter the GlcNAc salvage pathway. (B) GlcNAz and GlcNAlk bearing proteins can be co-
valently modified with detection tags using CuAAC.
Results
Fluorescent Detection of O-GlcNAc-Labeled Proteins.
Recent studies by ourselves and others have demonstrated improved detection of
metabolic chemical reporters with fluorescent tags under CuAAC reaction condi-
tions (34,41), suggesting that O-GlcNAz visualization could be improved. We
therefore synthesized Ac4GlcNAz (2.1, Figure 2-1A) and the corresponding
64
alkyne-bearing fluorescent detection tag, alkynyl-rhodamine (Alk-Rho, 2.2, Fig-
ure 2-2A) to explore the detection of O-GlcNAc-modified proteins by CuAAC.
HEK293 cells were metabolically labeled with various concentrations of Ac4Glc-
NAz for 16 h in low-glucose medium (1 g⁄L), thereby maximizing the uptake of our
chemical reporter through the GlcNAc salvage pathway. The cells were then
washed, lysed, and the soluble fraction was reacted with Alk-Rho under CuAAC
conditions. In-gel fluorescence scanning revealed robust labeling of a variety of
proteins at all concentrations of Ac4GlcNAz tested (Figure 2-2B). Previous com-
parative analyses of chemical reporters have demonstrated that alkynyl-chemical
reporters, in combination with the azido-detection tags, have improved sensitiv-
ity when compared to the reverse CuAAC orientation due to decreased back-
ground signal (34,48,51). Because of this observation, we next asked if the orien-
tation of the CuAAC azide and alkyne partners could be reversed (Figure 2-1B).
Toward this end, we synthesized the alkyne-modified GlcNAc analogue,
Ac4GlcNAlk (2.3, Figure 2-1A), and azide-bearing fluorescent detection tag (Az-
Rho, 2.4, Figure 2-2A). HEK293 cells were metabolically labeled under identical
conditions to GlcNAz and reacted with Az-Rho. In-gel fluorescence scanning
again revealed robust detection of a large number of proteins (Figure 2-2B). Im-
portantly the profile of GlcNAlk-labeled proteins was similar to GlcNAz, suggest-
ing that the two carbohydrates are metabolically equivalent. At high contrast, the
GlcNAlk orientation did indeed display lower background signal when compared
to GlcNAz (Figure 2-2C), which may prove critical at lower concentrations of
65
metabolic chemical reporters and for specific O-GlcNAc substrates that are modi-
fied at substoichiometric levels.
Figure 2-2. Fluorescence detection of O-GlcNAc-modified proteins by GlcNAz and
GlcNAlk. (A) Fluorescent-tags Alk-Rho and Az-Rho for CuAAC-dependent detection of
GlcNAz and GlcNAlk. (B) HEK293 cells were treated with the indicated concentrations of
Ac4GlcNAz or Ac4GlcNAlk for 16 h and analyzed by in-gel fluorescence scanning.
Coomassie blue staining demontrated equal protein loading. (C) Higher contrast of lanes
corresponding to 0 µM in B to allow for comparison of background levels. (D) HEK293
cells were treated with 200 µM Ac4GlcNAz or Ac4GlcNAlk for the indicated times in low-
glucose (1 g/mL) media for the indicated times, followed by in-gel fluorescence scanning.
(E) HEK293 cells were treated with 200 µM Ac4GlcNAz or Ac4GlcNAlk for the indicated
times in low-glucose (1 g/mL) media and chased with Ac4GlcNAc (200 µM) for the indi-
cated times, followed by in-gel fluorescence scanning.
66
GlcNAz and GlcNAlk Are Metabolically Incorporated and Removed at Similar
Rates.
Cellular analysis of O-GlcNAc modifications with chemical reporters requires that
they are efficiently metabolized by the HBP, added to proteins by OGT, and re-
moved by OGA (Figure 2-1A). The metabolic processing of GlcNAz by the en-
zymes of the GlcNAc salvage pathway has been previously analyzed in vitro (43),
demonstrating that GlcNAz is efficiently processed by the GlcNAc salvage path-
way and used by OGT and OGA. To qualitatively determine if GlcNAlk is metab-
olically incorporated as efficiently as GlcNAz, HEK293 cells were treated with
Ac4GlcNAz (200 µM) or Ac4GlcNAlk (200 µM) for different lengths of time. In-
gel fluorescence scanning after CuAAC revealed that proteins are modified with
GlcNAz and GlcNAlk at similar rates (Figure 2-2D), suggesting that GlcNAlk is
also an efficient substrate for the GlcNAc salvage pathway and OGT. A diverse
spectrum of labeled proteins was detectable in as little as 2 h with fluorescence,
representing an improvement compared to previous reports using the Staudinger
ligation or CuAAC with immunotags and Western blotting, where a time course
was not reported or only a subset of proteins was detected (43-46). To determine
if GlcNAlk is also a reliable substrate for OGA, HEK293 cells were labeled with
Ac4GlcNAz (200 µM) or Ac4GlcNAlk (200 µM) for 16 h. At this time, the cells
were washed with PBS and media containing Ac4GlcNAc (200 µM) was added.
Cells were then harvested at different times, lysed, and the soluble fractions re-
67
Type to enter text
acted with the appropriate rhodamine tag. Analysis by in-gel fluorescence scan-
ning showed that the chemical-reporter-dependent fluorescence signal was lost at
the same rate (Figure 2-2E), suggesting that O-GlcNAlk-modified proteins are
efficient substrates for OGA. Importantly the global half-life of the chemical re-
porter modification (t1⁄2 = 12–24 h) corresponds well with specific O-GlcNAc-
modified proteins that were analyzed by radioactive glucosamine treatment (α-
crystallin t1⁄2 = ∼10 h; cytokeratin t1⁄2 = ∼55 h) (52,53).
GlcNAz and GlcNAlk Have Multiple Metabolic Fates.
To take maximum advantage of environmental nutrients, mammalian cells are
equipped with enzymes that enable crosstalk between various saccharide biosyn-
thetic pathways including the HBP (Figure 2-3A). GlcNAz and GlcNAlk could
therefore have multiple metabolic fates and access multiple glycosylation path-
ways that could all contribute to fluorescent signal. In addition to O-GlcNAc,
GlcNAc is also directly incorporated into the conserved core pentasaccharide of
N-linked glycans or the branches of both N-linked and mucin-type O-linked oli-
gosaccharides (blue pathway, Figure 2-3A). Additionally, UDP-GlcNAc can be en-
zymatically converted to UDP-GalNAc and subsequently incorporated at the core
of mucin-type O-linked glycans (red pathway, Figure 2-3A). As stated above,
GlcNAz has been incorporated into O-GlcNAc (43) and other studies have shown
that GalNAz can be interconverted to GlcNAz and be similarly incorporated
(47,54). In addition, GalNAz is also incorporated into mucin-type O-linked gly-
68
cans (47,55). Despite previous experiments, these metabolic chemical reporters
have not been compared directly on both a specific cell-surface protein and a O-
GlcNAc-modified protein, and the alkyne analogs (GlcNAlk and GalNAlk) have
not been previously reported.
Figure 2-3. Characterizing GlcNAz and GlcNAlk protein labeling. (A) Monosaccharide
chemical reporters have several possible metabolic fates. GlcNAz and GlcNAlk can enter
the glucosamine salvage pathway (blue pathway) and potentially label O-GlcNAc-
modified proteins and N-linked glycans. They could also be reversibly converted to the
corresponding GalNAc analogs, GalNAz and GalNAlk (red pathway), resulting in the po-
tential labeling of mucin-type O-linked glycosylation. (B and C) COS-7 cells were trans-
fected with a plasmid encoding GlyCAM-IgG, treated with the indicated chemical re-
porter, and analyzed by in-gel fluorescence scanning. PNGase-F treatment prior to
CuAAC was performed to selectively remove N-linked glycans. (D - G) COS-7 cells were
transfected with a plasmid encoding FoxO1A, treated with the indicated chemical re-
porter, and analyzed by in-gel fluorescence. For comparison experiments D - G, fluores-
cence levels were measured and normalized simultaneously.
69
To perform this comprehensive analysis, we first used the chimeric glycoprotein,
GlyCAM-IgG (56), which contains both N-linked and mucin-type O-linked gly-
cans. GlyCAM-IgG was expressed in COS-7 cells treated with Ac4GlcNAz (200
µM) or Ac4GlcNAlk (200 µM) in high-glucose (4.5 g⁄mL) media. As a control for
GalNAc metabolic labeling, we also synthesized Ac4GalNAz (2.5) and Ac4GalNAlk
(2.6) and simultaneously expressed GlyCAM-IgG in the presence of these sugars
under identical conditions. Immunoprecipitation followed by CuAAC and in-gel
fluorescence scanning revealed robust chemical reporter dependent labeling in
the case of all sugars (Figure 2-3B). This result is somewhat contradictory to pre-
vious reports where significantly less Ac4GlcNAz incorporation into cell surfaces
compared to Ac4GalNAz was detected by the Staudinger ligation (57). These re-
sults could simply reflect a difference in the secondary labeling chemistry (Staud-
inger ligation vs. CuAAC), treatment time (3 d vs. 16 h), concentration of chemi-
cal reporter used (50 vs. 200 µM), and/or proteins analyzed (global cell surface
vs. GlyCAM-IgG) (55). To determine if GlcNAz or GlcNAlk were incorporated into
the N-linked glycan of the Ig domain, metabolically labeled GlyCAM-IgG was
treated with Peptide N-glycosidase F (PNGase-F) prior to CuAAC (Figure 2-3B).
Significant loss of signal was observed for both GlcNAz and GlcNAlk, revealing
that they are both incorporated into N-linked glycans, whereas much less signal
was lost from GalNAz in mucin-type glycans (55). Next, we expressed GlyCAM-
IgG in cells treated with our four chemical reporters (200 µM) in low-glucose (1
g⁄L) media. We observed significantly less metabolic labeling across all chemical
70
reporters (Figure 2-3C), suggesting that, under conditions of low glucose, both
GlcNAc and GalNAc derivatives may be funneled into different metabolic path-
ways or there may be a general decrease in cell surface glycosylation.
We next used an insulin-insensitive mutant of FoxO1, FoxO1A (58), that has been
previously shown to be constitutively O-GlcNAc modified. FLAG-tagged FoxO1A
was expressed in COS-7 cells treated with one of our four chemical reporters (200
µM) in low-glucose (1 g⁄L) media. FoxO1A was then enriched, subjected to
CuAAC, and analyzed by in-gel fluorescence scanning and Western blotting (Fig-
ure 2-3D and Figure 2-3F). As expected, both GlcNAz and GlcNAlk exhibit robust
labeling of FoxO1A. In addition, GalNAz shows labeling of FoxO1A, consistent
with interconversion from previous reports (47,54). In contrast, GalNAlk displays
very low global labeling of cellular proteins (Figure 2-3F) and modification of
FoxO1A. We attribute the multiple bands in our fluorescent gel and Western blot-
ting to phosphorylation (59) and/or ubiquitination (60) of FoxO1A. We next re-
peated this experiment in COS-7 cells in high-glucose (4.5 g⁄L) media, where the
biosynthesis of competitive UDP-GlcNAc should be greater, and essentially no
labeling of FoxO1A was observed (Figure 2-3E and Figure 2-3G). In addition,
global labeling of proteins by GlcNAz, GlcNAlk, and to a lesser extent GalNAz,
was reduced, whereas GalNAlk labeling remained unchanged (Figure 2-3G).
71
Taken together, these data suggest unique metabolic fates for each chemical re-
porter: GlcNAz and GlcNAlk are incorporated into both the IgG domain N-linked
glycan (Figure 2-3B) and O-GlcNAc on FoxO1A. However, because we observe
very low global levels of labeling of these reporters under high-glucose condi-
tions, which is required for robust GlyCAM-IgG labeling, this incorporation must
be limited to a subset of N-linked glycans. In addition, GalNAz labels both mucin-
type glycans on GlyCAMIgG and O-GlcNAc on FoxO1A confirming previous ex-
periments (47,54,55). Because this interconversion is reversible, GlcNAz could
result in metabolic conversion to GalNAz and labeling of mucin-type O-linked
glycans. In contrast, our second chemical reporter, GalNAlk, is distinct from the
three other monosaccharides tested. GalNAlk has notably lower global levels of
labeling (Figure 2-3F) but labels GlyCAM-IgG strongly and not FoxO1A, suggest-
ing UDP-GalNAlk can be generated in cell but not efficiently interconverted to
GlcNAlk.
Different Cell Types Have Diverse Labeling Patterns.
Given that our chemical reporters efficiently label a known O-GlcNAc-modified
protein, we tested the generality of GlcNAlk against a panel of cell lines. HEK293,
CHO, COS-7, HeLa, Jurkat, Mcf-7, and NIH3T3 cells were treated with
Ac4GlcNAlk (200 µM) for 16 h. In-gel fluorescence scanning revealed a striking
diversity in the intensity and pattern of O-GlcNAc-modified proteins (Figure 2-
4). Although some O-GlcNAc-modified proteins are common among the different
72
cell types, unique patterns are readily apparent (Mcf-7 and NIH3T3 cells). In ad-
dition, the global levels of O-GlcNAc modification vary greatly, even between cell
types with similar modification patterns (HEK293 and Jurkat cells). These data
demonstrate the ability of our chemical reporters and fluorescent detection
method to monitor specific changes in O-GlcNAc modification patterns and levels
that are key to understanding specific cellular outcomes.
Figure 2-4. The incorporation efficiency of GlcNAlk in a variety of cell lines. The indi-
cated cells types were treated with Ac4GlcNAlk (200 µM) for 16 h, lysed and reacted with
azido-rhodamine under CuAAC conditions, and analyzed by in-gel fluorescence scan-
ning. Coomassie blue staining demonstrated protein loading.
O-GlcNAc-Modified Proteins Can Be Identified with GlcNAlk.
To identify O-GlcNAc-modified proteins, we performed a large-scale enrichment
using GlcNAlk as a chemical handle. NIH3T3 cells in low-glucose media (1 g⁄L)
were treated with Ac4GlcNAlk (200 µM), lysed under denaturing conditions (4%
SDS), and reacted with an azido-biotin cleavable affinity tag (azido-azo-biotin,
2.10, Scheme 2-1 and Figure 2-5A) using CuAAC. Labeled proteins were enriched
73
with streptavidin beads and selectively eluted with sodium dithionite (Figure 2-
5B). The specificity of GlcNAlk-dependent retrieval was demonstrated by
Coomassie blue staining (Figure 2-5C), and the corresponding proteins were
identified by gel-based proteomic mass spectrometry.
Scheme 2-1. Synthesis of azido-azo-biotin. (2.10). (a) Imidazole-1-sulfonyl Azide HCl,
Na2CO3, CuSO4, H2O, MeOH, 16 h, RT, 99% yield; (b) i. NaNO2, 6M HCl, 4-
aminobenzoic acid ii. Et3N, THF, 19% yield over 2 steps. (c) DCC, N-hydroxysuccinimide,
THF, 99% yield. (d) biotin-PEG-NH2, DMF, 52% yield.
Identified proteins were compiled and categorized into high- and medium-
confidence lists based on the number of assigned spectra and the fold increase
above control (DMSO vehicle treated) samples (Tables 2-1 and 2-2). We identi-
fied 374 proteins by GlcNAlk labeling, representing proteins with diverse cellular
functions (Figure 2-5D), with 142 and 232 proteins falling into the high- and
medium-confidence lists respectively (Tables 2-1 and 2-2). Of these proteins, 63
(44%) of high- 32 (14%) of medium-confidence hits have been previously re-
ported using large-scale proteomic techniques including elimination-addition
chemistry (61), metabolic chemical reporters (44-47), enzymatic extension
74
(26,39-41,62), lectin affinity chromatography (63), and O-GlcNAc-specific anti-
bodies (28). We also identified 23 proteins that we determined could not be O-
GlcNAc modified due to their localization in the secretory pathway, the cell mem-
brane, or extracellular space. All of these proteins have confirmed (7 proteins) or
potential (16 proteins) N-linked glycosylation sites, confirming the PNGase-F
sensitive GlcNAlk labeling of Glycam-IgG (Figures 2-3B and C).
75
Figure 2-5. Selective enrichment and identification of O-GlcNAc-modified proteins. (A)
Structure of the chemically cleavable azido-azo-biotin tag. (B) GlcNAlk-modified pro-
teins are reacted under CuAAC conditions with azido-azo-biotin and enriched by incuba-
tion with streptavidin beads. After several washes, azido-azo-biotin can be cleaved with
Na2S2O4 (50 mM) and enriched proteins were identified by gel based proteomics. (C) In-
puts and biotin enriched proteins were stained with Coomassie blue. (D) O-GlcNAc-
modified proteins were enriched from NIH3T3 cells after treatment with Ac4GlcNAlk
(200 µM).
To confirm the proteins identified above, we performed Western blot analysis of
the GlcNAlk enriched proteome using antibodies against the known O-GlcNAc
substrate p62 (64) and three previously uncharacterized proteins (NEDD4-1,
HP1, and Lamin A), confirming their specific recovery (Figure 2-6A). In addition,
76
we transfected COS-7 cells with a construct encoding α-B crystallin, which is only
O-GlcNAc modified at 5–10%, and were able to use our biotin enrichment strat-
egy selectively recover this protein (Figure 2-6A). We next asked if we could visu-
alize the incorporation of GlcNAlk using our fluorescent tags. Nuclearporin p62
was immunoprecipitated from NIH3T3 cells, and fluorescent signal was selec-
tively detected from cells treated with Ac4GlcNAlk (Figure 2-6B). We also de-
tected fluorescent signal from α-B crystallin, from COS-7 cells, confirming our
ability to detect substoichiometrically modified proteins (Figure 2-6B).
Figure 2-6. Identification of O-GlcNAlk-modified proteins. (A) O-GlcNAlk-modified
proteins were enriched from NIH3T3 cells treated with Ac4GlcNAlk (200 µM) using
azido-azo-biotin and analyzed by Western blotting. (B) Known O-GlcNAcylated proteins
p62 and α-B crystallin were immunoprecipitated from cells treated with Ac4GlcNAlk
(200 µM) and analyzed by in-gel fluorescence. (C) NEDD4-1 was selectively enriched
from cells treated with Ac4GlcNAlk (200 µM) and analyzed by in-gel fluorescence. (D)
NEDD4-1 was immunoprecipitated from cells and analyzed by Western blotting.
We next focused on the O-GlcNAc modification of the ubiquitin ligase NEDD4-1.
The family of NEDD4-like E3 ligases represent a small but important subset of
enzymes capable of transferring ubiquitin to protein substrates. The founding
member of this class of E3 ligases, NEDD4-1, plays an important role in neuronal
development, cell metabolism, receptor endocytosis, and tumorigenesis and cell
growth (49,50). However, the regulation of NEDD4-1 remains poorly understood.
77
To confirm O-GlcNAc modification of NEDD4-1, HA-tagged NEDD4-1 was ex-
pressed in both HEK293 and COS-7 cells. After immunoprecipitation and CuAAC
with azido-rhodamine, GlcNAlk-dependent fluorescent signal was readily de-
tected on NEDD4-1 (Figure 2-6C). Finally, NEDD4-1 was expressed and im-
munoprecipitated from HEK293 cells and O-GlcNAc modification was detected
using an anti-O-GlcNAc antibody (CTD110.6) (Figure 2-6D), demonstrating that
NEDD4-1 is a bona fide O-GlcNAc substrate.
Discussion
The modification of cytosolic and nuclear proteins by O-GlcNAc is indispensable
for proper cellular function. To allow for the detection of O-GlcNAc modification,
we have developed chemical reporters that can metabolically enter the GlcNAc
salvage pathway, allowing for robust fluorescent detection of O-GlcNAc modified
proteins using CuAAC. Although the azide-modified GlcNAc analog, GlcNAz, has
been used previously to detect O-GlcNAc-modified proteins using immunoblot-
ting methods, our alkyne analog, GlcNAlk, in combination with an azide bearing
fluorophore, may yield a more specific mode for detection of O-GlcNAc-modified
proteins after CuAAC because it may not be converted to GalNAlk. Comparison of
GlcNAz and GlcNAlk revealed that the analogs are metabolically incorporated at
similar rates into a large collection of cellular proteins, and they are both re-
moved from proteins at rates consistent with published radioactive O-GlcNAc
probes.
78
We next analyzed the metabolic fates of GlcNAz and GlcNAlk. To determine if
GlcNAz or GlcNAlk could label N-linked or mucin-type O-linked glycans, we used
the chimeric, secreted protein GlyCAM-IgG. Under high-glucose culture condi-
tions, both GlcNAz and GlcNAlk are incorporated into GlyCAM-IgG. Much of this
labeling was PNGase-F sensitive, demonstrating that our chemical reporters are
incorporated into N-linked glycans. Under the same set of conditions, treatment
of cells with GlcNAz and GlcNAlk does not result in detectable labeling of a
known O-GlcNAc-modified protein, FoxO1A, and very little global protein label-
ing occurs. In contrast, under low-glucose conditions, GlyCAM-IgG labeling by
GlcNAz and GlcNAlk is greatly reduced, and the same chemical reporters robus-
tly label FoxO1A. As controls for metabolic conversion of GlcNAz and GlcNAlk to
the corresponding galactose analogs, we also synthesized GalNAz and GalNAlk.
Consistent with prior experiments, under high-glucose conditions the majority of
GalNAz is incorporated into mucin-type O-linked glycans (55). Under low-
glucose conditions, GlyCAMIgG labeling is reduced, and FoxO1A is labeled by
GalNAz, through transformation to GlcNAz and subsequent O-GlcNAc modifica-
tion (47,55). We hypothesize that GalNAz modifies a combination of mucin-type
O-linked glycans and O-GlcNAc-modified proteins and that the ratio of these
modifications can be adjusted by cell culture conditions. Our second chemical re-
porter, GalNAlk, also labels GlyCAM-IgG under high-glucose conditions. The
global levels of GalNAlk labeling are low, and it is not readily converted to
79
GlcNAlk as FoxO1A is not labeled by GalNAlk. Further experiments will be
needed to test the exact location of this monosaccharide.
Because GlcNAlk and GalNAlk appear to not be interconverted, GlcNAlk may be
a more specific reporter of O-GlcNAc modification when compared to GlcNAz
and GalNAz. We therefore used a cleavable biotin affinity tag (azido-azo-biotin)
in combination with GlcNAlk to identify 374 proteins, including 279 proteins that
had not been previously identified as potential O-GlcNAc substrates. Because
GlcNAlk is also potentially incorporated into N-linked glycans, we also identified
23 N-linked glycosylated proteins that we determined could not contain O-
GlcNAc modification sites due to their localization. Despite these contaminating
proteins, the over 10-fold difference in number of proteins identified supports
our conclusion that the majority of GlcNAlk-labeled proteins are potential O-
GlcNAc substrates. Additionally we confirmed the presence of O-GlcNAc as the
first glycosylation event on NEDD4-1. We are currently focusing on understand-
ing the affects of O-GlcNAcylation on NEDD4-1 stability, localization, and inter-
action with substrates.
Conclusion
In summary, the methods described here represent an improvement in the analy-
sis of O-GlcNAc-modified proteins with metabolic chemical reporters. The use of
fluorescent reporters, combined with CuAAC, allows for the dynamic analysis of
80
O-GlcNAc modification levels and patterns that cannot be readily determined by
published immunoblotting methods. Our analysis of different monosaccharide
chemical reporters reveals that, in contrast to previously reported GlcNAz(43)
and GalNAz(47), GlcNAlk may not be readily interconverted with GalNAlk, mak-
ing it more specific for O-GlcNAc modification. In addition, unlike methods that
measure steady-state levels of modification, the metabolic chemical reporters
could be used to examine the dynamic removal of O-GlcNAlk in a pulse–chase
format, which we are currently exploring.
Materials and Methods
All reagents used for chemical synthesis were purchased from Sigma-Aldrich un-
less otherwise specified and used without further purification. All anhydrous re-
actions were performed under argon atmosphere. Analytical TLC was conducted
on Silica Gel 60 F
254
plates (EMD Chemicals) with detection by ceric ammonium
molybdate (CAM), anisaldehyde, or UV. For flash chromatography, 60 Å silica gel
(EMD Chemicals) was utilized. Electrospray ionization mass spectrometry (ESI-
MS) was performed using a Shimadzu liquid chromatography (LC)-MS 2020.
1
H
spectra were obtained at 600 MHz on a Varian VNMRS-600 unless otherwise
specified. Chemical shifts are recorded in ppm (δ) relative to CHCl3 (7.26 ppm)
for spectra acquired in CDCl3.
13
C spectra were obtained at 200 MHz on the same
instrument.
81
Chemical Synthesis.
Known compounds peracetylated N-azidoacetylglucosamine (GlcNAz; (65)),
1,3,4,6-Tetra-O-Acetyl-N-4-pentynylglucosamine (GlcNAlk; (46), Peracetylated
N-azidoacetylgalactosamine (GalNAz; (55)), N-(6-(diethylamino)-9-(2-(4-hept-6-
ynoylpiperazine -1-carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-
ethylethanaminium (alk-rho; (34)), N-(9-(2-(4-(6-azidohexanoyl)piperazine-1-
carbonyl)phenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminiu
m (az-rho; (34)), and azido-azo-biotin (32) were synthesized according to litera-
ture procedures as described below.
Compound 2.1 1,3,4,6-Tetra-O-Acetyl-N-azidoacetylglucosamine
(Ac4GlcNAz). Known azidoacetic acid (343 mg, 3.40 mmol,
(66)) was dissolved in DMF (10 mL) under Argon. To this was
added pyridine (0.300 mL, 4.11 mmol) and pentafluorophenyl-
trifluoroacetate (0.707 mL, 4.11 mmol). The reaction was allowed to proceed for 1
h. Upon completion, as determined by TLC, the reaction was diluted with EtOAc,
washed with 1 M HCl, saturated NaHCO3, H2O and brine. The organic layer was
dried over MgSO4, filtered and concentrated to afford the crude pentaflouro-
phenyl ester that required no further purification. Known 1,3,4,5-Tetra-O-Acetyl-
glucosamine hydrochloride (1.00 g, 2.61 mmol, (67)) was coevaporated from
toluene 3 times and resuspended in DMF (20 mL) under Argon. DIEA (0.910 mL,
O
AcO
NH
OAc
N
3
O
OAc
AcO
82
5.21 mmol) was added and the reaction stirred for 15 min. The azidoacetyl-
pentaflourophenyl ester ( 886 mg, 3.13 mmol) was then added the reaction al-
lowed to proceed overnight. Upon completion, the reaction was concentrated, di-
luted in EtOAc, washed with saturated NaHCO3, H2O and brine. The organic
layer was dried over MgSO4, filtered and concentrated to afford the crude. Col-
umn chromatography(50% EtOAc in Hexanes) yielded the pure product (337 mg,
23 % yield).
1
H NMR (600 MHz, CDCl3) β-anomer δ 6.47 (d, J = 9.3 Hz, 1H), 5.79
(d, J = 8.7 Hz, 1H), 5.25 (dd, J = 10.5, 9.3 Hz, 1H), 5.14 (t, J = 19.2, 9.0 Hz, 1H),
4.28 (dd, J = 12.5, 4.7 Hz, 1H), 4.23 (ddd, J = 10.5, 9.3, 8.7 Hz, 1H), 4.13 (dd, J =
12.5, 2.3 Hz, 1H), 3.91 (d, J = 1.0 Hz, 2H), 3.84 (ddd, J = 9.9, 4.7, 2.3 Hz, 1H),
2.11 (s, 3H), 2.09 (s, 3H), 2.04 (d, J = 1.4 Hz, 6H).
Compound 2.2 N-(6-(diethylamino)-9-(2-(4-hept-6-ynoylpiperazine-1-
carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-
ethylethanaminium (alk-rho). This procedure was
adapted from Charron et al (34).6-Heptynoic acid
(56 µL, 0.220 mmol) was diluted in anhydrous DMF
(20 mL) under Argon and 1-1’-Carbonyl diimidazole (72 mg, 0.220 mmol) was
added. The reaction was allowed to proceed for 1 h after which time rhodamine B
piperazine amide ((68), 200 mg, 0.183 mmol) was added and the reaction was
allowed to proceed overnight. Upon completion, the reaction was concentrated to
afford crude. Column chromatography (80% EtOAc/13% MeOH/7% H2O)
O Et
2
N NEt
2
N
N
O
O
83
yielded the pure product (113 mg, 50% yield).
1
H NMR (600 MHz, CD3OD) δ 7.76
- 7.72 (m, 2H), 7.70 – 7.64 (m, 1H), 7.50 - 7.46 (m, 1H, 7.24 (dt, J = 6.7, 2.4 Hz,
2H), 7.04 (dd, J = 9.6, 2.5 Hz, 2H), 6.98 (dd, J = 9.6, 2.5 Hz, 2H), 3.65 (q, J = 7.2
Hz, 8H), 3.30 (d, J = 0.7 Hz, 1H), 3.27 (p, J = 1.6 Hz, 4H), 2.33 (dd, J = 8.1, 6.9
Hz, 1H), 2.18 - 2.12 (m, 2H), 1.27 (dt, J = 9.8, 5.4 Hz, 21H). ESI-MS calculated for
C39H47N4O3 [M]
+
619.36, found 619.10.
Compound 2.3 1,3,4,6-Tetra-O-Acetyl-N-4-pentynylglucosamine
(Ac4GlcNAlk). Known 1,3,4,5-Tetra-O-Acetyl-glucosamine
hydrochloride (1.00 g, 2.61 mmol, (67)) was coevaporated
from toluene 3 times and resuspended in CH2Cl2 (20 mL)
under Argon. To this was added 4-pentynoic acid (332 mg, 3.39 mmol) and TEA
(908 µL, 6.51 mmol) and the reaction stirred for 10 min, allowing the gluco-
samine to go into solution. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was
then added (999 mg, 5.21 mmol) and the reaction proceeded for 15 min prior to
addition of dimethylaminopyridine (3.2 mg, 0.0261 mmol) after which time the
reaction proceeded overnight. Upon completion, the reaction was diluted with
CH2Cl2 (100 mL), washed with 1 M HCl (100 mL), saturated NaHCO3, H2O and
brine. The organic layer was dried over MgSO4, filtered and concentrated to af-
ford the crude. Column chromatography (2% MeOH in CH2Cl2) yielded the pure
product (1.060 g, 73% yield). 1H NMR (400 MHz, CDCl3) β-anomer δ 5.82 (d, J =
9.4 Hz, 1H), 5.70 (d, J = 8.8 Hz, 1H), 5.22 – 5.08 (m, 2H), 4.37 – 4.23 (m, 2H),
O
AcO
NH
OAc
O
OAc
AcO
84
4.12 (dd, J = 12.5, 2.3 Hz, 1H), 3.85 - 3.81 (m, 1H), 2.51 – 2.45 (m, 2H), 2.35 –
2.29 (m, 2H), 2.11 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.97 (t, J = 2.6
Hz, 1H).
Compound 2.4 N-(9-(2-(4-(6-azidohexanoyl)piperazine-1-carbonyl)phenyl)-
6-(diethylamino)-3H-xanthen-3-ylidene)-N-
ethylethanaminium (az-rho). This procedure was
adapted from Charron et al (34). Azidohexanoic
acid ((66), 48 mg, 0.300 mmol) was diluted in an-
hydrous DMF (20 mL) under Argon and 1-1’-Carbonyl diimidazole (49 mg, 0.300
mmol) was added. The reaction was allowed to proceed for 1 h after which time
rhodamine B piperazine amide ((68), 150 mg, 0.276 mmol) was added and the
reaction was allowed to proceed overnight. Upon completion, the reaction was
concentrated to afford crude. Column chromatography (80% EtOAc/13% MeOH/
7% H2O) yielded the pure product (105 mg, 58% yield).
1
H NMR (600 MHz,
CD3OD) δ 7.71 – 7.65 (m, 2H), 7.63 – 7.58 (m, 1H), 7.46 – 7.39 (m, 1H), 7.18 (d, J
= 9.5 Hz, 2H), 6.87 (d, J = 2.5 Hz, 2H), 3.59 (q, J = 7.1 Hz, 8H), 3.35 – 3.25 (m,
8H), 2.26 (t, J = 7.5 Hz, 2H), 1.48 (pd, J = 7.5, 4.3 Hz, 5H), 1.33 – 1.25 (m, 3H),
1.21 (t, J = 7.1 Hz, 12H).
O Et
2
N NEt
2
N
N
O
O
N
3
85
Compound 2.5 1,3,4,6-Tetra-O-Acetyl-N-azidoacetylgalactosamine
(Ac4GalNAz). The synthesis was conducted according to Hang
et al (55). D-Galactosamine·HCl (1.00 g, 4.64 mmol) was coe-
vaporated from toluene and resuspended in anhydrous MeOH
(20 mL) under Argon. Freshly prepared 1 M NaOMe in MeOH
(4.64 mL, 4.64 mmol) was added dropwise and the solution stirred for 30 min at
room temperature. Chloroacetic anhydride (1.19 g, 6.96 mmol) was added to the
reaction, followed by TEA (970 µL, 6.96 mmol), and the reaction was allowed to
proceed overnight. Upon completion, the reaction was quenched with saturated
NaHCO3,if necessary, and concentrated to afford crude. The reaction was further
purified using column chromatography (20% MeOH in CHCl3). The purified ma-
terial was coevaporated with toluene and dissolved in DMF (30 mL) under Ar-
gon. To this, NaN3 (1.508 g, 23.3 mmol) was added and the reaction was warmed
to 50 ℃ under Argon and reacted for 48 h. Upon completion the reaction was
concentrated, resuspended in pyridine (20 mL) and acetic anhydride (2.19 mL,
23.2 mmol) added. The reaction stirred for 16 h, after which time it was concen-
trated, resuspended in CH2Cl2, washed with 1 M HCl , saturated NaHCO3, H2O
and brine. The organic layer was dried over MgSO4, filtered and concentrated to
afford the crude. Column chromatography (65% EtOAc in Hexanes) yielded the
pure product (754 mg, 38% yield)
1
H NMR (500 MHz, CDCl3) δ 6.49 (d, J = 9.3
Hz, 1H), 5.82 (d, J = 8.8 Hz, 1H), 5.40 – 5.38 (m, 1H), 5.24 (dd, J = 11.2, 3.4 Hz,
O
AcO
NH
OAc
N
3
O
OAc
AcO
86
1H), 4.36 (dt, J = 11.3, 9.0 Hz, 1H), 4.18 – 4.05 (m, 4H), 3.90 (s, 2H), 2.16 (s,
4H), 2.11 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H).
Compound 2.6 1,3,4,6-Tetra-O-Acetyl-N-4-pentynylgalactosamine
(Ac4GalNAlk). To a solution of 4-pentynoic acid (1.50 g,
15.29 mmol) in CH2Cl2 (20 mL) under Argon was added
N,N'-Dicyclohexylcarbodiimide (1.58 g, 7.65 mmol) and the
reaction stirred overnight. Upon completion, the reaction
was diluted with CH2Cl2, filtered and concentrated. This process was repeated 4
times to get rid of any remaining side products (dicyclohexylurea) and the anhy-
dride was used below with no further purification.
Galactosamine·HCl (806 mg, 3.74 mmol) was resuspended in anhydrous MeOH
(15 mL) under Argon. To the flask, freshly-prepared 1 M NaOMe in MeOH (3.74
mL, 3.74 mmol) was added dropwise and the reaction was allowed to stir 30 min
as the carbohydrate went into solution. 4-Pentynoic anhydride (1.00 g, 5.61
mmol) was added followed by TEA (1.56 mL, 11.2 mmol) and the reaction was al-
lowed to proceed overnight. Upon completion the reaction was quenched with
saturated NaHCO3 (200 µL) and concentrated to afford crude. Column chroma-
tography (10%-20% MeOH in CH2Cl2 with 1% acetic acid) afforded the purified
unprotected sugar N-4-pentynylgalactosamine (GalNAlk, 909 mg, 94% yield).
O
AcO
NH
OAc
O
OAc
AcO
87
In an ice bath, N-4-pentynylgalactosamine (909 mg, 3.51 mmol) was resus-
pended in pyridine (7.63 mL, 94.3 mmol) and acetic anhydride (4.22 mL, 44.8
mmol). A catalytic amount of dimethylaminopyridine was added and the reaction
was allowed to stir 16 h, slowly warming to room temperature. Upon completion
the reaction was concentrated to remove most of the pyridine. The reaction mix-
ture then resuspended in CH2Cl2 (1000 mL), washed with 1 M HCl (1x), neutral-
ized with a saturated sodium bicarbonate solution (1x), and washed with H2O
(3x) and brine. The organic layer was then dried over sodium sulfate and concen-
trated. Purification by silica gel column chromatography (2% MeOH⁄98% CH2Cl2)
afforded the product (909 mg, 61% yield, 57% yield over 2 steps).
1
H NMR (600
MHz, CDCl3) β-anomer: δ 6.04 (d, J = 8.4, 1H), 5.73 (d, J = 9.0, 1H), 5.36 (dd, J =
1.2, 3.6, 1H), 5.13 (dd, J = 3.0, 11.4, 1H), 4.48 (dt, J = 9.6, 10.8, 1H), 4.17 (m, 2H),
4.06 (t, J = 6.6, 1H), 2.47 (m, 2H), 2.33 (t, J = 7.2, 2H), 2.15 (s, 3H), 2.10 (s, 3H),
2.05 (s, 3H), 1.99 (s, 3H), 1.96 (s, 1H).
13
C NMR (125 MHz, CDCl3) β-anomer: δ
171.38, 170.64, 170.45, 170.22, 169.55, 92.81, 82.63, 71.70, 70.31, 69.47, 66.38,
61.37, 49.48, 35.45, 20.95, 20.70, 20.66, 20.65, 14.91. ESI-MS calculated for
C19H25NO10 [M + Na]
+
450.14, found 450.00.
Compound 2.7 4-(2-azidoethyl)phenol. Tyramine (3.00 g, 26.2 mmol) was dis-
solved in MeOH (100 mL) and stirred at room temperature. To
this was added sodium carbonate (4.64 g, 43.7 mmol) and
copper sulfate (66 mg, 0.262 mmol). Imidazole-1-sulfonyl
HO
N
3
88
Azide HCl ((66), 5.50 g, 26.2 mmol) was then added and reaction stirred over-
night. Upon completion, the mixture was concentrated, diluted with H2O (100
mL), acidified with concentrated HCl and extracted with EtOAc 3 x (100 mL).
The combined organic layers were then dried over Na2SO4, filtered and concen-
trated to afford crude. The crude material was further purified using column
chromatography (15% EtOAc in Hexanes) to afford the product (3.54 g, 99%
yield).
1
H NMR (600 MHz, CDCl3) δ 7.09 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.5 Hz,
2H), 3.46 (t, J = 7.2 Hz, 3H), 2.83 (t, J = 7.2 Hz, 3H).
Compound 2.8 (E)-4-((5-(2-azidoethyl)-2-hydroxyphenyl)azenyl)benzoic
acid. To an ice-cooled suspension of 4-
aminobenzoic acid (900 mg, 6.55 mmol) in 6 M
HCl (10 mL) was added NaNO2 and the solution
turned yellow. In a separate flask, 2.7 (1.19 g, 7.30
mmol) was resuspended in pre-chilled THF (5 mL) and the solution was placed in
an ice bath. K2CO3 was added to reach a desired pH of 8. After 40 min, the diazo-
nium salt was added dropwise to flask containing 2.7 and the pH was maintained
using K2CO3. Upon completion of addition, the reaction was allowed to stir for 30
min. Reaction completion was determined by TLC. Once the reaction was com-
plete, the mixture was concentrated, redissolved in EtOAc (100 mL), washed with
1M HCl 3x (100 mL) and dried over Na2SO4. The organic layer was then filtered
and concentrated to afford the crude. Purification by column chromatography
HO
N
N
O
N
3
HO
89
afforded the product (430 mg, 19% yield).
1
H NMR (500 MHz, CD3OD) δ 8.24 –
8.15 (m, 2H), 8.04 – 7.95 (m, 2H), 7.86 (q, J = 3.1 Hz, 1H), 7.35 (dd, J = 8.6, 5.7
Hz, 1H), 7.06 – 6.95 (m, 1H), 3.65 – 3.52 (m, 2H), 2.98 – 2.88 (m, 2H).
Compound 2.9 (E)-2,5-dioxopyrrolidin-1-yl4-((5-(2-azidoethyl)-2-hydroxy-
phenyl)azenyl) benzoate. Under Argon to 2.8
(100 mg, 0.321 mmol) dissolved in anhydrous
THF (12 mL) w as ad d e d N ,N ' -
Dicyclohexylcarbodiimide (73.0 mg, 0.354 mmol)
and N-hydroxysuccinimide (41.0 mg, 0.354 mmol). The reaction was stirred at
room temperature for 4 h or until complete as determined by TLC. Upon comple-
tion, the reaction was concentrated and the crude redissolved in chilled CH2Cl2.
The urea was then filtered off and the filtrate concentrated. This was repeated 2 x.
The crude was then purified by column chromatography (30% EtOAc in Hex-
anes) to afford the product (129 mg, 99% yield).
1
H NMR (600 MHz, CDCl3) δ
8.30 (d, J = 8.9 Hz, 2H), 7.99 (d, J = 8.9 Hz, 2H), 7.86 (d, J = 2.2 Hz, 1H), 7.30 –
7.28 (m, 1H), 7.03 (d, J = 8.5 Hz, 1H), 3.58 (t, J = 7.1 Hz, 2H), 2.95 (t, J = 7.1 Hz,
6H).
Compound 2.10 (E)-4-((5-(2-azidoethyl)-2-hydroxyphenyl)azenyl)-
N-(15-oxo-19-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14
-azanonadecyl)benzamide (azido-azo biotin). To 2.9 (32.0 mg, 0.080 mmol) in
HO
N
N
O
N
3
O N
O
O
90
anhydrous DMF (6 mL) was added biotin-PEG-amine (50.0 mg, 0.119 mmol) un-
der Argon. The reaction
stirred overnight at
room temperature.
Upon completion, the crude was concentrated, resuspended in a minimal amount
of CH3CN:H2O (1:1) and subjected to HPLC purification(CH3CN: 5% - 40% in 10
min, then 40% - 100% in 40 min). The product was collected at 33 min as a yel-
low solid (29.6 mg, 52 % yield).
1
H NMR (400 MHz, CD3OD) δ 8.04 (d, J = 0.7
Hz, 4H), 7.88 (d, J = 2.3 Hz, 1H), 7.38 (dd, J = 8.5, 2.3 Hz, 1H), 7.03 (d, J = 8.4
Hz, 1H), 4.51 – 4.46 (m, 1H), 4.29 (dd, J = 7.9, 4.5 Hz, 1H), 3.74 – 3.51 (m, 18H),
3.36 (d, J = 5.6 Hz, 3H), 3.22 – 3.15 (m, 1H), 2.96 (t, J = 7.0 Hz, 3H), 2.71 (d, J =
12.8 Hz, 1H), 2.20 (t, J = 7.4 Hz, 2H), 1.66 (tdt, J = 21.8, 13.8, 7.0 Hz, 5H), 1.43
(q, J = 7.5 Hz, 2H). ESI-MS calculated for C33H45N9NaO7S [M + Na]
+
734.8,
found 734.4.
Cell Culture.
HEK293, CHO, COS-7, HeLa, MCF-7, and NIH3T3 cells were cultured in high-
glucose DMEM media (HyClone, Thermo-Scientific) enriched with 10% FCS
(HyClone, Thermo-Scientific). Jurkat cells were cultured in RPMI medium 1640
enriched with 10% FCS. All cell lines were maintained in a humidified incubator
at 37 °C and 5.0% CO2.
O
O
O
H
N
HO
N
N
HN
O
N
3
O
S
HN
NH
O
91
Metabolic Labeling.
To cells at 80–85% confluency, high- or low-glucose media containing Ac4Glc-
NAz, Ac4GlcNAlk, Ac4GalNAz, Ac4GalNAlk (1,000x stock in DMSO), or DMSO
vehicle was added as indicated. For chase experiments, low-glucose media was
replaced with high-glucose media supplemented with 200 µM Ac4GlcNAc
(Sigma).
Preparation of Nonidet P-40 (NP-40)-Soluble Lysates.
Cells were then collected by trypsinization and pelleted by centrifugation 4 °C for
3 min at 2,000 × g, followed by washing with PBS (1 mL) two times. Cell pellets
were then resuspended in 100 µL of 1% NP-40 lysis buffer [1% NP-40, 150 mM
NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete EDTA-free Mini pro-
tease inhibitor cocktail (Roche Biosciences) for 10 min and then centrifuged at 4
°C for 10 min at 10,000 × g. The supernatant (soluble cell lysate) was collected
and the protein concentration was determined by bicinchoninic acid (BCA) assay
(Pierce, Thermo-Scientific).
Cu(I)-Catalyzed [3 + 2] Azide-Alkyne Cycloaddition.
Cell lysate (200 µg) was diluted with cold 1% NP-40 lysis buffer to obtain a de-
sired concentration of 1 µg⁄µL. Newly-made click chemistry cocktail (12 µL) was
added to each sample [azido- or alkynyl-rhodamine tag (100 µM, 10 mM stock
solution in DMSO); tris (2-carboxyethyl)phosphine hydrochloride (1 mM, 50 mM
92
freshly-prepared stock solution in water); tris[(1-benzyl-1-H-1,2,3-triazol-4-
yl)methyl]amine (100 µM, 10 mM stock solution in DMSO); CuSO4 •5H2O (1 mM,
50 mM freshly prepared stock solution in water)] for a total reaction volume of
200 µL. The reaction was gently vortexed and allowed to sit at room tempera-
ture for 1 h. Upon completion, 1 mL of ice-cold methanol was added to the reac-
tion, and it was placed at −80 °C for 2 h to precipitate proteins. The reactions
were then centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was re-
moved, the pellet was allowed to air dry for 5 min, and then 50 µL 4% SDS buffer
(4% SDS, 150 mM NaCl, 50 mM TEA pH 7.4) was added to each sample. The
mixture was sonicated in a bath sonicator to ensure complete dissolution, and 50
µL of 2x loading buffer (20% glycerol, 0.2% bromophenol blue, 1.4% β-
mercaptoethanol) was then added. The samples were boiled for 5 min at 97 °C,
and 30 µg of protein was then loaded per lane for SDS-PAGE gel separation (4–
20% Tris • HCl Criterion Gel, Bio-Rad).
In-Gel Fluorescence Scanning.
Following SDS-PAGE gel separation, the gel was incubated in destaining solution
(50% methanol, 40% H2O, 10% glacial acetic acid) for 5 min followed by H2O for
an additional 5 min prior to scanning. The gel was scanned on a Molecular Im-
ager FX (Bio-Rad) using a 580-nm laser for excitation and a 620-nm bandpass
filter for detection.
93
FoxO1A Labeling.
COS-7 cells in 150 × 25 mm plates at 95% confluency were transfected with cDNA
encoding FoxO1A (32 µg, Addgene plasmid 13508) using Lipofectamine 2000
(Invitrogen) according to manufacturer’s procedures. After 6 h, media was re-
placed with high-glucose DMEM with 10% FCS. After 16 h, media was replaced
with high- or low-glucose DMEM with 10% FCS and 200 µM Ac4GlcNAc,
Ac4GlcNAlk, Ac4GalNAz, Ac4GalNAlk, or Ac4GlcNAc (200 mM stock in DMSO).
After 10 h, media was replaced with fresh high- or low-glucose DMEM with 10%
FCS and 200 µM Ac4GlcNAc, Ac4GlcNAlk, Ac4GalNAz, Ac4GalNAlk, or Ac4Glc-
NAc (200 mM stock in DMSO). After 16 h, cells were washed with PBS,
trypsinized, and pelleted.
COS-7 cell pellets were resuspended in 20 µL H2O, 0.2 µL O-(2-acetamido-2-
deoxy-D-glucopyranosylidene)amino-N-phenyl carbamate (PUGNAc, 100 mM
stock solution in DMSO, Tocris), and 36 µL 0.05% SDS buffer (0.05% SDS, 5 mM
MgCl2, 10 mM TEA pH 7.4) with Complete EDTA-free Mini protease inhibitor
cocktail (Roche Biosciences). Samples were then treated with 1 µL Benzonase
(Sigma) and incubated on ice for 30 min. To this mixture was added 145 µL of 4%
SDS buffer (4% SDS, 5 mM MgCl2, 1 mM TEA pH 7.4). The samples were then
sonicated briefly in a bath sonicator and centrifuged at 10,000 × g for 10 min at
15 °C. Protein concentrations were then normalized using the BCA assay (Pierce,
Thermo-Scientific). Total cell lysate (1.5 mg) was diluted as necessary to a final
94
volume of 1 mL with 1% NP-40 buffer with Complete Mini protease inhibitor
cocktail (Roche Biosciences). EZview Red anti-FLAG M2 affinity gel (40 µL,
Sigma), washed with PBS three times, were added to each sam- ple, and the sam-
ples were placed on a rotator for 2 h at 4 °C.
Beads were collected by centrifugation at 3,000 × g for 2 min at 4 °C, and the su-
pernatant was carefully removed. Beads were then washed with 500 µL PBS three
times. The final PBS wash was carefully removed and the beads were suspended
in 40 µL 4% SDS buffer. Samples were then boiled for 5 min at 97 °C to elute pro-
teins. The appropriate amount of click chemistry cocktail was added and the reac-
tion was allowed to proceed for 1 h, after which time 40 µL of 2x loading buffer
was added. Samples were boiled for 5 min at 97 °C. Protein samples (40 µg) were
then loaded per lane for SDS-PAGE gel separation (4–20% Tris • HCl Criterion
Gel, Bio-Rad).
GlyCAM-IgG Labeling.
COS-7 cells in 150 × 25 mm plates at 95% confluency were transfected with cDNA
encoding GlyCAM-IgG (32 µg, Addgene plasmid 17841) using Lipofectamine
2000 (Invitrogen) according to manufacturer’s procedures. After 5 h, media was
changed. After 16 h, cells were trypsinized and split 4∶10 into 100 × 20 mm plates
in high- or low-glucose DMEM with 10% FCS and 200 µM Ac4GlcNAc,
95
Ac4GlcNAlk, Ac4GalNAz, Ac4GalNAlk, or Ac4GlcNAc in DMSO (1,000x stock in
DMSO).
After 24 h, the media from each sample was collected by centrifugation at 3,000
× g for 10 min at 4 °C to remove cell debris. The supernatant was incubated with
500 µL of recombinant protein G sepharose beads (Invitrogen) in 100 mM TEA,
pH 8 overnight. Beads were collected by centrifugation at 3,000 × g for 2 min at
4 °C. Beads were washed three times with 3 mL 100 mM TEA, pH 8 to remove
media. GlyCAM-Ig was eluted by addition of 500 µL 2% SDS buffer (2% SDS, 150
mM NaCl, 50 mM TEA pH 7.4) and boiling for 5 min at 97 °C. Protein concentra-
tion was determined by BCA assay (Pierce, Thermo-Scientific). Final SDS concen-
tration was diluted to 0.5% by addition of 50 mM TEA pH 7.4. Protein (60 µg)
was then treated with Peptide N-glycosidase F (PNGase-F, New England Bio-
labs), according to manufacturer’s procedures, or left untreated. The appropriate
amount of click chemistry cocktail was added and the reaction was allowed to
proceed for 1 h after which time 4x loading buffer (40% glycerol, 0.4% bromo-
phenol blue, 2.8% β-mercaptoethanol) was added. Samples were boiled for 5 min
at 97 °C and 50 µg were loaded for SDS-PAGE gel separation (4–20% Tris • HCl
Criterion Gel, Bio-Rad).
96
p62 Labeling.
Recombinant protein G sepharose beads (45 µL, Invitrogen) were incubated
overnight with MAb414 (Covance) in a solution of 3∶2 non-ionic buffer (1.66%
Triton X-100, 3.3% BSA, 2 mM EDTA, 100 mM NaCl, 50 mM TEA, pH 7.4) to
NP-40 buffer (1% NP-40, 150 mM NaCl, 50 mM TEA, pH 7.4) at a ratio of 1.2 mg
antibody/mL beads. Beads incubated overnight were collected by centrifugation
at 3,000 × g for 2 min at 4 °C. The supernatant was removed and beads resus-
pended and gently vortexed in 3∶2 nonionic to NP-40 buffers. The beads were
collected again, supernatant decanted, and beads resuspended in 45 µL of PBS.
NIH3T3 cell pellets were resuspended in 34 µL H2O and 66 µL 0.05% SDS buffer
(0.05% SDS, 5 mM MgCl2, 10 mM TEA, pH 7.4) with Complete Mini protease in-
hibitor cocktail (Roche Biosciences). Samples were then treated with 2 µL Benzo-
nase (Sigma) and incubated on ice for 30 min. To this mixture was added 100 µL
of 1% SDS buffer (1% SDS, 5 mM MgCl2, 1 mM TEA, pH 7.4). The samples were
then sonicated briefly in a bath sonicator and centrifuged at 20,000 × g for 10
min at 15 °C. Protein concentrations were then normalized using the BCA assay
(ThermoScientific). Total cell lysate (1 mg) was diluted as necessary to a final vol-
ume of 200 µL with 1% SDS buffer, and then 300 µL of nonionic buffer with pro-
tease inhibitor was added to each sample. Freshly prepared anti-p62 beads (7.5
µL) were then added to each sample, and samples were incubated on a rotator at
4°C for 2h.
97
Beads were collected by centrifugation at 2,000 × g for 2 min at 4 °C, and the su-
pernatant was carefully removed. Beads were then washed with 500 µL washing
buffer (0.1% Triton X-100, 300 mM NaCl, 10 mM TEA, pH 7.4) three times, fol-
lowed by PBS twice. The final PBS wash was carefully removed, and the beads
were suspended in 50 µL 4% SDS buffer. The appropriate amount of click chem-
istry cocktail was added, and the reaction was allowed to proceed for 1 h after
which time 50 µL of 2x loading buffer was added. Samples were boiled for 5 min
at 97 °C. Protein samples (40 µg) were then loaded per lane for SDS-PAGE gel
separation (4–20% Tris • HCl Criterion Gel, Bio-Rad).
α-B Crystallin Labeling.
COS-7 cells in 150 × 25 mm plates at 85% confluency were transfected with cDNA
encoding HA-α-B Crystallin (32 µg) using Lipofectamine 2000 (Invitrogen) ac-
cording to manufacturer’s procedures. After 5 h, media was replaced with high-
glucose DMEM with 10% FCS. After 16 h, media was replaced with low-glucose
DMEM with 10% FCS and 200 µM Ac4GlcNAlk or DMSO vehicle. After 8 h, me-
dia was again replaced with fresh DMEM with 10% FCS and 200 µM Ac4GlcNAlk
or DMSO vehicle. After 16 h, cells were washed with PBS, trypsinized, and pel-
leted.
98
COS-7 cell pellets were resuspended in 10 µL H2O, 0.2 µL PUGNAc (100 mM
stock solution in DMSO, Tocris) and 18 µL 0.05% SDS buffer (0.05% SDS, 5 mM
MgCl2, 10 mM TEA, pH 7.4) with Complete EDTA-free Mini protease inhibitor
cocktail (Roche Biosciences). Samples were then treated with 0.5 µL Benzonase
(Sigma) and incubated on ice for 30 min. To this mixture was added 72.5 µL of
4% SDS buffer (4% SDS, 5 mM MgCl2, 1 mM TEA, pH 7.4). The samples were
then sonicated briefly in a bath sonicator and centrifuged at 10,000 × g for 10
min at 15 °C. Protein concentrations were then normalized using the BCA assay
(Pierce, Thermo-Scientific). Total cell lysate (1 mg) was diluted as necessary to a
final volume of 1 mL with 1% NP-40 buffer with Complete Mini protease inhibitor
cocktail (Roche Biosciences) with a final concentration of 0.25% SDS. To each
sample was added 10 µL HA.11 clone 16B12 monoclonal antibody (Covance), and
samples were incubated on a rotator at 4 °C for 16 h.
Recombinant protein G sepharose beads (10 µL, Invitrogen), resuspended in an
equal volume of PBS were then added to each sample. The samples incubated on
a rotator at 4 °C for 2 h. Upon completion, beads were collected by centrifugation
at 2,000 × g for 2 min at 4 °C, and the supernatant was carefully removed. Beads
were then washed with 200 µL PBS three times. The final PBS wash was carefully
removed, the beads were suspended in 25 µL 4% SDS buffer and boiled for 5 min
at 97 °C. The appropriate amount of click chemistry cocktail was added, and the
reaction was allowed to proceed for 1 h after which time 25 µL of 2x loading
99
buffer was added. Samples were boiled for 5 min at 97 °C. Protein samples were
then loaded per lane for SDS-PAGE gel separation (4–20% Tris • HCl Criterion
Gel, Bio-Rad).
Western Blotting.
Proteins were separated by SDS-PAGE before being transferred to PVDF mem-
brane (Bio-Rad) using standard Western blotting procedures.
All Western blots besides anti-O-GlcNAc (see below) were blocked in TBST (0.1%
Tween-20, 150 mM NaCl, 10 mM Tris, pH 8.0) containing 5% nonfat milk for 1 h
at room temperature (RT), then incubated with the appropriate primary antibody
in blocking buffer overnight at 4 °C. The anti-HA antibody (Covance) and
MAb414 antibody (Covance) were used at a 1∶5,000 dilution for detection of α-B
crystallin and p62, respectively. The blots were then washed three times in TBST
and incubated with the HRP-conjugated secondary antibody for 1 h in blocking
buffer at RT. HRP-conjugated anti-mouse and anti-human antibodies (Jackson
ImmunoResearch) were used at 1∶10,000 dilutions. After being washed three
more times with TBST, the blots were developed using ECL reagents (Bio-Rad)
and the ChemiDoc XRS+ molecular imager (Bio-Rad).
100
Biotin Enrichment.
NIH3T3 cell pellets from 20 150 × 25 mm plates, labeled with Ac4GlcNAlk (200
µM) or DMSO were resuspended in 200 µL H2O, 60 µL PMSF in H2O (250 mM),
and 500 µL 0.05% SDS buffer (0.05% SDS, 10 mM TEA, pH 7.4, 150 mM NaCl)
with Complete Mini protease inhibitor cocktail (Roche Biosciences). To this was
added 8 µL Benzonase (Sigma), and the cells were incubated on ice for 30 min. At
this time, 4% SDS buffer (2000 µL) was added and the cells were briefly soni-
cated in a bath sonicator and collected by centrifugation at 20,000 × g for 10 min
at 15 °C. Protein concentration was normalized by BCA assay (Pierce, Thermo-
Scientific) to 1 mg⁄mL (10 mg total cell lysate). The appropriate amount of click
chemistry cocktail (substituting 5mM azido-azo-biotin for 10 mM rhodamine)
was added and the reaction was allowed to proceed for 1 h, after which time 10
volumes of ice-cold methanol were added. Precipitation proceeded overnight or 2
h at −80°C. Precipitated proteins were centrifuged at 5;200 × g for 30 min at 0
°C and washed three times with 40 mL ice-cold MeOH, with resuspension of the
pellet each time. The pellet was then air dried for 1 h. To capture the biotinylated
proteins by streptavidin beads, the air-dried protein pellet was resuspended in 4
mL of Hepes buffer (6 M urea, 2 M thiourea, 10 mM Hepes, pH 8.0) by bath
sonication.
The captured proteins were incubated with freshly made 1 mM dithiothreitol
(100 mM stock solution) for 40 min to reduce cystienes. Cystiene capping was
101
achieved after further incubation with freshly prepared 5.5 mM iodoacetamide
(550 mM stock solution) for 30 min in the dark. The beads were then washed
twice with PBS (250 µL) and once with 4% SDS buffer. Beads resuspended in
Hepes buffer were then incubated on a rotator for 2 h, washed twice with Hepes
buffer, twice with PBS, and twice with 1% SDS in PBS (10 mL per wash, 2,000 ×
g, 2 min). Samples were then transferred to 2-mL dolphin-nosed tubes. Beads
were then incubated in 250 µL of sodium dithionite solution (1% SDS, 25 mM so-
dium dithionite) for 30 min at RT to elute captured proteins. The beads were cen-
trifugated for 2 min at 2,000 × g and the eluent collected. The elution step was
repeated, and the eluents combined. Protein was concentrated using a YM-10
Centricon (Millipore), washed with 300 µL PBS and centrifuged at 10,000 × g for
30 min. The concentrated eluent was transferred to a microcentrifuge tube and
dried by SpeedVac.
Dried pellets were resuspended in 1x SDS-free loading buffer (10% glycerol, 0.1%
bromophenol blue, 0.7% β-mercaptoethanol) and boiled for 5 min. The majority
of this resuspended solution, 90%, was loaded onto SDS-PAGE for in-gel trypsin
digestion, and the remaining sample was loaded onto another SDS-PAGE for
validation of protein candidates by Western blot analysis.
102
LC-MS Analysis.
Each lane of the SDS-PAGE gel was sliced into 10 fractions, and each excised gel
slice was placed in a microcentrifuge tube. The gel slices were washed twice with
50 mM ammonium bicarbonate (ABC, 300 µL, 15 min), destained twice with a 1∶1
solution of 50 mM ABC/acetonitrile for 30 min, and then dehydrated in 100%
acetonitrile. After drying the gel pieces in a SpeedVac, gel pieces were rehydrated
in a trypsin solution (2 µg of trypsin per gel slice) and incubated at 37 °C in a wa-
ter bath for 18 h. The peptides were eluted in 50% acetonitrile in H2O with 0.1%
TFA (200 µL, twice), and SpeedVac dried. Samples were then subjected to nano-
HPLC/MS/MS analysis (Thermo Linear Trap Quadrupole-Orbitrap in the Pro-
teomic Resource Center at Rockefeller University).
LC-MS analysis was performed with a Dionex 3000 nano- HPLC coupled to an
Linear Trap Quadrupole-Orbitrap ion trap mass spectrometer (ThermoFisher).
Peptides were pressure loaded onto a custom-made 75-µm–diameter, 15-cm C18
reverse- phase column and separated with a gradient running from 95% buffer A
[HPLC water with 0.1% (vol⁄vol) formic acid] and 5% buffer B [HPLC-grade
CH3CN with 0.1% (vol⁄vol) formic acid] to 55% B over 30 min, next ramping to
95% B over 10 min and holding at 95% (vol⁄vol) B for 10 min. One full MS scan
(300–2,000 MW) was followed by three data-dependent scans of the nth most
intense ions with dynamic exclusion enabled. Peptides were identified using SE-
QUEST version 28 and were searched against the mouse International Protein
103
Index protein sequence database v3.45. Scaffold software (Proteome Software)
was used to compile data.
Neural Precursor Embryonically Developmentally Down-Regulated 4-1
(NEDD4-1) Labeling.
Cells in 150 × 25 mm plates at 85% confluency were transfected with cDNA en-
coding HA-NEDD4-1 (20 µg) using GenePORTER (Genlantis) according to
manufacturer’s procedures. After 5 h, media was replaced with high-glucose
DMEM with 10% FCS and allowed to incubate overnight. Cells were then split
10∶20 into fresh high-glucose media. After another 8 h, media was replaced with
low-glucose DMEM with 10% FCS and 200 µM Ac4GlcNAlk or DMSO vehicle. Af-
ter 16 h, media was again replaced with fresh low-glucose DMEM with 10% FCS
and 200 µM Ac4GlcNAlk or DMSO vehicle. After 8 h, cells were washed with PBS,
trypsinized, and pelleted.
Cell pellets were resuspended in 100 µL 1% NP-40 lysis buffer (1% NP-40, 150
mM NaCl, 50 mM TEA, pH 7.4) with Complete EDTA-free Mini protease inhibi-
tor cocktail (Roche Biosciences) and 50 µM MG-132 (Calbiochem), allowed to sit
for 15 min, and then centrifuged at 4 °C for 10 min at 10,000 × g. The super-
natant (soluble cell lysate) was collected and the protein concentration was de-
termined by BCA assay (Pierce, Thermo-Scientific). Total cell lysate (1.5 mg) was
diluted as necessary to a final concentration of 3 mg⁄mL. To each sample was
104
added 3.33 µL HA.11 clone 16B12 monoclonal antibody (Covance), and samples
were incubated on a rotator at 4 °C for 16 h.
Recombinant protein G sepharose beads (30 µL, Invitrogen), resuspended in an
equal volume of PBS were then added to each sample. The samples incubated on
a rotator at 4 °C for 2 h. Upon completion, beads were collected by centrifugation
at 2,000 × g for 2 min at 4 °C, and the supernatant was carefully removed. Beads
were then washed with 200 µL PBS three times. The final PBS wash was carefully
removed, the beads were suspended in 25 µL 4% SDS buffer, and boiled for 5 min
at 97 °C. The appropriate amount of click chemistry cocktail was added, and the
reaction was allowed to proceed for 1 h, after which time 25 µL of 2x loading
buffer was added. Samples were boiled for 5 min at 97 °C. Protein samples were
then loaded per lane for SDS-PAGE gel separation (4–20% Tris • HCl Criterion
Gel, Bio-Rad).
NEDD4-1 Anti-O-GlcNAc Western Blotting.
Cells in 150 × 25 mm plates at 85% confluency were transfected with cDNA en-
coding HA-NEDD4-1 using GenePORTER (Genlantis) according to manufac-
turer’s procedures. After 5 h, media was replaced with high-glucose DMEM with
10% FCS and allowed to incubate overnight. Cells were then split 10∶20 into fresh
high-glucose media. After another 8 h, media was replaced with high-glucose
DMEM supplemented with 10% FCS and 200 µM Ac4GlcNAc. After another 16 h,
105
media was again refreshed with high-glucose DMEM supplemented with 10%
FCS and 200 µM Ac4GlcNAc. After 8 h, cells were washed with PBS, trypsinized,
and pelleted.
Cell pellets were resuspended in 100 µL 1% NP-40 lysis buffer (1% NP-40, 150
mM NaCl, 50 mM TEA, pH 7.4) with Complete Mini protease inhibitor cocktail
(Roche Biosciences) and 50 µM MG-132 (Calbiochem), allowed to sit for 15 min,
and then centrifuged at 4 °C for 10 min at 10,000 × g. The supernatant (soluble
cell lysate) was collected and the protein concentration was determined by BCA
assay (Pierce, Thermo-Scientific). Total cell lysate (1.5 mg) was diluted as neces-
sary to a final concentration of 3 mg⁄mL. To each sample was added 3.33 µL
HA.11 clone 16B12 monoclonal antibody (Covance), and samples were incubated
on a rotator at 4 °C for 16 h.
Recombinant protein G sepharose beads (30 µL, Invitrogen), resuspended in an
equal volume of PBS were then added to each sample. The samples incubated on
a rotator at 4 °C for 2 h. Upon completion, beads were collected by centrifugation
at 2,000 × g for 2 min at 4 °C, and the supernatant was carefully removed. Beads
were then washed with 200 µL PBS three times. The final PBS wash was carefully
removed, the beads were suspended in 25 µL 4% SDS buffer, and boiled for 5 min
at 97 °C. Following elution, 25 µL of 2x loading buffer was added and samples
were again boiled for 5 min at 97 °C. Protein samples were then loaded per lane
106
for SDS-PAGE gel separation (4–20% Tris • HCl Criterion Gel, Bio-Rad) and sub-
sequently transferred to PVDF membrane (Bio-Rad) using standard Western
blotting procedures.
The blot was then blocked in TBST (0.1% Tween-20, 150 mM NaCl, 10 mM Tris,
pH 8.0) containing 5% BSA overnight at 4 °C, then incubated with the primary
antibody CTD110.6 (Covance) at a 1∶1,000 dilution. The blots were then washed
three times in TBST and incubated with the HRP-conjugated anti-mouse anti-
body (Jackson ImmunoResearch) for 1 h in BSA blocking buffer at RT at a
1∶10,000 dilution. After being washed three more times with TBST, the blots were
developed using ECL reagents (Bio-Rad) and the ChemiDoc XRS+ molecular im-
ager (Bio-Rad).
107
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Chapter Three. Robust In-Gel Fluorescence Detection of
Mucin-Type O-linked Glycosylation
*
Introduction
Mucin-type O-linked glycosylation is an abundant post-translational modifica-
tion that occurs in the secretory pathway of higher eukaryotes (1). The modifica-
tion is initiated by a family of polypeptide N-acetylgalactosaminyl transferases
(ppGalNAcTs), which modify serine and threonine residues with an α-linked N-
acetylgalactosamine residue (Figure 3-1) (2). This glycoprotein product can then
be enzymatically elaborated to produce complex and heterogeneous oligosaccha-
ride structures that impact several biological processes including protein struc-
ture, trafficking and cell-cell communication (3). Accordingly, changes in mucin-
type glycosylation have been implicated in human diseases ranging from immu-
nodeficiency to cancer (2). Because of the importance of mucin-type O-linked
glycosylation, several methods have been developed for its visualization and iden-
tification (4). However, the heterogeneity of mucin-type O-linked glycosylation
has hampered the robustness of some of these techniques. For example, despite
O-linked glycosylation prediction software, the lack of a characteristic amino acid
substrate sequence makes site identification challenging (5,6). In addition, unlike
N-linked glycosylation, there is no pan-specific lectin which can be used to enrich
120
*
Leslie A. Bateman (University of Southern California) contributed to the work presented in this
chapter.
for complex O-linked glycans, which instead require the enlistment of several gly-
cosidases that can unveil the core GalNAc residue for recognition by Helix poma-
tia agglutinin (7). Furthermore unlike N-linked glycans, which can be selectively
liberated by the enzyme Peptide N-Glycosidase F (PNGase-F), there is no avail-
able glycanase for mucin-type glycan cleavage. As a chemical alternative, cleavage
of the core GalNAc residue can be carried out via β-elimination (8). However, the
alkaline reaction conditions necessary for O-linked glycan removal are not selec-
tive and can cleave other post-translational modifications as well as degrade the
parent protein (9-13).
121
Figure 3-1. The GalNAc salvage pathway. UDP-GalNAc is synthesized from GalNAc by
GalNAc 1-kinase and UDP-GalNAc pyrophosphorylase. The donor sugar is then trans-
ported into the Golgi lumen where ppGalNAcTs installs the monosaccharide onto serine
and threonine residues of glycoproteins. As the protein of interest (POI) continues to
travel through the secretory pathway, elaborating glycosyltransferases further modify the
α-GalNAc resulting in a mucin-type O-linked glycan.
122
Bioorthogonal labeling reactions have emerged as an alternative to these ap-
proaches. Notably, the Bertozzi laboratory has developed a metabolic labeling
strategy which utilizes the fully-protected, azide-bearing GalNAc chemical re-
porter Ac4GalNAz. Ac4GalNAz can enter cells, where it is deacetylated and con-
verted to UDP-GalNAz by the enzymes of the GalNAc salvage pathway, GalNAc 1-
kinase and UDP-GalNAc phosphorylase (14). This azide-bearing UDP sugar do-
nor is a good substrate for ppGalNAcTs resulting in addition of GalNAz onto ser-
ine and threonine residues. GalNAz-modified proteins were first labeled with a
FLAG-tagged phosphine probe via Staudinger ligation (15). This approach selec-
tively labels azide-modified proteins for visualization via immunoblotting tech-
niques. In the same study, a FITC-conjugated α-FLAG antibody was used for
fluorescent quantitation of azide-modified proteins by flow cytometry (14). The
same techniques were subsequently used to visualize GalNAz incorporation in
mice (16). To improve upon these conditions, a difluorinated cyclooctyne (DIFO)
was also developed to allow for strain promoted, Cu-free [3+2] cycloaddition (17).
Fluorophore-conjugated DIFO analogs were subsequently exploited for imaging
of GalNAz-modified proteins on living cells and in Caenorhabditis elegans (18)
and zebrafish (19,20). Finally, GalNAz has been used in conjunction with Staud-
inger ligation for the identification of 18 glycoproteins via mass spectroscopy-
based proteomics (21).
123
Recent studies have suggested that Cu(I)-catalyzed [3+2] azide-alkyne cycloaddi-
tion (CuAAC), in combination with fluorescent and biotin tags, provides the most
robust method for visualization and identification of azide-modified proteins by
in-gel fluorescence and proteomics, respectively (22-24). This method has been
applied to glycosylation (25), acetylation (26), lipidation (22,27,28), and
covalently-linked small molecule inhibitors (29,30). Herein we describe the use
of CuAAC conditions in combination with GalNAz and an alkynyl fluorescent
probe for the rapid in-gel visualization of mucin-type glycoproteins. These condi-
tions allow for labeling in as little as 4 hours, visualization of glycosylation levels
in a variety of cell lines and can be used as a specific chemical reporter to label
known O-linked glycoproteins. Importantly, under our labeling conditions we see
preferential incorporation of the chemical reporter into mucin-type glycoproteins
in comparison to previous conditions in which GalNAz is also incorporated onto
O-GlcNAc-modified proteins.
124
Figure 3-2. Fluorescent detection of mucin-type O-linked glycoproteins. (A) The fluo-
rescent tag alkynyl-rhodamine (Alk-Rho) for CuAAC. (B) Metabolic incorporation of
Ac4GalNAz. HEK-293T cells were treated with 50-200 µM Ac4GalNAz or DMSO vehicle
for 16 h and analyzed by in-gel fluorescent scanning.
Results
Fluorescent visualization of mucin-type O-linked glycoproteins.
In order to visualize putative mucin-type O-linked glycoproteins using CuAAC
conditions, we synthesized Ac4GalNAz and the alkyne-bearing fluorescent tag,
alkynyl rhodamine (Alk-Rho) (Figure 3-2A). HEK293T cells were treated with
Ac4GalNAz (50-200 µM) or DMSO vehicle for 16 h, after which time cells were
washed and lysed in 1% NP-40 buffer. The soluble fractions were diluted to a
concentration of 1 mg/mL and reacted with a rhodamine-derived Alk-Rho under
CuAAC conditions for 1 h. At this time, the reaction was quenched with 1 mL ice-
cold methanol and placed at -80 ℃ for 2 h to precipitate proteins. The precipi-
tated proteins were resuspended in 4% SDS buffer and 2x SDS-free loading
buffer was added. Samples were boiled for 5 min, and proteins were resolved by
125
SDS-PAGE. In-gel fluorescence scanning revealed robust labeling at all concen-
trations tested (Figure 3-2B).
Because the synthesis, glycosylation, and turnover of glycoproteins is a dynamic
process, analysis of these events requires that GalNAz be efficiently incorporated
into mucin-type glycans. Previous in vitro analysis demonstrated that a subset of
the ppGalNAcTs (1-5 and 11) utilized UDP-GalNAz at ~15 to 50% efficiency when
compared to UDP-GalNAc (15); however, the minimal treatment time for GalNAz
to be incorporated into living cells was unknown. To qualitatively determine the
rate of GalNAz incorporation, we treated HEK293T cells with Ac4GalNAz (200
µM) for 0-12 h. Following CuAAC, in-gel fluorescence demonstrated incorpora-
tion of the chemical reporter in as little as 4 h and significant labeling after 8 h
(Figure 3-3A). This is an improvement over previous reports using Staudinger
ligation and immuno-tags where treatment times were on the order of days (14).
We also conducted a pulse-chase experiment to visualize the decrease in fluores-
cence signal from GalNAz-modified proteins over time (Figure 3-3B). HEK293T
cells were labeled with Ac4GalNAz (200 µM) for 16 h after which time cells were
washed with PBS and media containing Ac4GalNAc (200 µM) was added. Cells
were treated for 0 - 72 h, lysed, and subjected to CuAAC. In-gel fluorescence ex-
hibited a steady decrease in labeling over 3 days. Importantly this corresponds
well with global cell surface turnover experiments that were performed with ra-
dioactive methionine pulse-chase (31).
126
Figure 3-3. Characterization of Ac4GalNAz labeling. (A) Incorporation of Ac4GalNAz.
HEK-293T cells were labeled with 200 µM Ac4GalNAz for 0-12 h and analyzed by in-gel
fluorescence scanning. (B) Pulse-chase analysis of Ac4GalNAz. HEK-293T cells were la-
beled with 200 µM Ac4GalNAz for 16 h after which time the media was replaced with
media containing 200 µM Ac4GalNAc for 0-72 h. In-gel fluorescence scanning was then
conducted.
GalNAz is preferentially incorporated onto mucin-type O-linked glycoproteins.
To exploit available environmental nutrients, mammalian cells have enzymes
which facilitate crosstalk between carbohydrate biosynthetic pathways including
the GalNAc salvage pathway. GalNAz could therefore have several metabolic
fates, resulting in different types of glycosylation that contribute to total fluores-
cent signal. For example UDP-GalNAc from the GalNAc salvage pathway can be
converted to UDP-GlcNAc by the action of the enzyme UDP-galactose 4’-
epimerase (32). Previous results have demonstrated that UDP-GalNAz is also
substrate for UDP-galactose 4’ epimerase, resulting in the generation of UDP-
GlcNAz and subsequent modification of nuclear and cytoplasmic proteins
127
through O-GlcNAc on serine and threonine residues (21). Because of this inter-
conversion, cellular fractionation was required to selectively visualize mucin O-
linked glycoproteins or O-GlcNAc modified proteins. To determine if our condi-
tions also result in a mixture of azide labeled mucin and O-GlcNAcylated pro-
teins, specific proteins with known glycosylation patterns were analyzed. Specifi-
cally, we first visualized GalNAz labeling of GlyCAM-IgG, a fusion protein con-
taining both N-linked and mucin O-linked glycans. GlyCAM-IgG was expressed
in COS-7 cells treated with GalNAz or DMSO vehicle for 24 h, resulting in secre-
tion of the protein (14). The media was collected and incubated with protein G
sepharose beads to enrich the IgG domain. The samples were then subject to
CuAAC, and in-gel fluorescence scanning revealed robust labeling of the glyco-
protein by GalNAz, consistent with previous results (Figure 3-4A) (14). To deter-
mine if GalNAz was incorporated into the N-linked glycan of the IgG domain,
GlyCAM-IgG was treated with PNGase-F. Subsequent analysis by CuAAC and in-
gel fluorescence revealed only a slight decrease in signal, consistent with previous
results that the majority of GalNAz is incorporated into mucin-type O-linked gly-
cosylation at the cell surface (Figure 3-4A) (14).
128
Type to enter text
Figure 3-4. Characterizing the metabolic fate of Ac4GalNAz. (A) Incorporation of
Ac4GalNAz onto GlyCAM-IgG. COS-7 cells expressing GlyCAM-IgG were treated with
200 µM Ac4GalNAz for 24 h. In-gel fluorescence analysis followed. (B) Incorporation of
Ac4GalNAz onto FoxO1A. COS-7 cells expressing FoxO1A were treated with 200 µM
Ac4GalNAz for 24 h followed by in-gel fluorescent scanning.
As stated above, UDP-GalNAz can be converted to UDP-GlcNAz resulting in the
modification of serine and threonine residues of nuclear and cytosolic proteins.
In order to determine whether GalNAz was being incorporated into O-GlcNAc
proteins under our labeling conditions, we used FLAG-tagged FoxO1A, a mutant
of the transcriptional regulator FoxO1, which is known to be heavily O-GlcNAc-
modified (33). FLAG-tagged FoxO1A was expressed in COS-7 cells treated with
Ac4GalNAz or DMSO vehicle for 24 h. Cells were then lysed and FoxO1A im-
munoprecipitated with anti-FLAG beads. Reaction of the azide under CuAAC
conditions and subsequent in-gel fluorescence scanning revealed no labeling of
the protein of interest under standard labeling conditions (Figure 3-4B). This re-
sult is contradictory to the report by the Bertozzi lab; however, our conditions dif-
fer in labeling time (24 h vs 3 days) (21). In addition, we performed GalNAz label-
ing in media containing lower levels of glucose, and thus presumably lower en-
129
dogenous levels of UDP-GlcNAc, and observed conversion to GlcNAz and FoxO1A
labeling (Figure 3-4B) (34). Under these lower glucose conditions, we were able
to visualize FoxO1A labeling using GalNAz. These data suggest that mucin-type
O-linked glycosylation can be selectively visualized using GalNAz under certain
conditions. We attribute the higher molecular weight bands to ubiquitination
(35) and/or phosphorylation (36) of FoxO1A.
Figure 3-5. GalNAz labels proteins in a variety of cell lines. NIH3T3, CHO, COS-7,
HEK-293T, HeLa, Mcf-7 and SH-SY5Y cells were treated with Ac4GalNAz (200 µM) for
16 h followed by in-gel fluorescent scanning.
GalNAz labels mucin-type O-linked glycoproteins in a variety of cell lines.
Given that under our labeling conditions GalNAz is a metabolic chemical reporter
of mucin-type glycosylation, we tested its generality in a variety of cell lines. Ac-
cordingly, NIH3T3, CHO, COS-7, HEK-293T, HeLa, Mcf-7 and SH-SY5Y cells
were treated with Ac4GalNAz (200 µM) for 16 h. Following lysis and CuAAC, in-
gel fluorescence scanning revealed distinct labeling patterns and intensities for
130
all cell lines tested (Figure 3-5). These data demonstrate that GalNAz in combina-
tion with our CuAAC conditions and fluorescent tag can monitor changes in
mucin-type glycosylation that are key to understanding cellular processes.
Discussion
Mucin-type O-linked glycosylation is a key post-translational modification that
has been implicated in a variety of biological functions that are indispensable for
survival. However, the visualization and identification of mucin glycoproteins is
hampered by their heterogeneity and complexity. We have developed Cu(I)-
catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC) conditions that can be used
in combination with a fluorescent tag and the metabolic chemical reporter Gal-
NAz to robustly visualize mucin-type O-linked glycoproteins. In contrast to pre-
vious reports using immunoblotting tags and treatment times of 3 days, GalNAz
at concentrations as low as 50 µM can be readily visualized after only 16 h. In ad-
dition, at higher concentrations (200 µM) GalNAz labeling can be visualized in as
little as 4h, potentially enabling the analysis of dynamic changes in mucin glyco-
proteins on short timescales. We next conducted a pulse-chase experiment in or-
der to visualize the global loss of signal from GalNAz-labeled proteins over time.
We observed the loss of signal over the course of 3 days, which coincides well
with the reported half-life of cell-surface proteins (31).
131
One possible caveat to our approach is the recently reported data showing the
metabolic conversion of UDP-GalNAz to UDP-GlcNAz and subsequent labeling of
O-GlcNAc modified proteins in the cytosol and nucleus. To examine the meta-
bolic fate of GalNAz under our conditions, we used known glycoproteins. We first
exploited the chimeric fusion protein GlyCAM-IgG to confirm preferential label-
ing of mucin type O-linked glycans with GalNAz. Consistent with previous re-
ports, GalNAz treatment resulted in robust labeling of GlyCAM-IgG and pre-
treatment of the sample with PNGase-F did not result in a significant decrease in
labeling, confirming that the majority of GalNAz resides in mucin-type glycans at
the cell surface (14). To examine the possible interconversion of UDP-GalNAz to
UDP-GlcNAz we used the known O-GlcNAc substrate FoxO1A. We observed no
fluorescence labeling of FoxO1A treated with GalNAz, suggesting that under our
conditions GalNAz is selectively incorporated into mucin-type O-linked glycans.
Importantly, we can visualize GlcNAz and GalNAz labeling of FoxO1A under low-
glucose conditions, confirming their interconversion (34). We attribute our selec-
tivity to short treatment times (16 to 24 h) when compared to previous results (3
d) (14). While it is possible that GalNAz can label other O-GlcNAc modified pro-
teins under our labeling conditions, we suspect that this prospect is unlikely, as
FoxO1A is known to be constitutively and heavily glycosylated. Therefore, if Gal-
NAz was susceptible to interconversion and thus capable of modifying O-
GlcNAcylated proteins, we feel that we would see at least some labeling of
FoxO1A. Finally, we explored the generality of our in-gel fluorescence detection.
132
Analysis of GalNAz-modified proteins in a variety of cell lines demonstrates the
ability of GalNAz, in combination with in-gel fluorescence, to detect not only
changes in mucin levels but also in different patterns of glycoprotein expression
and/or modification.
Conclusion
In summary, we described the rapid and robust fluorescent visualization of
mucin-type O-linked glycoproteins using the chemical reporter GalNAz and in-
gel fluorescence scanning. These conditions allow for the visualization of modifi-
cation levels and patterns, which are not readily determined using published im-
munoblotting techniques. Additionally, unlike methods that measure steady state
levels of glycosylation, metabolic chemical reporters such as GalNAz can be used
to monitor dynamic changes on defined timescales. Finally, we anticipate that
this technology combined with published affinity tags will assist in developing a
proteomic approach to identifying mucin-type O-linked glycoproteins.
Materials and Methods
All reagents used for chemical synthesis were purchased from Sigma-Aldrich un-
less otherwise specified and used without further purification. All anhydrous re-
actions were performed under argon atmosphere. Analytical TLC was conducted
on Silica Gel 60 F254 plates (EMD Chemicals) with detection by ceric ammonium
molybdate (CAM), anisaldehyde, or UV. For flash chromatography, 60 Å silica gel
133
(EMD Chemicals) was utilized. Electrospray ionization mass spectrometry (ESI-
MS) was performed using a Shimadzu liquid chromatography (LC)-MS 2020.
1
H
spectra were obtained at 600 MHz on a Varian VNMRS-600 unless otherwise
specified. Chemical shifts are recorded in ppm (δ) relative to CHCl3 (7.26 ppm)
for spectra acquired in CDCl3.
13
C spectra were obtained at 200 MHz on the same
instrument.
Chemical Synthesis.
Known compounds peracetylated N-azidoacetylgalactosamine (2.5, GalNAz;
(14)) and N-(6-(diethylamino)-9-(2-(4-hept-6-ynoylpiperazine -1-
carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium (2.2, alk-rho;
(22)) were synthesized according to literature procedures as described in Chapter
2.
Cell Culture.
HEK293, CHO, COS-7, HeLa, MCF-7, and NIH3T3 cells were cultured in high-
glucose DMEM media (HyClone, Thermo-Scientific) enriched with 10% FCS, in
the case of NIH3T3, or FBS (HyClone, Thermo-Scientific). SH-SY5Y cells were
cultured in 1:1 DMEM/F-12K medium enriched with 10% FBS. All cell lines were
maintained in a humidified incubator at 37 °C and 5.0% CO2.
134
Metabolic Labeling.
To cells at 80–85% confluency, high- or low-glucose media containing Ac4Gal-
NAz (1,000x stock in DMSO), or DMSO vehicle was added as indicated. For chase
experiments, media was replaced with media supplemented with 200 µM
Ac4GalNAc (Sigma).
Preparation of Nonidet P-40 (NP-40)-Soluble Lysates.
Cells were then collected by trypsinization and pelleted by centrifugation 4 °C for
3 min at 2,000 × g, followed by washing with PBS (1 mL) two times. Cell pellets
were then resuspended in 100 µL of 1% NP-40 lysis buffer [1% NP-40, 150 mM
NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete EDTA-free Mini pro-
tease inhibitor cocktail (Roche Biosciences) for 10 min and then centrifuged at 4
°C for 10 min at 10,000 × g. The supernatant (soluble cell lysate) was collected
and the protein concentration was determined by bicinchoninic acid (BCA) assay
(Pierce, Thermo-Scientific).
Cu(I)-Catalyzed [3 + 2] Azide-Alkyne Cycloaddition.
Cell lysate (200 µg) was diluted with cold 1% NP-40 lysis buffer to obtain a de-
sired concentration of 1 µg⁄µL. Newly-made click chemistry cocktail (12 µL) was
added to each sample [alkynyl- rhodamine tag (100 µM, 10 mM stock solution in
DMSO); tris (2-carboxyethyl)phosphine hydrochloride (1 mM, 50 mM freshly-
prepared stock solution in water); tris[(1-benzyl-1-H-1,2,3-triazol-4-
135
yl)methyl]amine (100 µM, 10 mM stock solution in DMSO); CuSO4 •5H2O (1 mM,
50 mM freshly prepared stock solution in water)] for a total reaction volume of
200 µL. The reaction was gently vortexed and allowed to sit at room tempera-
ture for 1 h. Upon completion, 1 mL of ice-cold methanol was added to the reac-
tion, and it was placed at −80 °C for 2 h to precipitate proteins. The reactions
were then centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was re-
moved, the pellet was allowed to air dry for 5 min, and then 50 µL 4% SDS buffer
(4% SDS, 150 mM NaCl, 50 mM TEA pH 7.4) was added to each sample. The
mixture was sonicated in a bath sonicator to ensure complete dissolution, and 50
µL of 2x loading buffer (20% glycerol, 0.2% bromophenol blue, 1.4% β-
mercaptoethanol) was then added. The samples were boiled for 5 min at 97 °C,
and 30 µg of protein was then loaded per lane for SDS-PAGE gel separation (4–
20% Tris • HCl Criterion Gel, Bio-Rad).
In-Gel Fluorescence Scanning.
Following SDS-PAGE gel separation, the gel was incubated in destaining solution
(50% methanol, 40% H2O, 10% glacial acetic acid) for 5 min followed by H2O for
an additional 5 min prior to scanning. The gel was scanned on a Molecular Im-
ager FX (Bio-Rad) using a 580-nm laser for excitation and a 620-nm bandpass
filter for detection.
136
FoxO1A Labeling.
COS-7 cells in 150 × 25 mm plates at 95% confluency were transfected with cDNA
encoding FoxO1A (32 µg, Addgene plasmid 13508) using Lipofectamine 2000
(Invitrogen) according to manufacturer’s procedures. After 6 h, media was re-
placed with DMEM with 10% FCS. After 16 h, media was replaced with DMEM
with 10% FCS and Ac4GalNAz or Ac4GalNAc (200 mM stock in DMSO). After 10
h, media was replaced with DMEM with 10% FCS and Ac4GalNAz or Ac4GalNAc
(200 mM stock in DMSO). After 16 h, cells were washed with PBS, trypsinized,
and pelleted.
COS-7 cell pellets were resuspended in 20 µL H2O, 0.2 µL O-(2-acetamido-2-
deoxy-D-glucopyranosylidene)amino-N-phenyl carbamate (PUGNAc, 100 mM
stock solution in DMSO, Tocris), and 36 µL 0.05% SDS buffer (0.05% SDS, 5 mM
MgCl2, 10 mM TEA pH 7.4) with Complete EDTA-free Mini protease inhibitor
cocktail (Roche Biosciences). Samples were then treated with 1 µL Benzonase
(Sigma) and incubated on ice for 30 min. To this mixture was added 145 µL of 4%
SDS buffer (4% SDS, 5 mM MgCl2, 1 mM TEA pH 7.4). The samples were then
sonicated briefly in a bath sonicator and centrifuged at 10,000 × g for 10 min at
15 °C. Protein concentrations were then normalized using the BCA assay (Pierce,
Thermo-Scientific). Total cell lysate (1.5 mg) was diluted as necessary to a final
volume of 1 mL with 1% NP-40 buffer with Complete Mini protease inhibitor
cocktail (Roche Biosciences). EZview Red anti-FLAG M2 affinity gel (40 µL,
137
Sigma), washed with PBS three times, were added to each sample, and the sam-
ples were placed on a rotator for 2 h at 4 °C.
Beads were collected by centrifugation at 3,000 × g for 2 min at 4 °C, and the su-
pernatant was carefully removed. Beads were then washed with 500 µL PBS three
times. The final PBS wash was carefully removed and the beads were suspended
in 40 µL 4% SDS buffer. Samples were then boiled for 5 min at 97 °C to elute pro-
teins. The appropriate amount of click chemistry cocktail was added and the reac-
tion was allowed to proceed for 1 h, after which time 40 µL of 2x loading buffer
was added. Samples were boiled for 5 min at 97 °C. Protein samples (40 µg) were
then loaded per lane for SDS-PAGE gel separation (4–20% Tris • HCl Criterion
Gel, Bio-Rad).
GlyCAM-IgG Labeling.
COS-7 cells in 150 × 25 mm plates at 95% confluency were transfected with cDNA
encoding GlyCAM-IgG (32 µg, Addgene plasmid 17841) using Lipofectamine
2000 (Invitrogen) according to manufacturer’s procedures. After 5 h, media was
changed. After 16 h, cells were trypsinized and split 4∶10 into 100 × 20 mm plates
in high- or low-glucose DMEM with 10% FCS and 200 µM Ac4GalNAc or
Ac4GalNAz in DMSO (1,000x stock in DMSO).
138
After 24 h, the media from each sample was collected by centrifugation at 3,000
× g for 10 min at 4 °C to remove cell debris. The supernatant was incubated with
500 µL of recombinant protein G sepharose beads (Invitrogen) in 100 mM TEA,
pH 8 overnight. Beads were collected by centrifugation at 3,000 × g for 2 min at
4 °C. Beads were washed three times with 3 mL 100 mM TEA, pH 8 to remove
media. GlyCAM-Ig was eluted by addition of 500 µL 2% SDS buffer (2% SDS, 150
mM NaCl, 50 mM TEA pH 7.4) and boiling for 5 min at 97 °C. Protein concentra-
tion was determined by BCA assay (Pierce, Thermo-Scientific). Final SDS concen-
tration was diluted to 0.5% by addition of 50 mM TEA pH 7.4. Protein (60 µg)
was then treated with Peptide N-glycosidase F (PNGase-F, New England Bio-
labs), according to manufacturer’s procedures, or left untreated. The appropriate
amount of click chemistry cocktail was added and the reaction was allowed to
proceed for 1 h after which time 4x loading buffer (40% glycerol, 0.4% bromo-
phenol blue, 2.8% β-mercaptoethanol) was added. Samples were boiled for 5 min
at 97 °C and 50 µg were loaded for SDS-PAGE gel separation (4–20% Tris • HCl
Criterion Gel, Bio-Rad).
Western Blotting.
Proteins were separated by SDS-PAGE before being transferred to PVDF mem-
brane (Bio-Rad) using standard Western blotting procedures.
139
Western blots besides were blocked in TBST (0.1% Tween-20, 150 mM NaCl, 10
mM Tris, pH 8.0) containing 5% nonfat milk for 1 h at room temperature (RT),
then incubated with the appropriate primary antibody in blocking buffer over-
night at 4 °C. The anti-FLAG antibody (Sigma Aldrich) was used at a 1:1,000 dilu-
tion. The blots were then washed three times in TBST and incubated with the
HRP-conjugated secondary antibody for 1 h in blocking buffer at RT. A HRP-
conjugated anti-mouse antibody (Jackson ImmunoResearch) was used at
a1∶10,000 dilution. After being washed three more times with TBST, the blots
were developed using ECL reagents (Bio-Rad) and the ChemiDoc XRS+ molecu-
lar imager (Bio-Rad).
140
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Chapter Four. N-Propargyloxycarbamate Monosaccharides
as Metabolic Chemical Reporters of Carbohydrate Salvage
Pathways and Protein Glycosylation
*
Introduction
An ever expanding repertoire of bioorthogonal reactions has enabled the specific
labeling of reporter molecules in a range of biological contexts (1,2). In many ap-
plications, this two-step detection strategy relies upon the metabolic delivery and
subsequent enzymatic installation of a chemical reporter. Emblematic of this
technology, metabolic chemical reporters of glycosylation have been in use for
over a decade and contain the founding member of azide-containing reporters an
analogue of N-acetyl-mannosamine (ManNAc) termed N-azidoacetyl- manno-
samine (ManNAz), which was developed for the visualization of sialic acid-
containing carbohydrates upon reaction with an immuno-tag using the Staud-
inger ligation (3). Inspired by this result, a variety of other metabolic chemical
reporters have been developed to target sialic acid modification (4-6), mucin O-
linked glycosylation (7,8), fucosylation (6,9), and intracellular O-GlcNAc modifi-
cation (O-GlcNAcylation) (10,11). These chemical reporters take advantage of
carbohydrate scavenging pathways that convert them into the corresponding nu-
cleotide sugar-donors for utilization by glycosyltransferases. Until recently, these
147
*
Leslie A. Bateman and Kelly N. Chuh (University of Southern California) contributed to the work
presented in this chapter.
metabolic chemical reporters were thought to function largely in a one-input one-
output paradigm, where treatment with one chemical reporter would read-out on
one type of glycoconjugate (e.g. ManNAz treatment results in sialic acid labeling).
However, cells are armed with metabolic pathways that can enzymatically inter-
convert monosaccharides and uridine diphosphate (UDP) sugar donors (12). For
example, N-acetyl-glucosamine (GlcNAc) can be reversibly converted to both N-
acetyl-galactosamine (GalNAc) (13) and N-acetyl-mannosamine (ManNAc) (14).
Therefore, it was not entirely surprising when multiple reports demonstrated that
the azide-containing chemical reporter, N-azidoacetyl-galactosamine (GalNAz),
can be transformed to N-azidoacetyl-glucosamine (GlcNAz), resulting in the la-
beling of a combination of mucin O-linked, O-GlcNAcylation, and some N-linked
glycans (11,15,16). While these different types of glycosylation can be separated
using biochemical methods, a more ideal metabolic chemical reporter would spe-
cifically read-out on only one type of glycosylation. Because each of the carbohy-
drate scavenging pathways and glycosyltransferases is likely to display unique
tolerance to chemically-modified monosaccharides, it should be possible to create
chemical reporters that discriminate between glycosylation pathways. In support
of this, we previously reported on alkyne-containing chemical reporters, N-
butynyl-glucosamine (GlcNAlk) and N-butynyl-galactosamine (GalNAlk), which
are not interconverted, enabling the more selective visualization and identifica-
tion of O-GlcNAc modified proteins using the Cu(I)-catalyzed azide–alkyne cy-
cloaddition (CuAAC) (11).
148
Herein, we continue to examine the chemical tolerance of mammalian glycosyla-
tion pathways through the synthesis and characterization of the N-
propargyloxycarbamate (Poc) containing chemical reporters GlcPoc (17), GalPoc,
and ManPoc (18) (Figure 4-1A). Interestingly, we find that each chemical reporter
displays unique labeling efficiencies and that qualitatively GlcPoc and GalPoc are
incorporated into the same proteins while ManPoc labels a different pattern. Fi-
nally, we show that all three chemical reporters allow for the selective enrichment
of a known O-GlcNAc modified protein NEDD4, suggesting that they all can be
metabolically converted to enter the O-GlcNAcylation pathway.
Figure 4-1. N-Propargylcarbamate-containing metabolic chemical reporters incorpo-
rated onto proteins. (A) Metabolic chemical reporters Ac 4GlcPoc: N-
propargylcarbamate-1,3,4,6-tetra-O-acetyl-glucosamine, Ac 4GalPoc: N-
propargylcarbamate-1,3,4,6-tetra-O-acetyl-galactosamine, Ac 4ManPoc: N-
propargylcarbamate-1,3,4,6-tetra-O-acetyl-mannosamine. (B) NIH3T3 cells were treated
with each chemical reporter (200 µM) for 16 hours before reaction with azido-
rhodamine under CuAAC conditions and analysis by in-gel fluorescence. Coomassie blue
shows protein loading.
149
Results
Fluorescence labeling of proteins by Poc analog.
To test the ability of Poc-modified monosaccharides to transit carbohydrate sal-
vage pathways and serve as substrates for glycosyltransferases, the hydrochloride
salts of glucosamine, galactosamine, and mannosamine were reacted with
propargyl-chloroformate. The remaining free hydroxyl groups were subsequently
acetylated to give Ac4GlcPoc, Ac4GalPoc, and Ac4ManPoc (Figure 4-1A). NIH3T3
cells were metabolically labeled at 200 µM for 16 hours. The cells were washed,
lysed, and the soluble protein fraction was reacted with a previously reported
azide-containing rhodamine fluorescent dye (Az-Rho) using CuAAC (11). In-gel
fluorescent scanning showed labeling of a variety of proteins with all three
chemical reporters (Figure 4-1B). ManPoc labels cells at the highest level followed
by GlcPoc and, finally, GalPoc. Interestingly, GlcPoc and GalPoc qualitatively la-
bel the same subset of proteins, while ManPoc enables visualization of a non-
overlapping population, suggesting that the Poc group is tolerated to different
degrees by all the monosaccharide salvage pathways but discriminated against at
the level of the metabolic interconversion and/or glycosyltransferases. To test the
concentration dependence of the chemical reporters, NIH3T3 cells were treated
with various concentrations of each molecule for 16 hours, followed by lysis,
CuAAC with Az-Rho, and analysis by in-gel fluorescent scanning (Figure 4-2A).
All three reporters labeled proteins at concentrations as low as 50 µM.
150
Figure 4-2. Characterization of N-propargyloxycarbamate (Poc) bearing metabolic
chemical reporters. A) NIH3T3 cells were treated with the indicated concentrations of
Ac4GlcPoc, Ac4GalPoc, or Ac4ManPoc for 16 hours, followed by analysis by in-gel fluores-
cence scanning. B) NIH3T3 cells were treated with 150 µM Ac4GlcPoc, Ac4GalPoc, or
Ac4ManPoc for the indicated times before analysis by in-gel fluorescence scanning. C)
NIH3T3 cells were treated with 150 µM Ac4GlcPoc, Ac4GalPoc, or Ac4ManPoc and
chased with 150 µM Ac4GlcNAc, Ac4GalNAc, or Ac4ManNAc, respectively, followed by in-
gel fluorescence scanning. Coomassie blue staining demonstrates protein loading.
151
A key feature of all metabolic chemical reporters is their ability to label a newly-
generated protein fraction in a pulse experiment; however, this requires that they
are efficiently incorporated on short time-scales. To qualitatively determine the
rate of chemical reporter labeling, NIH3T3 cells were treated with Ac4GlcPoc (150
µM), Ac4GalPoc (200 µM), or Ac4ManPoc (150 µM) for different amounts of time.
Fluorescent modification using CuAAC and in-gel fluorescence demonstrated in-
corporation of all three chemical reporters in as little as 4 h (Figure 4-2B), consis-
tent with the labeling times of other reporters including GlcNAz and GlcNAlk
(11). Additionally, metabolic chemical reporters can be used to determine the sta-
bility of a modification or protein in a pulse-chase format by measuring the de-
crease in fluorescence signal over time. Accordingly, NIH3T3 cells were treated
with Ac4GlcPoc (150 µM), Ac4GalPoc (200 µM), or Ac4ManPoc (150 µM) for 8,
16, and 16 hours, respectively. At these times, the cells were washed with PBS and
media containing the corresponding unmodified sugar (Ac4GlcNAc, Ac4GalNAc,
or Ac4ManNAc) at 150 mM was added. Cells were treated for 0–72 hours before
analysis by in-gel fluorescence as above (Figure 4-2C). Interestingly, GlcPoc and
GalPoc displayed similar signal decay rates, again suggesting that they label the
same protein substrates. In contrast, loss of ManPoc signal was somewhat slower,
supporting its incorporation into a different type of glycosylation.
152
GlcPoc and ManPoc label cell surface proteins
We further analyzed the efficiency of the Poc metabolic chemical reporters using
flow-cytometry. Accordingly, NIH3T3 cells were treated with each chemical re-
porter at 150 mM concentration for 8 hours. At this time, the cells were fixed with
paraformaldehyde (PFA) and permeabilized with Triton X-100 before CuAAC in
the presence of Az-Rho. Consistent with our fluorescence scanning, ManPoc
showed the highest level of signal, followed by GlcPoc and finally GalPoc (Fig. 4-
3). Again this demonstrates that the Poc group is differentially tolerated by the
carbohydrate salvage pathways and/or glycosyltransferases.
Figure 4-3. Flow cytometry analysis of metabolic chemical reporter incorporation.
NIH3T3 cells were treated with each chemical reporter (150 mM) for 16 hours. Cells
were then fixed with 3.7% PFA and permeabilized with 0.1% Triton X-100 before reac-
tion with azido-rhodamine under CuAAC conditions and analysis by flow cytometry. Er-
ror bars represent S.E.M. of three experiments.
Finally, to examine the metabolic interconversion of the Poc-bearing chemical
reporters we examined their ability to read out on O-GlcNAc modifications. Ac-
153
cordingly, NIH3T3 cells were treated with Ac4GlcPoc (150 µM), Ac4GalPoc (200
µM), or Ac4ManPoc (150 µM) for 8 h. The corresponding cell-lysates were then
reacted under CuAAC conditions with a known azide-containing biotin affinity
probe (azido-azo-biotin) (11). Labeled proteins were enriched with streptavidin
beads, washed, and eluted with sodium dithionite. Western blot analysis of these
enriched proteomes was then performed using an antibody against a known O-
GlcNAc substrate NEDD4 (Figure 4-4) (11). Interestingly, NEDD4 was enriched
by all three chemical reporters, with GlcPoc and ManPoc displaying approxi-
mately the same level of enrichment followed by GalPoc, demonstrating that both
ManPoc and GalPoc can be metabolically converted to GlcPoc and enter the O-
GlcNAc modification pathway.
Figure 4-4. Incorporation of metabolic chemical reporters into the O-GlcNAcylation
pathway. NIH3T3 cells were treated with each chemical reporter (150 mM) for 16 hours.
Labelled proteins were reacted with azido-azo-biotin using CuAAC before streptavidin
enrichment and analysis of O-GlcNAc modification by anti-NEDD4 Western blotting.
Discussion and Conclusion
The continually expanding range of metabolic chemical reporters of glycosylation
has enabled the visualization and identification of many types of glycans and un-
derlying protein substrates. We and others have demonstrated that many chemi-
cal reporters can be metabolically transformed and therefore label multiple gly-
154
cosylation pathways. However, given the complete chemical control of these
small molecules, it might be possible to structurally bias their metabolic fates to
generate more specific reporters. To further test this possibility, we have devel-
oped the N-propargyloxycarbonyl (Poc) bearing metabolic chemical reporters
GlcPoc, GalPoc, and ManPoc. Comparison of these reporters revealed that they
are all incorporated into large numbers of cellular proteins, which can be readily
visualized using both in-gel fluorescence scanning and flow cytometry. The pat-
tern of GlcPoc and GalPoc labeled proteins are approximately identical, suggest-
ing that they are incorporated into the same type of glycosylation. In contrast to
the azide-bearing chemical reporter GalNAz when compared to GlcNAz (15,19),
GalPoc is utilized at a much lower efficiency than GlcPoc, consistent with our
previous analysis of the structurally similar alkyne-containing derivative GalNAlk
(11). However, based on the enrichment of NEDD4, GalPoc can be interconverted
to GlcPoc, which does not occur with GalNAlk and other chemical reporters with
larger substituents at the N-acyl position (11,20). This demonstrates that even
small alterations to the structure of these chemical reporters can influence their
metabolism and final destination. Likewise, ManPoc can be converted to GlcPoc
and subsequently participate in O-GlcNAc modification of NEDD4. Interestingly,
the level of NEDD4 enrichment is similar in both GlcPoc and ManPoc treated
cells. Together with the much higher global levels of ManPoc incorporation and
its distinct labeling pattern, we believe that only a minority of ManPoc enters the
155
Type to enter text
O-GlcNAcylation pathway, while the majority is transformed to sialic acid, sup-
porting previous in vitro experiments (17).
Materials and Methods
All reagents used for chemical synthesis were purchased from Sigma-Aldrich un-
less otherwise specified and used without further purification. All anhydrous re-
actions were performed under argon atmosphere. Analytical thin-layer chroma-
tography (TLC) was conducted on EMD Silica Gel 60 F254 plates with detection by
potassium permanganate (KMnO4), anisaldehyde or UV. For flash chromatogra-
phy, 60 Å silica gel (EMD) was utilized.
1
H spectra were obtained at 600 MHz or
500 MHz on a Varian VNMRS-600 or AMX-500. Chemical shifts are recorded in
ppm (δ) relative to CHCl3 (7.26 ppm) for spectra acquired in CDCl3 or methanol.
13
C spectra were obtained at 150 or 125 MHz on the same instruments.
Chemical Synthesis.
K n o w n c o m p o u n d s , N - ( 9 - ( 2 - ( 4 - ( 6 - a z i d o h e x a n o y l ) p i p e r a z i n e - 1 -
carbonyl)phenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminiu
m (2.4, az-rho; (21)), and azido-azo-biotin (2.10, (22)) were synthesized accord-
ing to literature procedures as described as described in Chapter 2.
156
Compound 4.1 N-propargyloxycarbamate-1,3,4,6-tetra-O-acetyl-glucosamine
(Ac4GlcPoc). Glucosamine HCl (1 g, 4.6 mmol), and sodium
bicarbonate (0.7 g, 7.88 mmol, Mallinckrodt) were dissolved
in H2O (10 mL). To the stirring solution, propargyl chloro-
formate (679 µL, 6.96 mmol) was added dropwise. The solution was allowed to
stir for 16 h at room temperature. The reaction mixture was concentrated,
washed with methanol (10 mL) and filtered. Resulting filtrate was concentrated
and dissolved in pyridine (10 mL) and stirred. Acetic anhydride (1.8 mL, 19.14
mmol) was then added and allowed to stir for 16 h at room temperature. Purifica-
tion by silica gel column chromatography (45% EtOAc in Hexanes) afforded the
product (853.2 mg, 52% yield) as a white solid.
1
H NMR (600 MHz, CDCl3) δ 5.70
(d, J = 8.3 Hz, 1H), 5.18 (t, J = 9.8 Hz, 1H), 5.11 (t, J = 9.6 Hz, 1H), 4.96 (d, J =
8.1 Hz, 1H), 4.66 (s, 1H), 4.28 (dd, J = 12.5, 4.4 Hz, 1H), 4.11 (dd, J = 12.4, 1.7 Hz,
1H), 4.06 (t, J = 6.7 Hz, 1H), 3.93 (dd, J = 19.0, 9.4 Hz, 1H), 3.81 (dd, J = 9.9, 4.6,
2.2 Hz, 1H), 2.45 (bs, 1H), 2.13 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H).
13
C NMR (150 MHz, CDCl3) δ 170.78, 169.48, 155.00, 92.60, 77.96, 73.00, 72.36,
68.01, 64.51, 61.76, 55.16, 53.07, 21.02, 20.87, 20.78, 20.73.
Compound 4.2 N-propargyloxycarbamate-1,3,4,6-tetra-O-acetyl-
galactosamine (Ac4GalPoc). Galactosamine HCl (100 mg,
0.46 mmol, Carbosynth), and sodium bicarbonate (133 mg,
1.58 mmol, Mallinckrodt) were dissolved in H2O (2.3 mL)
O
AcO
NH
OAc
O
O
OAc
AcO
O
AcO
NH
OAc
O
O
OAc
AcO
157
and 1,4-dioxane (4 mL). To the stirring solution, propargyl chloroformate (68 µL,
0.7 mmol) was added dropwise. The solution was allowed to stir for 16 h at room
temperature. The reaction mixture was concentrated, washed with methanol (10
mL) and filtered. Resulting filtrate was concentrated and purified by silica gel
column chromatography (20:80, methanol, methylene chloride). Resulting prod-
uct was dissolved in pyridine (704 µL, 8.7 mmol) and stirred. Acetic anhydride
(328 µL, 3.48 mmol) was then added and allowed to stir for 16 h at room tem-
perature. Reaction mixture was concentrated and pyridine was removed. The
crude was then resuspended in CH2Cl2 and extracted with 1 M HCl, saturated so-
dium bicarbonate, water and brine. Purification by silica gel column chromatog-
raphy (45% EtOAc in Hexanes) afforded the product (118 mg, 79% yield) as a
white solid.
1
H NMR (500 MHz, CDCl3) δ 6.22 (d, 1H), 5.41 (d, J = 2.6 Hz, 1H),
5.17 (dd, J = 11.6, 2.9 Hz, 1H), 4.88 (d, J = 9.7 Hz, 1H), 4.73 – 4.58 (m, 2H), 4.41
(td, J = 11.4, 3.5 Hz, 1H), 4.22 (dd, J = 13.5, 6.7 Hz, 1H), 4.10 – 4.02 (m, 2H),
2.48 (t, J = 2.0 Hz, 1H), 2.16 (s, 6H), 2.01 (s, 6H).
13
C NMR (125 MHz, CDCl3) δ
170.93, 170.47, 170.27, 169.96, 168.93, 155.01, 91.45, 75.16, 68.67, 68.09, 66.83,
61.35, 53.14, 48.92, 21.03, 20.81, 20.76, 20.74.
Compound 4.3 N-propargyloxycarbamate-1,3,4,6-tetra-O-acetyl-
manosamine (Ac4ManPoc). Mannosamine HCl (100 mg,
0.46 mmol. Carbosynth), and sodium bicarbonate (133
mg, 1.58 mmol, Mallinckrodt) were dissolved in H2O (2.3
O
AcO
AcO
OAc
AcO
HN O
O
158
mL) and 1,4-dioxane (4 mL). To the stirring solution, propargyl chloroformate
(68 µL, 0.7 mmol) was added dropwise. The solution was allowed to stir for 16 h
at room temperature. The reaction mixture was concentrated and washed with
methanol (10 mL) and filtered. Resulting filtrate was concentrated and purified
by silica gel column chromatography (15% MeOH in CH2Cl2). Resulting product
was dissolved in pyridine (671 µL, 8.3 mmol) and stirred. Acetic anhydride (314
µL, 3.3 mmol) and DMAP (0.4 mg, 0.003 mmol) were then added and allowed to
stir for 16 h at room temperature. Reaction mixture was concentrated and pyri-
dine was removed. The cruse was then resuspended in CH2Cl2 and extracted with
1 M HCl, saturated sodium bicarbonate, water and brine. Purification by silica gel
column chromatography (50% EtOAc in Hexanes) afforded the product (123.5
mg, 86% yield) as a white solid.
1
H NMR (500 MHz, CDCl3) δ 6.07 (d, J = 1.8 Hz,
1H), 5.41 (d, J = 9.5 Hz, 1H), 5.29 (dd, J = 10.2, 4.3 Hz, 1H), 5.20 (d, J = 10.2 Hz,
1H), 4.68 (dt, J = 4.9, 2.5 Hz, 2H), 4.24 (dd, J = 12.7, 5.0 Hz, 1H), 4.11 – 3.96 (m,
3H), 2.49 (d, J = 2.2 Hz, 1H), 2.16 (d, J = 0.6 Hz, 3H), 2.08 (s, 4H), 2.04 (s, 4H),
2.01 (d, J = 3.7 Hz, 3H).
13
C NMR (125 MHz, CDCl3) δ 170.78, 170.19, 169.71,
168.20, 155.18, 91.82, 75.22, 73.46, 70.28, 69.12, 65.39, 62.12, 53.14, 51.39,
20.95, 20.83, 20.72.
159
Cell culture
NIH3T3 cells were cultured in high glucose DMEM media (Cellgro) with 10% fe-
tal calf serum (FCS, Cellgro) and were maintained in a humidified incubator at 37
°C and 5.0% CO2.
Metabolic labeling
To cells at 80-85% confluency, high glucose media containing Poc analog (1,000
x stock in DMSO), or DMSO vehicle was added as indicated. For chase experi-
ments, media was supplemented with 150 µM GlcNAc, GalNAc or ManNAc.
Preparation of NP-40-soluble lysates
The cells were collected by scraping and pelleted by centrifugation at at 4 °C for 2
min at 2,000 x g, followed by washing with PBS (1 mL) two times. Cell pellets
were then resuspended and lysed in 75 µl of 1% NP-40 lysis buffer [1% NP-40,
150 mM NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete Mini prote-
ase inhibitor cocktail (Roche Biosciences) for 15 min and followed by centrifuga-
tion at 4 °C for 10 min at 10,000 x g. The resulting supernatant (soluble cell
lysate) was collected and separated to determine protein concentration via BCA
assay (Pierce, ThermoScientific).
Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC)
Soluble cell lysate (200 µg) was diluted with cold 1% NP-40 lysis buffer to a con-
160
centration of 1 µg/µL. Newly made click chemistry cocktail (12 µL) was added to
each sample [azido-rhodamine tag (100 µM, 10 mM stock solution in DMSO);
tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (1 mM, 50 mM freshly pre-
pared stock solution in water); tris[(1-benzyl-1-H- 1,2,3-triazol-4-
yl)methyl]amine (TBTA) (100 µM, 10 mM stock solution in DMSO); Cu-
SO4•5H2O (1 mM, 50 mM freshly prepared stock solution in water) for a total re-
action volume of 200 µL. The reaction was gently vortexed and allowed to sit at
room temperature for 1 h. Upon completion, 1 mL of ice cold methanol was added
to the reaction, and proteins were precipitated at -20 °C for 2 h. The reactions
were then centrifuged at 4 °C for 10 min at 10,000 x g. The supernatant was re-
moved, the pellet was allowed to air dry for 5 min, and then 50 µL 4% SDS buffer
(4% SDS, 150 mM NaCl, 50 mM TEA pH 7.4) was added to each sample. The
mixture was sonicated in a bath sonicator to ensure complete dissolution, and 50
µL of 2x loading buffer (20% glycerol, 0.2% bromophenol blue, 1.4% β-
mercaptoethanol) was then added. The samples were boiled for 5 min at 98 °C,
and 40 µg of protein was then loaded per lane for SDS- PAGE separation (Any kD
Criterion Gel, Bio-Rad).
In-gel Fluorescence Scanning
The gel was scanned on a Molecular Imager FX (Bio-Rad) using a 580 nm laser
for excitation and a 620 nm bandpass filter for detection.
161
Biotin Enrichment
NIH3T3 cell pellets labeled with GlcPoc, GalPoc, ManPoc (150 µM) or DMSO
were resuspended in 13 µL H2O, and 25 µL 0.05% SDS buffer (0.05% SDS, 10
mM TEA pH 7.4, 150 mM MgCl2) with Complete Mini protease inhibitor cocktail
(Roche Biosciences). To this was added 1 µL Benzonase (Sigma), and the cells
were incubated on ice for 30 min. At this time, 4% SDS buffer (100 µL) was
added, and the cells were briefly sonicated in a bath sonicator and collected by
centrifugation at 20,000 x g for 10 min at 15 °C. Protein concentration was nor-
malized by BCA assay (Pierce, ThermoScientific) to 1 mg/mL (1 mg total cell
lysate). The appropriate amount of click chemistry cocktail was added and the
reaction was allowed to proceed for 1 h, after which time 10 volumes of ice-cold
methanol were added. Precipitation proceeded 2 hours at -20 °C. Precipitated
proteins were centrifuged at 5,200 x g for 30 min at 0 °C and washed 3x with 10
mL ice-cold MeOH, with resuspension of the pellet each time. The pellet was then
air-dried for 1 h. To capture the biotinylated proteins by streptavidin beads, the
air-dried protein pellet was resuspended in 400 µL of resuspension buffer (6 M
urea, 2 M thiourea, 10 mM HEPES pH 8.0) by bath sonication. Samples were
then transferred to 2 mL dolphin-nosed tubes containing streptavidin beads (25
µL) that were pre-washed (2x with PBS (1 ml) and 1x with resuspension buffer
(2,000 x g, 2 min)). Samples were then incubated on a rotator for 2 h. Beads were
washed 2x with resuspension buffer (1 mL), 2x in PBS (1 mL) and 2x with 1% SDS
in PBS buffer (1 mL) and collected by centrifugation (2,000 x g, 2 min). Beads
162
were then incubated in 25 µL of sodium dithionite solution (1% SDS, 25 mM so-
dium dithionite) for 30 min at room temperature to elute captured proteins. The
beads were centrifuged for 2 min at 2,000 x g and the eluent collected. The elu-
tion step was repeated and the eluents combined. Protein was precipitated in ice
cold methanol (1 mL) overnight in -20 °C. Protein was collected by centrifugation
(10 min, 10,000 x g, 4 °C), and the pellet was allowed to air dry for 5 min, and
then 30 µL 4% SDS buffer (4% SDS, 150 mM NaCl, 50 mM TEA pH 7.4) was
added to each sample. The mixture was sonicated in a bath sonicator to ensure
complete dissolution, and 30 µL of 2x loading buffer (20% glycerol, 0.2% bromo-
phenol blue, 1.4% β-mercaptoethanol) was then added. The samples were boiled
for 5 min at 98 °C, and samples were loaded into a gel for SDS-PAGE separation
(Any kD Criterion Gel, Bio-Rad).
Western Blotting
Proteins were separated by SDS-PAGE before being transferred to PVDF mem-
brane (Bio-Rad) using standard Western blotting procedures. Briefly, all Western
blots were blocked in TBST (0.1% Tween-20, 150 mM NaCl, 10mM Tris pH 8.0)
containing 5% non-fat milk for 1 h at rt. They were then incubated with the ap-
propriate primary antibody in blocking buffer overnight at 4 °C. The anti-NEDD4
WW2 antibody (Millipore) was used at a 1:10,000 dilution. The blots were then
washed three times in TBST and incubated with the horseradish
peroxidase(HRP)-conjugated secondary antibody for 1 h in blocking buffer at RT.
163
HRP-conjugated anti-mouse and anti-human antibodies (Jackson ImmunoRe-
search) were used at 1:10,000 dilutions. After being washed three more times
with TBST, the blots were developed using ECL reagents (Bio-Rad) and the
ChemiDoc XRS+ molecular imager (Bio-Rad).
Flow Cytometry
NIH3T3 cells were treated with GlcPoc, GalPoc, ManPoc (150 µM) or DMSO for
16 h. Cells were collected and washed 2X with cold PBS and fixed with 3.7% PFA
in PBS for 10 min. Cells were then washed 1x with 2% FCS in PBS and permeabi-
lized (0.1% Triton X-100 in PBS for 10 min at room temperature). Cell were
washed with PBS and blocked for 10 min with 2% FCS in PBS. Cells were resus-
pended in 100 µL PBS that contained 100 µM Az-Rho, 1 mM TCEP, 100 µM
TBTA and 1 mM CuSO4•5H2O. Samples were incubated in the dark for 1 h and
washed 5x with 1% Tween-20 and 0.5 mM EDTA in PBS and 1x with 2% FCS in
PBS. Flow cytometry analysis was then performed on a Beckman Coulter LSR II
at USC Flow Cytometry Core.
164
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168
Chapter Five. An Alkyne-Aspirin Chemical Reporter for the
Detection of Aspirin-Dependent Protein Modification in
Living Cells
*
Introduction
Aspirin, a non-steroidal anti-inflammatory drug (NSAID), is one of the most
common small-molecule treatments in the world, and has been utilized in differ-
ent forms for over a thousand years for the reduction of inflammation, pain, and
fever. Aspirin has at least two biochemical mechanisms by which it produces
these anti-inflammatory effects. First, the salicylate moiety of aspirin has been
shown to down-regulate the NF-κB signaling pathway through inhibition of IKK-
β (1,2). Second, the acetate group of aspirin is directly transferred to and thereby
inhibits the cyclooxygenase (Cox) enzymes, preventing the production of pro-
inflammatory prostaglandins (3). Specifically, the acetylation occurs in a 1:1 stoi-
chiometry on serine 530 in Cox1 and serine 516 in Cox2 preventing the binding of
the lipid substrate arachidonic acid. This acetylation is irreversible, requiring the
translation of new Cox enzyme for prostaglandin production. Interestingly, low-
dose aspirin has also been shown to lower the rates of heart attack and stroke in
patients with cardiovascular disease (4,5), and more recently, a variety of obser-
vational studies and trials have demonstrated that chronic aspirin use greatly re-
169
*
Leslie A. Bateman and Stephanie M. Miller (University of Southern California) contributed to
the work presented in this chapter.
duces the incidence of cancer and cancer mortality, with the largest decrease in
gastrointestinal cancers (6-10).
While the majority of effort towards understanding aspirin cardioprotection and
chemoprevention has focused on the NFκB and Cox pathways, and much of the
observed effects are clearly due to inhibition of these pathways (7,11-13), aspirin
is known to modify other proteins by chemical transfer of its acetate group to
amino acid side-chains (14). This modification, which we have termed aspirin-
dependent acetylation, raises the possibility that other protein modification
events could contribute to the pharmacological effects of aspirin. The first ex-
periments to demonstrate this used radiolabeled aspirin (H
3
, C
14
) to show that
human serum albumin is acetylated both in vitro and in vivo and that a variety of
other proteins including immunoglobulins, enzymes, and histones are acetylated
in vitro (15,16), followed by in vivo demonstration using similar techniques
(17,18). Additional studies demonstrated that the acetylation occurs on the ε-
amine side-chains of lysine residues on fibrinogen (19). More recently, mass
spectrometry was used to identify aspirin-dependent acetylation of cysteine side-
chains of lens γ-crystallins in vitro, and this acetylation prevents cataract-
promoting carbamylation of the same residues (20). Finally, specific antibodies
were used to identify acetylation on lysine 382 of p53 resulting from aspirin
treatment of MDA-MB-231 cells (14). Aspirin treatment increased p53 nuclear
localization and expression of the target gene p21CIP1, although acetylation of
170
lysine 382 was not unambiguously identified as the mechanism. Although these
radioactivity-based techniques have allowed for the characterization of aspirin-
dependent acetylation events, they cannot be used to visualize and identify the
entire spectrum of acetate modifications in a complex proteome. The Bhat and
Hagen laboratories addressed this limitation using an anti-acetyl-lysine antibody
that allowed for the visualization of increased cellular lysine-acetylation upon as-
pirin treatment and enabled the enrichment and identification of 33 of these pro-
teins using mass spectrometry (21). However, this approach necessarily suffers
from contamination by endogenous lysine-acetylated proteins and no identifica-
tion of other acetylation events (e.g. serine and cysteine).
Given the crucial role of protein-acetylation in almost every area of biology, we
expect that understanding aspirin-dependent acetylation will be uniquely fruitful
for uncovering other cellular pathways affected by aspirin. However, research
into this area has languished since the 1960s and 70s due to a lack of tools that
could enable the robust and specific visualization and identification of these
aspirin-dependent modification events. Bioorthogonal reactions, such as the
Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC), have been used in a
variety of contexts to overcome the limitations of traditional biological technolo-
gies (e.g. antibodies) (22-24). In fact, alkyne-bearing probes have been applied to
investigate protein glycosylation, acetylation, methylation, lipidation, other post-
translational modifications, and covalent small molecule inhibitors (25-34).
171
Herein, we describe the synthesis and characterization of the CuAAC compatible
aspirin-analog containing an alkyne (AspAlk). AspAlk allowed for the robust fluo-
rescent visualization of aspirin-dependent acetylation events in a variety of cells.
In addition, treatment of cells with AspAlk in combination with CuAAC resulted
in the identification of ~400 potentially aspirin-acetylated proteins, representing
a range of biological functions.
Figure 5-1. AspAlk is a chemical reporter of aspirin-dependent protein modification. A)
Experimental Details B) The indicated panel of cell-lines were treated with AspAlk (1
mM) for 6 hours before reaction with az-rho under CuAAC conditions and visualization
by in-gel fluorescence. Coomassie blue shows protein loading.
172
Results
Fluorescent Detection of asprin-dependent acetylation
To generate our chemical reporter of aspirin-dependent acetylation, we reasoned
that it should structurally mimic aspirin as closely as possible. The smallest struc-
tural perturbation would incorporate a 3-butynoic ester onto salicylic acid to gen-
erate the corresponding alkyne-containing aspirin analog. However, we found
that activation of 3-butynoic acid for subsequent ester formation [e.g., N,N’-
dicyclohexylcarbodiimide (DCC)] resulted in decomposition of the intermediate.
Therefore, the symmetric anhydride of 4-pentynoic acid was synthesized by DCC
mediated dehydration as previously described (35) and subsequently reacted
with salicylic acid to yield AspAlk (Figure 5-1A). A small panel of cell-lines (HCT-
15, PC-3, Cos-7, H1299, HEK293, HeLa, MCF7, and NIH3T3) were treated with
AspAlk (1 mM) for 6 hours. In-gel fluorescence, following CuAAC with with a
previously reported azide-containing rhodamine fluorescent dye (az-rho) (34),
enabled robust visualization of a large variety of proteins (Figure 5-1B). Notably,
the pattern of labeled proteins was largely consistent between the different cell
types. However, certain cell-types (PC-3, HeLa, and MCF7) had somewhat lower
levels, and interestingly, HeLa and NIH3T3 cells have unique protein-targets that
are qualitatively not present at high levels in the other cell-lines. Importantly, the
pattern of visualized proteins does not simply correspond to protein abundance
as judged by Coomassie blue staining (Figure 5-1B), suggesting that modification
occurs on specific protein-substrates that are expressed in most cell-lines.
173
Figure 5-2. Characterization of AspAlk labeling. A) HCT-15 colorectal-cancer cells were
labeled with the indicated concentrations of AspAlk for 6 hours before reaction with az-
rho and analysis by in-gel fluorescent scanning. B) HCT-15 cells were treated with either
AspAlk (250 µM) or AspAlk and aspirin (each at 250 µM) for 6 hours before visualization
by fluorescence.
AspAlk labels the same proteins as aspirin in colorectal-cancer cells
Because aspirin has the largest effect on the incidence of gastrointestinal cancers,
we next characterized AspAlk labeling in HCT-15 colorectal-cancer cells. These
cells were treated with different concentrations of AspAlk for 6 hours, before
washing, lysis, and reaction of the soluble protein fraction with az-rho using
CuAAC (34). In-gel fluorescent scanning showed dose-dependent labeling of a
variety of proteins in as little as 50 to 100 µM (Figure 5-2A), which represents a
notable increase in sensitivity over a published immunoblotting method (14,21).
Importantly, a range of aspirin concentrations used (100 to 300 µM) is achiev-
able in the plasma of patients treated with a short analgesic dose (600 mg) and
174
others undergoing chronic aspirin-treatment of rheumatoid arthritis (36,37).
While the concentrations of aspirin in tissues such as the colon are unknown,
even higher concentrations of aspirin (2.5 to 10 mM) are routinely used in pheno-
typic cell-based experiments (38). To qualitatively determine if AspAlk treatment
resulted in the acetylation of the same proteins that are modified by aspirin,
HCT-15 cells were treated with AspAlk (250 µM) in the presence of an equal con-
centration of aspirin (250 µM) before CuAAC with az-rho and analysis by in-gel
fluorescence. Co-treatment with aspirin resulted in an approximately 50% reduc-
tion in labeling signal (Figure 5-2B), suggesting that AspAlk modifies the same
proteins as aspirin.
Figure 5-3. Kinetic analysis of AspAlk labeling. (A) HCT-15 cells were treated with As-
pAlk (1 mM) for the indicated lengths of time before reaction with az-rho under CuAAC
conditions and visualization by in- gel fluorescence scanning. (B) HCT-15 cells were
treated with AspAlk (1 mM) for 2 h before fresh media containing aspirin (1 mM) was
added. Cells were collected after the indicated lengths of time, subjected to CuAAC, and
visualization by in-gel fluorescence scanning. Coomassie-blue staining shows protein
loading.
175
A significant property of many chemical reporters is the ability to visualize label-
ing dynamics in a pulse-labeling experiment. To visualize the rate of AspAlk la-
beling, HCT-15 cells were incubated with AspAlk (1 mM) for different amounts of
time. In-gel fluorescent scanning, after lysis and reaction with az-rho by CuAAC,
demonstrated robust labeling in as little as 30 minutes and near-maximal signal
in 4 hours (Figure 5-3A), potentially representing an improvement compared to
previous Western-blotting based reports, where time courses were not reported
(37). Next, to determine how long aspirin-dependent modifications persist in
cells, HCT-15 cells were treated with AspAlk (1 mM) for 2 hours. At this time, the
cells were washed with PBS and media containing aspirin (1 mM) was added. Cell
were then harvested at different times, lysed, and subjected to CuAAC with az-
rho. Analysis by in-gel fluorescence scanning showed that significant chemical-
reporter signal was lost within the first 12 hours while some signal persisted for
48 hours (Figure 5-3B). To examine this in more detail we treated HCT-15 cells as
above, followed by harvesting after shorter lengths of time (Figure 5-3B). As in
the previous pulse-chase, more than half of the fluorescent signal was progres-
sively lost within the first 12 hours. This fraction of signal is consistent with other
chemical reporters of enzymatically-reversible protein acylation pathways (39);
however, the persistence of the remaining signal suggests that at a percentage of
AspAlk modifications are not removed by protein deacetylases.
176
Figure 5-4. Identification of potential aspirin-dependent acetylation substrates. A)
HCT-15 cells were treated in triplicate with AspAlk (1 mM) for 6 hours before CuAAC
with azido-PEG3-biotin and enrichment of modified proteins. Tryptic peptides were
eluted and proteins identified by mass spectrometry and grouped by cellular function. B)
Core histones were acid precipitated from HCT-15 cells treated with AspAlk (1 mM) or
DMSO vehicle for 6 hours, followed by CuAAC with az-rho and analysis by in-gel fluores-
cence scanning. Coomassie blue shows protein loading.
Identification of AspAlk labelled proteins by biotin enrichment and MS
Finally, to we used AspAlk to identify proteins that are potential substrates of
aspirin-dependent acetylation. HCT-15 cells were treated in triplicate with As-
pAlk (1 mM) or DMSO vehicle for 2 hours and lysed under denaturing conditions
(4% SDS). CuAAC was then performed with an azido-biotin affinity tag (azido-
PEG3-biotin), followed by incubation with streptavidin beads, washing, and on-
bead trypsinolysis. Eluted peptides were then concentrated, identified by LC-MS/
MS, and quantified by spectral counting. Proteins were considered aspirin-
dependent acetylation substrates using the following criteria: 1) proteins much
have been identified by at least one unique peptide and 2 spectral counts in each
177
Type to enter text
of the three replicate experiments, and 2) the average spectral counts for each
identified protein in the AspAlk-treated sample must have been significantly
higher than the corresponding average counts in the DMSO negative control (un-
paired student’s t-test, p<0.05). Proteins meeting these conditions were then
rank-ordered based on the number of spectral counts (Table 5-1). We identified
120 proteins with diverse cellular functions, which importantly contained 12 pro-
teins previously known to be acetylated by aspirin (Figure 5-4A)(15,16,21,40). In
this list of identified proteins were several of the core histone proteins, including
H2B, H3, and H4. Given the crucial role of histone acetylation in transcriptional
regulation (41,42), we next confirmed the aspirin-dependent modification of
these proteins. Accordingly, histones were enriched from HCT-15 cells treated
with either AspAlk (1 mM) or DMSO. After reaction with az-rho under CuAAC
conditions, AspAlk-dependent fluorescent signal was readily detected on all three
histones identified by proteomics (Figure 5-4B).
Discussion
Aspirin-dependent acetylation and subsequent inhibition of cyclooxygenase en-
zymes (Cox-1 & -2) is well established as one molecular mechanism contributing
to cardioprotection and chemoprevention (7,11-13,43). Notably, aspirin is known
to acetylate a variety of additional proteins, giving it the potential to simultane-
ously affect multiple other cellular pathways that could also contribute to the ef-
fects of chronic aspirin-treatment. However, understanding the molecular conse-
178
quences of aspirin-dependent acetylation has been hampered by a lack of tools to
visualize and identify protein targets. To enable the robust visualization and iden-
tification of these modifications, we have developed an aspirin chemical-reporter
(AspAlk) that transfers an alkyne functionality to proteins that can be subse-
quently detected using bioorthogonal chemistries such as CuAAC. Treatment of a
small panel of mammalian cell-lines with AspAlk, followed by CuAAC with an
azide-bearing fluorophore (az-rho), enabled the visualization of a range of pro-
teins by in-gel fluorescence scanning. Interestingly, the qualitative pattern of
these labeled-proteins was similar in most cell-lines, suggesting that the major
targets of aspirin-dependent acetylation may be broadly expressed proteins.
To test the limitations of AspAlk, we next focused on HCT-15 colorectal-cancer
cells and aspirin use has the most dramatic effect on gastrointestinal cancers. As-
pAlk displayed dose-dependent labeling of proteins up to a 1 mM concentration.
Notably, this labeling could be competed by the equimolar addition of aspirin at
levels consistent with other chemical reporters (30). As noted above, aspirin con-
centrations in the plasma of patients receiving an analgesic dose (600 to 650 mg)
can reach 100 to 300 µM (36,37). However, to our knowledge, the concentrations
of aspirin in the gastrointestinal tract have not been precisely measured, and mil-
limolar concentrations (2.5 to 10 mM) of aspirin have been routinely used in cell-
culture experiments in the past (38). Additionally, other studies have found that
repetitive administration of aspirin results in a disproportionately large increase
179
in serum levels (44). Therefore, although 1 mM AspAlk is somewhat higher than
the observed plasma concentrations, we utilized it to maximize our labeling effi-
ciency for AspAlk characterization in subsequent experiments. In cells treated
with a concentration of 1 mM AspAlk, protein-labeling could be visualized in as
little as 30 minutes, a large improvement over previously reported studies using
anti-acetyl lysine antibodies, which treated cells for 12 hours with aspirin (14,21).
Crucially, our short labeling-times correspond well to the measured stability of
aspirin in the plasma of human patients (36). A pulse-chase experiment showed
that AspAlk-dependent modifications are largely turned over in 12 hours, a length
of time consistent with the enzymatic removal of other chemical reporters of pro-
tein acylation (39). However some in-gel fluorescent signal persisted over 48
hours and could represent protein degradation and/or dilution through cell divi-
sion.
Given that the AspAlk chemical reporter improved the visualization of aspirin-
dependent acetylation substrates compared to previous methods, is was used in
combination with a chemically-cleavable affinity tag enabled the identification of
120 potential protein targets for modification by aspirin. Again, we used AspAlk
at a concentration of 1 mM to robustly identify as many potential protein sub-
strates as possible that could be validated in cell-culture and animal model ex-
periments in the future. Finally, we validated our proteomics data by visualizing
180
the aspirin-dependent acetylation of several core histones, suggesting a possible
role for aspirin in transcriptional regulation.
Conclusion
In summary, the experiments described here represent the first application of
chemical-reporter technology to aspirin-dependent acetylation and a significant
improvement in the analysis of this modification. The use of azide-containing
fluorescent tags, in combination with CuAAC, allows for the visualization of
modification patterns and dynamics not accessible to immunoblotting methods.
Furthermore, AspAlk enabled the identification of 120 potentially acetylated pro-
teins from HCT-15. We anticipate that this discovery tool will complement anti-
acetyl lysine antibodies in efforts to functionally characterize aspirin-dependent
acetylation, which will be crucial for a complete understanding of the role of aspi-
rin in cardioprotection and chemoprevention.
Materials and Methods
All reagents used for chemical synthesis were purchased from Sigma-Aldrich un-
less otherwise specified and used without further purification. All anhydrous re-
actions were performed under argon atmosphere. Analytical thin-layer chroma-
tography (TLC) was conducted on EMD Silica Gel 60 F254 plates with detection by
potassium permanganate (KMnO4), anisaldehyde or UV. Flash chromatography
was performed on 60 Å silica gel (EMD).
1
H and
13
C spectra were obtained Varian
181
VNMRS-600 at 600 and 125 MHz. Chemical shifts are recorded in ppm (δ) rela-
tive to solvent. Coupling constants (J) are reported in Hz.
Chemical Synthesis.
K n o w n c o m p o u n d s , N - ( 9 - ( 2 - ( 4 - ( 6 - a z i d o h e x a n o y l ) p i p e r a z i n e - 1 -
carbonyl)phenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminiu
m (2.4, az-rho; (30)), and azido-azo-biotin (2.10, (45)) were synthesized accord-
ing to literature procedures as described as described in Chapter 2.
Compound 5.1 2-Acetylphenyl pent-4-ynoate (AspAlk). Pentynoic acid (500
mg, 5.10 mmol) and N,N’-dicyclohexylcarbodiimide (526
mg, 2.55 mmol) were dissolved in anhydrous CH2Cl2 (20
mL), and solution was allowed to stir for 16 h at room
temperature under an argon atmosphere. The reaction
mixture was then diluted with CH2Cl 2 (10 mL), and filtered to remove N,N’-
dicylohexylurea. The resulting 4-pentynoic anhydride was dissolved in pyridine
(20 mL), and salicylic acid (Alfa Aesar, 211 mg, 1.53 mmol) was added. After stir-
ring for 16 h under an argon atmosphere, the reaction mixture was concentrated
and subjected to silica gel column chromatography (90:2.5:1, ethyl
acetate:methanol:water). The resulting mixture was resuspended in CH2Cl2
(5mL), filtered to removed contaminating 4-pentynoic acid, and concentrated to
yield 188 mg, (57% yield) of 2-acetylphenyl pent-4-ynate as a white solid.
1
H
O OH
O
O
182
NMR (400 MHz, CD3OD) δ 8.04 (dd, J = 7.8, 1.7 Hz, 1H), 7.63 (ddd, J = 8.0, 7.4,
1.8 Hz, 1H), 7.38 (td, J = 7.6, 1.2 Hz, 1H), 7.17 (dd, J = 8.1, 1.2 Hz, 1H), 2.89 –
2.82 (m, 2H), 2.60 (ddd, J = 8.5, 6.7, 2.7 Hz, 2H), 2.34 (t, J = 2.7 Hz, 1H).
13
C
NMR (101 MHz, D6-Acetone) δ 170.71, 165.89, 151.69, 134.60, 132.53, 126.73,
124.60, 83.10, 70.44, 33.99, 14.43. ESI-MS calculated for C12H10O4 [M+Na]
+

241.05, found 240.00.
Cell Culture.
CHO, COS-7, HEK293, HeLa, and MCF-7 cells were cultured in high glucose
DMEM media (CellGro) enriched with 10% fetal bovine serum (FBS, CellGro,).
NIH3T3 cells were cultured in high glucose DMEM media (CellGro) with 10% fe-
tal calf serum (FCS, CellGro). H1299 cells were cultured in RPMI 1640 (CellGro)
medium enriched with 10% FBS. All cell lines were maintained in a humidified
incubator at 37 ℃ and 5.0% CO2.
Metabolic Labeling.
To cells at 80-85% confluency, high glucose media containing AspAlk (1,000 x
stock in DMSO), or DMSO vehicle was added as indicated. For competition and
chase experiments, the indicated media was supplemented with O-acetyl salicylic
acid (Alfa Aesar) at the indicated concentrations.
183
Preparation of Nonidet P-40 (NP-40)-Soluble Lysates.
The cells were collected by trypsinization and pelleted by centrifugation at 4 ℃
for 2 min at 2,000 x g, followed by washing with PBS (1 mL) two times. Cell pel-
lets were then resuspended and lysed in 75 µl of 1% NP-40 lysis buffer [1% NP-
40, 150 mM NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete Mini
protease inhibitor cocktail (Roche Biosciences) for 15 min and followed by cen-
trifugation at 4 ℃ for 10 min at 10,000 x g. The resulting supernatant (soluble
cell lysate) was collected and protein concentration was determined via BCA as-
say (Pierce, ThermoScientific).
Cu(I)-Catalyzed [3 + 2] Azide-Alkyne Cycloaddition.
Soluble cell lysate (200 µg) was diluted with cold 1% NP-40 lysis buffer to a con-
centration of 1 µg/µL. Newly made click chemistry cocktail (12 µL) was added to
each sample [az-rhodamine tag (100 µM, 10 mM stock solution in DMSO); tris(2-
carboxyethyl)phosphine hydrochloride (TCEP) (1 mM, 50 mM freshly prepared
stock solution in water); tris[(1-benzyl-1-H-1,2,3-triazol-4-yl)methyl]amine
(TBTA) (100 µM, 10 mM stock solution in DMSO); CuSO4•5H2O (1 mM, 50 mM
freshly prepared stock solution in water) for a total reaction volume of 200 µL.
The reaction was gently vortexed and allowed to sit at room temperature for 1 h.
Upon completion, 1 mL of ice cold methanol was added to the reaction, and pro-
teins were precipitated at -20 ℃ for 2 h. The reactions were then centrifuged at 4
℃ for 10 min at 10,000 x g. The supernatant was removed, the pellet was allowed
184
to air dry for 5 min, and then 50 µL 4% SDS buffer (4% SDS, 150 mM NaCl, 50
mM TEA pH 7.4) was added to each sample. The mixture was sonicated in a bath
sonicator to ensure complete dissolution, and 50 µL of 2x loading buffer (20%
glycerol, 0.2% bromophenol blue, 1.4% β-mercaptoethanol) was then added. The
samples were boiled for 5 min at 98 ℃, and 40 µg of protein was then loaded per
lane for SDS-PAGE separation (Any kD Criterion Gel, Bio-Rad).
In-Gel Fluorescence Scanning.
Following SDS-PAGE, gels were incubated in H2O for 5 min prior to scanning on
a Typhoon 9400 (GE Healthcare) using a 580 nm laser for excitation and a 610
nm bandpass (30 nm) filter for detection.
Biotin enrichment and On-bead trypsinolysis
HCT-15 cell pellets labeled with AspAlk (1 mM) or DMSO were resuspended in
200 µL H2O, 60 µL PMSF in H2O (250 mM), and 500 µL 0.05% SDS buffer
(0.05% SDS, 10 mM TEA pH 7.4, 150 mM NaCl) with Complete Mini protease
inhibitor cocktail (Roche Biosciences). To this was added 8 µL Benzonase
(Sigma), and the cells were incubated on ice for 30 min. Then, 4% SDS buffer
(2000 µL) was added, and the cells were briefly sonicated in a bath sonicator and
collected by centrifugation (20,000 x g for 10 min at 15 °C). Protein concentra-
tion was normalized by BCA assay (Pierce, ThermoScientific) to 1 mg/mL, and 10
mg of total protein was subjected to the appropriate amount of click chemistry
185
cocktail containing azido-PEG3-biotin (5 mM, Click Chemistry Tools) for 1 h, af-
ter which time 10 volumes of ice-cold MeOH were added. Precipitation proceeded
2 hours at -20 °C. Precipitated proteins were centrifuged at 5,200 x g for 30 min
at 0 ℃ and washed 3 times with 40 mL ice-cold MeOH, with resuspension of the
pellet each time. The pellet was then air-dried for 1 h. To capture the biotinylated
proteins by streptavidin beads, the air-dried protein pellet was resuspended in 2
mL of resuspension buffer (6 M urea, 2 M thiourea, 10 mM HEPES pH 8.0) by
bath sonication. To cap cysteine residues, 100 µl of freshly-made TCEP (200 mM
stock solution, Thermo) was then added and the mixture incubated for 30 min,
followed by 40 µl of freshly prepared iodoacetamide (1 M stock solution, Sigma)
and incubation for a further 30 min in the dark. Streptavidin beads (250 µL of a
50% slurry per sample, Thermo) were washed 2x with 1 mL PBS and 1x with 1 mL
resuspension buffer and resuspended in resuspension buffer (200 µL). Each
sample was combined with streptavidin beads and incubated on a rotator for 2 h.
These mixtures were then transferred to Mini Bio-Spin
®
columns (Bio-Rad) and
placed on a vacuum manifold. Captured proteins were then washed 5x with re-
suspension buffer, 30x with 1% SDS in PBS, and 30x with PBS (1 mL per wash,
vacuum applied between each wash). Beads were then resuspended in 2 M urea
in PBS (1 mL), transferred to screw-top tubes, and pelleted by centrifugation
(2000 x g for 2 min). At this time, 800 µL of the supernatant was removed, leav-
ing a volume of 200 µL. To this bead-mixture was added 2 µL of CaCl2 (200 mM
stock, 1 mM final concentration) and 4 µL of 0.5 mg/mL sequence grade trypsin
186
(Promega) and incubated at 37 ℃ for 18 hours. The resulting mixtures of tryptic
peptides and beads were transferred to Mini Bio-Spin® columns (Bio-Rad) and
the eluent was collected by centrifugation (1,000 x g for 2 min). Any remaining
peptides were eluted by addition of 100 µL of 2 M urea in PBS followed by cen-
trifugation as immediately above. The tryptic peptides were then desalted using
C18 spin columns (Pierce) according to manufacturer's instructions, eluted with
70% acetonitrile in H2O, and concentrated to dryness on a speedvac.
LC-MS Analysis.
Extracted peptides were desalted on a trap column following separation on a 12
cm/75um reversed phase C18 column (Nikkyo Technos Co., Ltd. Japan). A 3 h
gradient increasing from 10% B to 45% B in 3 h (A, 0.1% formic acid; B,
acetonitrile/0.1% formic acid) was delivered at 150 nL/min. The liquid chroma-
tography setup (Dionex, Boston, MA, USA) was connected to an Orbitrap XL
(Thermo, San Jose, CA, USA) operated in top-5 mode. Acquired tandem MS spec-
tra (CID) were extracted using ProteomeDiscoverer, version 1.3 (Thermo, Bre-
men, Germany) and queried against the human Uniprot protein database using
MASCOT 2.3.02 (Matrixscience, London, UK). Peptides fulfilling a Percolator
calculated 1% false discovery rate threshold were reported. All LC−MS/MS analy-
sis were carried out at the Proteomics Resource Center at The Rockefeller Uni-
versity, New York, NY, USA. A total of 810 proteins were identified, with 120 ful-
filling our criteria as a “hit.”
187
Acid extraction of histones.
HCT-15 cells were treated with DMSO or 1 mM AspAlk for 6 hr. Cells were col-
lected by trypsinization and pelleted by centrifugation at 4 ℃ for 2 min at 2,000
x g, followed by washing with PBS (1 mL) two times. Cell pellets were then resus-
pended in ice-cold hypotonic lysis buffer [10 mM triethanolamine (TEA), 1 mM
KCl, 1.5 mM MgCl2, 1 mM PMSF, pH 7.4 with Complete Mini protease inhibitor
cocktail (Roche Biosciences)]. The resuspended cells were homogenized by
Dounce homogenizer and lysed in 3 cycles of freeze-thaw. Intact nuclei were pel-
leted at 4 ℃ for 10 min at 10,000 x g and washed 2x with ice-cold hypotonic lysis
buffer. The nuclear pellet was resuspended in 0.4 N H2SO4 and agitated over-
night on a rotator at 4 ℃. Nuclear debris was pelleted at 4 ℃ for 10 min at
16,000 x g and the supernatant containing histones was collected and precipi-
tated in ice-cold MeOH in -80 ℃ overnight. Precipitated histones were collected
at 4 ℃ for 10 min at 16,000 x g and washed 2x with ice-cold MeOH. The resulting
protein pellet was air dried and resuspended in water. Concentration was deter-
mined by BCA Assay and normalized with 1% NP-40 lysis buffer [1% NP-40, 150
mM NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete Mini protease
inhibitor cocktail] for CuAAC.
188
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Chapter Six. Changes in Metabolic Chemical-Reporter
Structure Yield a Selective Probe of O-GlcNAc Modification
*
Introduction
There are three common types of protein glycosylation that modify large num-
bers of protein substrates in mammalian cells. Proteins localized to the secretory
pathway and the cell surface or secreted into the extracellular space can be modi-
fied by oligosaccharide structures, such as N-linked glycosylation (linked through
asparagine) or mucin O-linked glycosylation (linked through serine and threo-
nine). Additionally, cytoplasmic, nuclear, and mitochondrial proteins can be sub-
strates for the addition of the single monosaccharide N-acetyl-glucosamine,
termed O-GlcNAc modification (O-GlcNAcylation, linked through serine and
threonine) (1-4). Unlike other forms of glycosylation, O-GlcNAcylation is dy-
namic. It is added to protein substrates by one of three isoforms of O-GlcNAc
transferase (OGT) and removed by two isoforms of O-GlcNAcase (OGA) (5). The
expression of these enzymes is also required for embryonic development in mice
and Drosophila (6-8). O-GlcNAc modification displays significant crosstalk with
other posttranslational modifications (PTMs), most significantly phosphorylation
and ubiquitination, setting up O-GlcNAcylation as a key regulator of cellular
pathways (9). A wide variety of proteins have been shown to be O-GlcNAc modi-
197
*
Kelly N. Chuh (University of Southern California) contributed to the work presented in this
chapter.
fied, including regulators of transcription and translation, cytoskeletal proteins,
signaling proteins, and metabolic enzymes. The specific consequences of most of
these modifications are unknown; however, limited biochemical analyses demon-
strate that O-GlcNAc modification can change protein localization, stability, mo-
lecular interactions, and activity. Critically, O-GlcNAcylation is also misregulated
in Alzheimer’s disease and cancer. For example, in neurodegenerative disorders
such as Alzheimer’s disease, O-GlcNAcylation levels are diminished directly lead-
ing to protein aggregation and cell death (10), and we have demonstrated that it
likely plays a similar role in Parkinson’s disease (11). Finally, higher levels of
O-GlcNAc modification are a common feature of many cancers and are necessary
for tumorigenesis and proliferation (12-15).
To identify and characterize O-GlcNAc modifications, complementary chemical
methods have been developed (16,17). In general, these technologies take
advantage of bioorthogonal chemistries, such as the copper(I)-catalyzed azide-
alkyne cycloaddition (CuAAC or “click chemistry”, Figure 6-1A) (18-20). This re-
action relies upon small, abiotic chemical-reporters (azides and alkynes) that can
be selectively reacted with alkyne- and azido-probes, respectively, for the installa-
tion of visualization- and affinity-tags. One of these methods, initiated by the Ber-
tozzi laboratory, takes advantage of monosaccharide analogs that directly incor-
porate azides or alkynes into their structures (21). These analogs, termed meta-
bolic chemical-reporters (MCRs) (22), are taken up by cells through carbohydrate
198
salvage pathways and subsequently feed into the biosynthesis of nucleotide
sugar-donors for use by glycosyltransferases. For example, the first O-GlcNAc-
targeted MCR, N-azidoacetyl-glucosamine (GlcNAz, Figure 6-1B), has been used
for the visualization and proteomic identification of labeled proteins (23-25). Un-
like other methods (e.g., Western blotting), MCRs do not necessarily read-out on
endogenous levels of O-GlcNAcylation, as they must compete with GlcNAc in the
cell. However, because they must be metabolically transformed before their in-
corporation onto proteins, they not only report on O-GlcNAc modification but
also on the integration of upstream metabolic pathways. Additionally, they can be
used much like radioactivity to isolate new modification events and subsequent
rates of removal in pulse and pulse-chase labeling experiments. Despite the clear
utility of this technology, the previous iterations have limitations. Until recently,
GlcNAz and other MCRs were presumed to label only one type of glycosylation
(i.e., GlcNAz treatment results in O-GlcNAcylation labeling), but several enzy-
matic pathways exist that can interconvert different monosaccharides, raising the
possibility that MCRs are converted in the same manner (26). Upon careful char-
acterization, it was demonstrated that GlcNAz can be readily transformed to N-
azidoacetyl-galactosamine (GalNAz, Figure 1B) and vice versa, resulting in the
labeling of both O-GlcNAcylated and mucin O-linked glycosylated proteins (25-
28). Furthermore, we showed that GlcNAz treatment leads to labeling of N-linked
glycosylation (25). This can be overcome using cellular fractionation (28); how-
ever, since we have complete chemical control over the MCR, we predicted that
199
structural alterations can limit this “off-target” labeling and produce an O-
GlcNAcylation-specific reporter. Indeed, we previously demonstrated that an al-
ternative MCR, N-pentynyl-glucosamine (GlcNAlk), which contains a larger func-
tional group at the N-acetyl position, is not converted to the galactosamine de-
rivative and therefore could not label mucin O-linked glycoproteins (25). Unfor-
tunately, GlcNAlk was still incorporated into N-linked glycans, preventing its use
as a completely-selective O-GlcNAcylation reporter.
Figure 6-1. Metabolic chemical reporters (MCRs). (A) Copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC). (B) Peracetylated MCRs used in this study.
We report here the development and application of 6-azido-6-deoxy-N-acetyl-
glucosamine (6AzGlcNAc, Figure 6-1B and Scheme 6-1) as a MCR in living cells.
Cellular analysis of this MCR using CuAAC and fluorescent probes demonstrated
that, unlike previous reporters, it is highly-selective for O-GlcNAcylated proteins,
allowing for the robust visualization of O-GlcNAc modifications using in-gel fluo-
rescence scanning. Furthermore, comparative proteomics using 6AzGlcNAc,
GlcNAz, and GalNAz confirmed the specificity of 6AzGlcNAc towards O-GlcNAc
modifications. 6AzGlcNAc-labeling resulted in the enrichment of zero proteins,
200
out of 367, which are annotated to have exclusively extracellular or lumenal lo-
calization. In contrast, GlcNAz and GalNAz identified 9 and 72 such proteins, re-
spectively. Finally, we also demonstrate that 6AzGlcNAc can bypass an assumed
biosynthetic roadblock by being phosphorylated by the enzyme phosphoace-
tylglucosamine mutase.
Scheme 6-1. Synthesis of Ac46AzGlcNAc (6.1). (a) 4-toluenesulfonyl chloride, pyridine,
-20 ℃, 18 h; (b) NaN3, DMF, 50 ℃, 3 d; Ac2O, pyridine, rt, 16 h, 60% over three steps.
Results
6AzGlcNAc is a robust metabolic chemical-reporter in living cells.
Our previous data using MCRs demonstrated that even small alterations in
chemical structure can have dramatic effects on the distribution of chemical re-
porters into different types of glycosylation (25,29). Therefore, to find a specific
MCR of O-GlcNAcylation, we synthesized a small panel of O-acetylated N-acetyl-
glucosamine analogs bearing azides at different positions; the acetate protecting-
groups allow diffusion across the cell membrane and are subsequently removed
by endogenous lipases/hydrolases. NIH3T3 cells were treated with these com-
pounds at 200 µM concentrations for 16 hours, followed by lysis, CuAAC with an
alkyne-containing rhodamine dye (alk-rho), and analysis by in-gel fluorescent
scanning. One of these compounds, Ac36AzGlcNAc (Figure 6-1B), gave a protein-
201
labeling pattern that was subjectively similar in both intensity and pattern to
Ac4GlcNAz (Figure 6-2A).
Figure 6-2. Ac36AzGlcNAc labels proteins in living cells. (A) NIH3T3 cells were treated
with Ac4GlcNAz (200 µM), Ac36AzGlcNAc (200 µM), or DMSO vehicle for 16 h, followed
by CuAAC and analysis by in-gel fluorescence scanning. (B) NIH3T3 cells were treated
with varying concentrations of Ac4GlcNAz or Ac36AzGlcNAc for 16 h, followed by CuAAC
and analysis by in-gel fluorescence scanning. (C) NIH3T3 cells were treated with
Ac36AzGlcNAc (200 µM), or DMSO vehicle for the times indicated and were tested for
toxicity using an MTS assay. (D) Proteins modified by 6AzGlcNAc were enriched from
NIH3T3 cells treated with Ac36AzGlcNAc (200 µM) or DMSO vehicle using CuAAC with
alkyne-azo-biotin and analyzed by Western blotting.
To further characterize this MCR, NIH3T3 cells were treated with various con-
centrations of Ac36AzGlcNAc or Ac4GlcNAz for 16 hours before reaction with alk-
rho. In-gel fluorescence scanning revealed labeling of a wide-range of proteins in
concentrations as low as 50 µM and maximal labeling achieved at approximately
200 µM (Figure 6-2B), consistent with other MCRs of glycosylation(25,29,30). To
examine the limits of treatment time with our MCR, the viability of NIH3T3 cells
was tested after treatment with 200 µM Ac36AzGlcNAc for 16 hours or 72 hours
202
using a cell proliferation assay. Only minimal loss of cell growth/survival was
seen even after 72 hours of treatment (Figure 6-2C). To determine if 6AzGlcNAc
could report on O-GlcNAc modifications, we treated NIH3T3 cells with
Ac36AzGlcNAc (200 µM) for 16 hours. The cells were then lysed and reacted with
an alkyne-containing cleavable affinity tag (alk-azo-biotin, Scheme 6-2) using
CuAAC. Labeled proteins were enriched using streptavidin beads before elution
with sodium dithionite. Enriched proteins were then subjected to Western blot-
ting using antibodies against the known O-GlcNAc modified proteins NEDD4
(25,31), pyruvate kinase (25), and nucleoporin 62 (nup62) (32). All three proteins
were selectively enriched using 6AzGlcNAc (Figure 6-2D), showing that the MCR
does label known O-GlcNAcylated proteins.
Scheme 6-2. Synthesis of alkyne-azo-biotin. (a) propargyl chloride, 0.1 M KOH, EtOH,
reflux, 20 h, 28%; (b) i.) NaNO2, methyl-4-amino-benzoate, 6 M HCl, K2CO3, H2O:THF
(2:1), 0 °C, 30 min; ii.) rt, 18 h, 90%; (c) NaOH, rt, 24 h, 70%; (d) N-hydroxysuccinimide,
N,N’-dicyclohexylcarbodiimide, THF, rt, 18 h, 56%; (e) EZ-link Amine PEG3-biotin,
DMF, rt, 18 h, 31%.
203
Figure 6-3. The GlcNAc salvage pathway. (A) Peracetylated GlcNAc accesses the HBP
through the GlcNAc salvage pathway. Ac4GlcNAz is accepted by these enzymes and is
ultimately transformed into the UDP donor sugar the which is utilized by OGT to modify
protein substrates. (B) 6AzGlcNAc cannot be phosphorylated at the 6-position by GNK.
We demonstrate that 6AzGlcNAc can be directly transformed to 6AzGlcNAc-1-phosphate
by AGM1 and subsequently transformed to UDP-6AzGlcNAc by AGX1.
6AzGlcNAc is metabolically incorporated by bypassing GlcNAc-6-kinase.
MCRs are enzymatically transformed into their nucleotide sugar-donors by
monosaccharide salvage pathways. Previous O-GlcNAc MCRs are thought to
largely utilize the GlcNAc salvage pathway (Figure 6-3A) (23). The first step of
this pathway is the phosphorylation of MCRs at the 6-position of the carbohy-
drate ring by N-acetylglucosamine kinase (GNK). This is followed by enzymatic
204
mutation of the phosphate to the 1-position and conversion to the uridine-
diphosphate (UDP) sugar donor by N-acetylglucosamine-phosphate mutase
(AGM1) and uridine-diphosphate-N-acetylglucosamine pyrophosphorylase
(AGX1/2), respectively. Although UDP-6AzGlcNAc is known to be accepted by
OGT (33), 6AzGlcNAc cannot be phosphorylated at the 6-position, as we have re-
placed the 6-hydroxyl functionality with an azide. Therefore, we first took a
candidate-based approach to identify a kinase that could directly phosphorylated
6AzGlcNAc at the 1-position and chose N-acetyl-galactosamine kinase (GalK2),
which performs this reaction on N-acetylgalactosamine (GalNAc) and poorly on
GlcNAc (34). To test this possibility, NIH3T3 cells were stably transformed with
five different short-hairpin RNA vectors targeting GalK2 using retroviral infec-
tion and then treated with Ac36AzGlcNAc (200 µM) for 16 hours. Subsequent
CuAAC with alk-rho and in-gel fluorescent scanning showed no loss of fluores-
cent signal, despite a clear reduction of GalK2 mRNA as measured by semi-
quantitative RT-PCR (Figure 6-4A), suggesting that GalK2 is not the enzyme re-
sponsible for 6AzGlcNAc metabolism. To confirm this result, we subjected
6AzGlcNAc (6.8, Scheme 6-3A) to in vitro phosphorylation by recombinant
GalK2 (35). Specifically, GalK2 was incubated with 40 mM concentrations of
GalNAc, GlcNAc, or 6AzGlcNAc and [
32
P]ɣATP (5 mM). At these elevated
substrate-concentrations, GalK2 readily phosphorylated both GalNAc and
GlcNAc but gave no detectable modification of 6AzGlcNAc (Figure 6-4B).
205
Type to enter text
Figure 6-4. Investigation of 6AzGlcNAc metabolism. (A) Cell-lines with stable knock-
down (shRNA) of galactosamine kinase (GalK2) were treated with 6AzGlcNAc (200 µM)
for 16 hours before visualization by in-gel fluorescence. (B) The indicated monosaccha-
rides (40 mM concentration) were tested as sub- strates for purified GalK2 in vitro. (C)
Proposed mechanism by-which AGM1 directly phosphorylates 6AzGlcNAc in the pres-
ence of GlcNAc-6-phosphate. (D) Kinetic constants for the enzymatic production of UDP
sugar donors from GlcNAc-1-phosphate and 6AzGlcNAc-1-phosphate by the enzyme
UDP-N- acetylhexosamine pyrophosphorylase (AGX1).
Next, we tested whether phosphoacetylglucosamine mutase (AGM1) could di-
rectly generate 6AzGlcNAc-1-phosphate. AGM1 typically converts GlcNAc-6-
phosphate to GlcNAc-1-phosphate during the biosynthesis of UDP-GlcNAc. As
part of its enzymatic cycle, AGM1 removes the 6-phosphate from substrate sug-
ars, resulting in a phosphoenzyme intermediate (36). Therefore, once loaded,
phosphorylated AGM1 might be capable of phosphorylating 6AzGlcNAc. To test
this possibility, human AGM1 was heterologously expressed in E. coli and puri-
fied. The enzyme was then incubated with 6AzGlcNAc (2.25 mM) with or without
different “cofactors” that could generate phosphorylated AGM1, specifically
glucose-6-phosphate or glucose-1,6-bisphosphate or GlcNAc-6-phosphate (all at 1
206
mM). To isolate any 6AzGlcNAc-1-phosphate that had been produced, the enzy-
matic reactions were first subjected to copper-free click chemistry with a
fluorescein-conjugated cyclooctyne tag. Fluorescein-labeled compounds (i.e.,
6AzGlcNAc and 6AzGlcNAc-1-phosphate) were then separated from the
phosphorylated-cofactors by paper chromatography. Finally, any fluorescent-
spots were eluted and analyzed by mass spectrometry (LC-MS, Figure 6-5). Incu-
bation of AGM1 with 6AzGlcNAc alone, or with glucose-6-phosphate or glucose-
1,6-bisphosphate, resulted in no detectable formation of 6AzGlcNAc-1-
phosphate. However, in the presence of GlcNAc-6-phosphate as a cofactor, the
formation of 6AzGlcNAc-1-phosphate was unambiguously detected. This demon-
strates that direct phosphorylation of 6AzGlcNAc by AGM1 represents one path-
way that circumvents the GNK biosynthetic-roadblock. However, because the
conversion is very low (< 1% conversion to product based on ion-intensities in
ESI-MS), AGM1 may not be the only enzyme that can produce 6AzGlcNAc-1-
phosphate in living cells. We next analyzed the final enzyme in the biosynthetic
pathway, UDP-N-acetylhexosamine pyrophosphorylase (AGX1), by first synthe-
sizing 6AzGlcNAc-1-phosphate (6.16, Scheme 6-3B). Recombinant AGX1 was
then incubated with different concentrations of GlcNAc-1-phosphate or
6AzGlcNAc-1-phosphate and [
3
H]UTP. Subsequent Michaelis-Menten kinetic
analysis demonstrated that 6AzGlcNAc-1-phosphate is a substrate of AGX1, al-
though at a significantly lower efficiency than GlcNAc-1-phosphate (Figure 6-
4D). Taken together, these data suggest that in living cells 6AzGlcNAc can be di-
207
rectly phosphorylated by AGM1 and enter the remainder of the GlcNAc salvage
pathway to generate UDP-6AzGlcNAc (Figure 6-2).
Scheme 6-3. Synthesis of 6AzGlcNAc-1-phosphate. (A) Reagents: (a) p-Toluenesulfonyl
chloride, pyridine, -20 °C, 16 h; (b) NaN3, DMF, 50 °C, 3 d, 14% over two steps. (B) Rea-
gents: (a) benzyl alcohol, concentrated HCl, 75 ℃, 4 h, 35%; (b) p-toluenesulfonyl chlo-
ride, pyridine, -20 ℃, 1 h, 52%; (c) acetic anhydride, pyridine, 3 h, quantitative yield; (d)
Pd(OH)2/C (10% Pd), H2, MeOH, 48 h; (e) i) 5-(ethylthio)-1H-tetrazole, diallyl-N,N’-
diisopropylphosphoramidite, CH2Cl2, 2 h; ii) m-chloroperoxybenzoic acid, CH2Cl2; -78
℃, 10 min, 74% over 2 steps; (f) sodium methoxide, MeOH, 1.5 h, 57%; (g) sodium azide,
DMF, 48 h, 71%; (h) p-toluenesulfinic acid sodium salt, tetrakis(triphenylphosphine)-
Palladium(0), 4 d, 99%.
208

Extrait d’ion correspondant au composé phosphorylé (isotope le plus intense de l’ion dichargé).
Extraction of the ions corresponding to the phosphorylated compound (most intense isotope of the double-charged ion)
X014864CYC.d
X014865CYC.d
X014956CYC.d
X014957CYC.d
X014958CYC.d
X014959CYC.d
0
20
40
60
Intens.
0
1
2
5
x10
0
50
100
150
200
0
500
1000
0
100
200
0
100
200
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Time [min]
Fraction 0.2
Fraction 1.2
Fraction 3.2
Fraction 2.2
Fraction 4.2
Fraction 5.2
6 azidoGlcNAc-Bicyclooctyn-POE3-Fluorescein (control)
6 azidoGlcNAc-1P-Bicyclooctyn-POE3-Fluorescein (control)
PGM3 + 6 azidoGlcNAc
PGM3 + 6 azidoGlcNAc + GlcNAc6P
PGM3 + 6 azidoGlcNAc + GlcP6P
PGM3 + 6 azidoGlcNAc + Glc1,6diP
Figure 6-5. LC-MS analysis of 6AzGlcNAc-1-phosphate production by AGM1. AGM1
enzymatic reactions were subjected to copper-free click chemistry with bicyclooctyn-
POE3-fluorescein and subjected to separation by paper chromatography. Fluorescent
spots were eluted and analyzed using LC-ESI-MS. Ions corresponding to the fluorescein-
conjugated 6AzGlcNAc-1-phosphate (most intense isotope of the double-charged ion)
were extracted (blue trace). Only in the presence of AGM1 (PGM3) and GlcNAc-6-
phosphate is the formation of 6AzGlcNAc-1-phosphate observed. Units on the y-axis are
not uniform.
6AzGlcNAc is a general and dynamic metabolic chemical-reporter in living
cells.
Next, to explore the generality of 6AzGlcNAc as a MCR, we labeled a panel of dif-
ferent cell lines. Specifically, Cos-7, H1299, HEK293, HeLa, MCF7, mouse em-
bryonic fibroblasts (MEFs), and NIH3T3 cells were treated with Ac36AzGlcNAc
(200 µM) for 16 hours. In-gel fluorescence scanning after CuAAC with alk-rho
showed labeling in all the cell lines examined and a diversity of the pattern and
intensity of modified proteins (Figure 6-6). To qualitatively compare 6AzGlcNAc
209
to previous MCRs of O-GlcNAcylation, the same panel of cell lines was treated
with 200 µM Ac4GlcNAz or Ac4GalNAz for 16 hours (Figure 6-6). In-gel fluores-
cence scanning showed incorporation of previously characterized MCRs in each
cell line with varying intensities and patterns, which were more pronouncedly
different for GalNAz, when compared to 6AzGlcNAc and GlcNAz.
210
Figure 6-6. Fluorescence incorporation of MCRs in a variety of cell lines. The indicated
cell-lines were treated with 200 µM Ac36AzGlcNAc, Ac4GlcNAz or Ac4GalNAz for 16
hours before modified proteins were subjected to CuAAC with alk-rho and analysis by in-
gel fluorescence scanning.
211
As stated above, MCRs only report on modifications that occur during the label-
ing time, raising the possibility that they can be used to isolate O-GlcNAcylation
events in a short time frame via a pulse-labeling experiment. To determine the
kinetics of protein labeling by 6AzGlcNAc, NIH3T3 cells were treated with
Ac36AzGlcNAc (200 µM) for different lengths of time. The cells were then lysed,
reacted with alk-rho using CuAAC, and analyzed by in-gel fluorescence scanning
(Figure 6-7A). Modified proteins can be clearly visualized over background in 2 to
4 hours, similar to the kinetics of protein labeling by Ac4GlcNAz at 200 µM (Fig-
ure 6-7A). MCRs also have the ability to read-out on the turnover of protein
modifications using a pulse-chase format. Accordingly, we treated NIH3T3 cells
with either Ac36AzGlcNAc or Ac4GlcNAz at concentrations of 200 µM. After 16
hours, the cells were washed and fresh media containing Ac4GlcNAc (200 µM)
was added. Cells were collected after different lengths of time, lysed, and sub-
jected to CuAAC with alk-rho. In-gel fluorescence scanning showed a steady loss
of protein labeling over the course of 48 hours (Figure 6-7B), in line with radioac-
tive (tritiated glucosamine) pulse-chase experiments of O-GlcNAc on α-crystallin
and cytokeratin (t1/2 of ~10 and 55 hours, respectively) (37,38). O-GlcNAcase
(OGA) is responsible for the dynamic removal of O-GlcNAc from substrate pro-
teins. To demonstrate that 6AzGlcNAc is incorporated into O-GlcNAcylation and
a substrate for OGA, cells were first treated with Ac36AzGlcNAc (200 µM) or
DMSO for 5 hours. Media was then exchanged for fresh media containing 200
µM Ac4GlcNAc with or without Thiamet-G (10 µM), a potent and highly-selective
212
OGA inhibitor (39). After 12 hours, cells were harvested and subjected to CuAAC
with alk-rho. In-gel fluorescence scanning shows that cells that were treated with
Thiamet-G maintain higher levels of 6AzGlcNAc labeling compared to those
without (Figure 6-7C), demonstrating that 6AzGlcNAc is incorporated into O-
GlcNAc modifications that can be subsequently removed by OGA.
Figure 6-7. Characterization of Ac36AzGlcNAc. (A) NIH3T3 cells were treated with 200
µM Ac36AzGlcNAc or Ac4GlcNAz for the indicated times, followed by CuAAC and analy-
sis by in-gel fluorescence scanning. (B) NIH3T3 cells were treated with 200 µM
Ac36AzGlcNAc or Ac4GlcNAz for 16 h at which time media was exchanged for fresh me-
dia containing 200 µM Ac4GlcNAc. Cells were harvested after the indicated lengths of
time, subjected to CuAAC and analyzed by in-gel fluorescence scanning. (C) HeLa cells
were treated with 200 µM Ac36AzGlcNAc or Ac4GlcNAz for 16 h at which time media was
exchanged for fresh media containing 200 µM Ac4GlcNAc and 10 µM of the OGA inhibi-
tor Thiamet-G or DMSO. Cells were harvested at the times indicated and subjected to
CuAAC before being analyzed by in-gel fluorescence scanning.
213
6AzGlcNAc is a specific metabolic chemical-reporter of O-GlcNAc modification.
As noted above, previous MCRs of O-GlcNAcylation are not selective for O-
GlcNAc modifications because they are also incorporated into either N-linked or
mucin O-linked glycans or both (25,27,28). To determine if 6AzGlcNAc specifi-
cally modifies O-GlcNAcylated proteins, we first took advantage of the chimeric,
secreted protein GlyCAM-IgG that contains both an N-linked and multiple mucin
O-linked glycosylation sites (25). NIH3T3 cells that stably express GlyCAM-IgG
via retroviral transformation were treated with Ac36AzGlcNAc, Ac4GlcNAz, or
Ac4GlcNAc at 200 µM concentrations for 48 hours. At this time, GlyCAM-IgG
was immunoprecipitated from the media using protein-A-conjugated beads. In-
gel fluorescence scanning, following CuAAC with alk-rho, showed that while
GlcNAz robustly labels GlyCAM-IgG, as expected based on our previous re-
sults(25), 6AzGlcNAc does not (Figure 6-8A). This demonstrates that while
GlcNAz does label the major types of cell-surface glycosylation, 6AzGlcNAc does
not. Next, to confirm that 6AzGlcNAc labels O-GlcNAcylated proteins, we treated
NIH3T3 cells that were stably transfected with the FLAG-tagged transcription
factor FoxO1 with Ac36AzGlcNAc, Ac4GlcNAz, or Ac4GlcNAc at 200 µM concen-
trations for 24 hours. In contrast to GlyCAM-IgG, in-gel fluorescence showed that
both MCRs robustly labeled FoxO1A (Figure 6-8B), demonstrating that 6AzGlc-
NAc is a highly-selective MCR of O-GlcNAc modifications.
214
To rule out the possibility that 6AzGlcNAc was excluded from GlyCAM-IgG but
labeled other cell-surface glycoproteins, NIH3T3 cells were treated with
Ac36AzGlcNAc, Ac4GlcNAz, or Ac4GalNAz at 200 µM for 16 hours before being
harvested and submitted to copper-free click chemistry using commercially avail-
able DBCO-biotin. After subsequent incubation with FITC-conjugated avidin,
cell-surface glycoprotein labeling by each chemical reporter was analyzed using
flow cytometry. No labeling over background was observed with Ac36AzGlcNAc
while labeling was observed with Ac4GlcNAz and Ac4GalNAz (Figure 6-8C). No-
tably, this corroborates live-cell flow cytometry data from the Bertozzi lab where
they observed some cell-surface labeling with GlcNAz and no labeling with
6AzGlcNAc (40).
215
Figure 6-8. Glycoprotein specificity of 6AzGlcNAc. NIH3T3 cells stably expressing ei-
ther GlyCAM-IgG (A) or Flag-tagged FoxO1 (B) were treated with the indicated MCRs or
Ac4GlcNAc, followed by immunoprecipitation, CuAAC, and analysis by in-gel fluores-
cence scanning. (C) NIH3T3 cells were treated with Ac36AzGlcNAc, Ac4GlcNAz, Ac4Gal-
NAz, or Ac4GlcNAc (all at 200 µM) for 16 hours at which time cells were harvested and
subjected to copper-free click chemistry with DBCO-biotin. After incubation with FITC-
avidin, live-cell surface labeling was analyzed by flow cytometry.
216
Direct comparison of 6AzGlcNAc, GlcNAz, and GalNAz as metabolic chemical-
reporters of glycosylation.
To identify the proteins labeled by 6AzGlcNAc and compare them to those en-
riched by the previous MCRs GlcNAz and GalNAz, NIH3T3 cells were treated in
triplicate with either Ac36AzGlcNAc, Ac4GlcNAz, Ac4GalNAz, or Ac4GlcNAc as a
control (all at 200 µM) for 16 hours. At this time cells were lysed using denatur-
ing conditions (4% SDS) and subjected to CuAAC conditions with an alkyne-
bearing biotin tag. The proteomes were then reduced, alkylated, and subjected to
biotin-enrichment using streptavidin-conjugated beads. After extensive washing
to remove unlabeled proteins, on-bead trypsinolysis afforded peptides that were
analyzed using LC-MS/MS, identified using Proteome Discoverer and Mascot,
and quantified by spectral counting. Labeled proteins were identified as those
that met the following threshold criteria: First, proteins must have been identi-
fied by at least 1 unique peptide in each of the three data sets and a total of 3
spectral counts in the sum of three replicate data sets. Second, the sum of spectral
counts of the MCR-treated samples must be 3-times greater than those in the
GlcNAc labeled samples. Finally, the number of spectral counts in the MCR
treated sample compared to the control must be statistically significant (p < 0.05,
t-test). Using these criteria, 366 proteins were identified as being labeled by
6AzGlcNAc (Table 6-1), including many known O-GlcNAcylated proteins, such as
the three annotated in black in Figure 6-9A (MAP4 , NEDD4, and HCF1). GlcNAz
and GalNAz labeling identified 359 proteins (Table 6-2) and 348 proteins (Table
217
6-3), respectively. In contrast to 6AzGlcNAc, these lists included both known O-
GlcNAcylated proteins and proteins that are exclusively localized to the extracel-
lular space or the lumen of the secretory pathway and lysosome, such as the three
annotated in red in Figure 6-9A (fibronectin, calumenin, and α-glucosidase).
Comparison of the three proteomics lists showed that 6AzGlcNAc has greater
overlap with GlcNAz than GalNAz (Figure 6-9B), consistent with with previous
studies that show more efficient global-incorporation of GalNAz versus GlcNAz
into cell-surface glycoproteins (40,41). Importantly, many of the proteins that
were identified by 6AzGlcNAc have been previously identified in other O-GlcNAc
proteomic studies (42,43).
218
Figure 6-9. Identification of O-GlcNAcylated proteins using 6AzGlcNAc. (A) NIH3T3
cells were treated with Ac36AzGlcNAc, Ac4GlcNAz, Ac4GalNAz, or Ac4GlcNAc (all at 200
µM) for 16 hours. At this time, the corresponding cell-lysates were subjected to CuAAC
with alkyne-biotin, enrichment with streptavidin-coated beads, and on-bead trypsinoly-
sis. Proteins identified by LC-MS/MS are graphically presented as total number of posi-
tive minus total number of control spectral counts. Three known O-GlcNAcylated pro-
teins are annotated in black and three known extracellular/lumenal proteins are anno-
tated in red. (B) Overlap between proteins identified using 6AzGlcNAc, GlcNAz and,
GalNAz. (C) Graphical representation of enriched proteins based on whether their local-
ization is exclusively intracellular (i.e., cytoplasmic, nuclear, or mitochondrial), exclu-
sively extracellular or lumenal (i.e., ER, Golgi, lysosome), or have domains in both (e.g.,
transmembrane protein).
We next annotated the proteins in our lists based on their characterized localiza-
tions (Figure 6-9C). Proteins with uncharacterized localizations were omitted.
Consistent with specific labeling of O-GlcNAcylated proteins, 6AzGlcNAc treat-
ment enriched 350 exclusively intracellular proteins (i.e., nuclear, cytosolic, and
mitochondrial) and 8 proteins that can be localized to both the cytosol and extra-
cellular space or lumenal compartments (e.g., transmembrane proteins). Notably,
219
only 1 exclusively extracellular or lumenal protein (galectin-1) was found in this
list. In contrast, 10 and 72 exclusively extracellular or lumenal proteins were
found using GlcNAz and GalNAz treatment, respectively (Figure 6-9C), reenforc-
ing the data demonstrating the non-specific labeling of multiple types of glycosy-
lation by GlcNAz and GalNAz.
Discussion/Conclusion
The use of MCRs for the visualization and identification of protein glycosylation
has expanded the ability to investigate these key post-translational modifications.
However, recent evidence from our lab and others has demonstrated that many
MCRs of protein glycosylation lack specificity, as they are incorporated into mul-
tiple types of glycans (25,27,28). We previously showed that small changes to the
chemical structure of MCRs can have a large impact on their distribution into dif-
ferent glycans (25,29). Following this chemical-optimization theme further, we
identified an MCR (6AzGlcNAc) that robustly labeled a variety of proteins in liv-
ing mammalian cells. Using a fluorescent alkyne-tag, we compared 6AzGlcNAc to
the previous MCR, GlcNAz, and demonstrated that 6AzGlcNAc is efficiently in-
corporated onto proteins allowing visualization in as little as 2 to 4 hours after
treatment. Furthermore, 6AzGlcNAc removal from protein is dependent on the
activity of OGA, demonstrating that it is dynamically incorporated into O-
GlcNAcylated proteins. Using two reporter proteins, we next demonstrated that
while GlcNAz labels both secreted glycoproteins and O-GlcNAcylated proteins,
220
6AzGlcNAc is specific for O-GlcNAc modifications. This is consistent with our
flow cytometry data and previous reports (40) that both showed essentially no
cell-surface labeling by 6AzGlcNAc and that chemically-synthesized UDP-
6AzGlcNAc is a substrate for recombinant O-GlcNAc transferase (33).
Unlike GlcNAz, 6AzGlcNAc cannot be metabolized to the corresponding UDP-
sugar donor by the canonical GlcNAc salvage-pathway (Figure 6-3), as the first
step involves phosphorylation at the 6-hydroxyl of the monosaccharide. There-
fore, an alternative enzyme must directly phosphorylate 6AzGlcNAc at the 1-
hydroxyl to bypass this roadblock. Taking a candidate-based approach, we tested
GalK2 and AGM1 in vitro to determine if they could generate 6AzGlcNAc-1-
phosphate. We did not observe any product formation using GalK2, and knock-
down of GalK2 in living cells using shRNA did not result in reduced protein-
labeling by 6AzGlcNAc (Figure 6-4A). Notably, however, we found that AGM1 is
capable of directly generating 6AzGlcNAc-1-phosphate when its normal sub-
strate, GlcNAc-6-phosphate is added to the reaction mixture (Figure 6-5). Based
on the enzymatic mechanism, we conclude that AGM1 removes the phosphate
from GlcNAc-6-phosphate to generate the known phosphoenzyme intermediate
(36), followed by binding of 6AzGlcNAc and phosphorylation of the 1-hydroxyl.
This is consistent with the reversible nature of AGM1’s activity, where the mono-
saccharide substrates can bind the active site with either the 1- or 6-hydroxyl
groups oriented towards the catalytic serine. Additionally, we also showed that
221
once 6AzGlcNAc-1-phosphate is formed it can be enzymatically transformed to
UDP-6AzGlcNAc by AGX1 (Figure 6-4C). We do not know if UDP-6AzGlcNAc can
be epimerized to UDP-6AzGalNAc in cells; however, even if this metabolite is
formed, previous studies by Bertozzi and co-workers demonstrated that UDP-
6AzGalNAc is not a substrate for the polypeptide-N-acetyl-galactosamine transfe-
rases (41). Together, these results suggest an unappreciated metabolic flexibility
in mammalian cells. AGM1 and potentially other, yet unidentified, small-
molecule phosphotransferases may contribute to the salvaging of natural mono-
saccharides from the environment. Furthermore, they have potentially important
implications for the metabolism of bacterial or abiotic carbohydrates that would
otherwise be assumed to not enter mammalian biosynthetic pathways. Finally,
our results challenge a dogma in MCR design, which relies on well-established
metabolic pathways and directly resulted in the previous dismissal of 6AzGlcNAc
as a viable MCR in living cells (33,40).
To further confirm the specificity of 6AzGlcNAc and demonstrate any advantages
over other MCRs previously used to study O-GlcNAcylated proteins, we per-
formed a proteomics experiment using 6AzGlcNAc, GlcNAz, and GalNAz in com-
bination with alkyne-biotin and on-bead trypsinolysis. We found that enrichment
with 6AzGlcNAc resulted in the identification of essentially only intracellular pro-
teins that cannot contain glycans (e.g., N-linked or mucin O-linked) that are
added in the secretory pathway. This confirms the high degree of specificity of
222
6AzGlcNAc for O-GlcNAcylated proteins. Consistent with our fluorescence data,
GlcNAz was less selective, resulting in the enrichment of 28 proteins that poten-
tially bear secretory-pathway glycans and 10 exclusively extracellular or lumenal
proteins. Finally, GalNAz was the least selective, since it enriched only 226 exclu-
sively intracellular proteins and 72 proteins that are only extracellular or lume-
nal. We believe that this lack of selectivity is one reason why a recent study using
GalNAz required subcellular fractionation and two-dimensional electrophoresis
to identify the potential O-GlcNAcylation of the voltage-dependent anion-
selective channel protein 2 (VDAC2) (44), while the same protein was readily
identified by 6AzGlcNAc labeling without any biochemical manipulations. This
specificity is a significant improvement over other MCRs that require biochemical
manipulations (e.g., cell fractionation) to exclude cell-surface glycoproteins(28).
Previous direct-comparisons of the selectivity of different glycoprotein MCRs are
somewhat limited (25,27-29,40). The Bertozzi lab reported that GalNAz has su-
perior O-GlcNAc labeling-efficiency compared to GlcNAz due to more efficient
metabolic conversion of GalNAz to UDP-GalNAz and subsequent epimerization
to UDP-GlcNAz (28). Our in-gel fluorescence data do not support these data, as
GlcNAz and GalNAz resulted in qualitatively similar levels of protein labeling in a
variety of cell lines (Figure 6-6). Interestingly, only a minority of cell-lines show
similar global-patterns of labeling between GlcNAz and GalNAz (e.g., MCF7),
while most are significantly different. This also is true of 6AzGlcNAc, which often
223
shows different labeling patterns and intensities from both GlcNAz and GalNAz
(Figure 6-6). This supports our results that each of the MCRs is incorporated into
different types of glycoproteins and utilizes independent metabolic enzymes for
the generation of the corresponding UDP donor-sugars. Notably, while 6AzGlc-
NAc is the most selective reporter of O-GlcNAcylation, it requires longer labeling-
times to achieve the same signal-to-noise as GlcNAz (Figure 6-7A), highlighting a
potential tradeoff between labeling efficiency and specificity. However, based on
our results, we predict that any bottlenecks in the metabolism of 6AzGlcNAc will
not dramatically hamper the visualization and identification of O-GlcNAcylated
proteins. Furthermore, the extent of O-GlcNAcylation and identity of modified
proteins has been shown to be dependent on the cellular concentration of UDP-
GlcNAc (45-47). Therefore, it could be advantageous to have limited metabolic
conversion of an MCR to minimize the chances of altering the endogenous reper-
toire of O-GlcNAcylated proteins, as long as the labeling is above the detection
limit. We are currently exploring if different concentrations of MCR treatment
change the overall levels of O-GlcNAcylation. Despite the increased labeling-
efficiency of GlcNAz and GalNAz compared to 6AzGlcNAc, approximately the
same total number of spectral counts were found in our comparative proteomics
experiment. We believe that this could be due to an excess of input that exceeded
the capacity of the streptavidin beads, resulting in equal total levels of protein en-
richment prior to trypsinolysis and identification.
224
Coupled with the ever-growing toolkit of commercially available and custom
azide-reactive tags, including terminal alkynes, cyclooctynes, and phosphines, we
predict that 6AzGlcNAc will become the most powerful and readily used MCR for
the study of O-GlcNAcylation. In particular, metabolic labeling strategies have
the unique ability to isolate time-resolved protein modifications that only occur
during cell labeling. Furthermore, MCRs can be used in pulse-chase experiments
to measure the dynamic removal of O-GlcNAc modifications in living cells. Fi-
nally, the successful application of synthetic chemistry to identify a selective MCR
of O-GlcNAcylation suggests that the same chemical-strategy could be used to
create reporters that are specific for other types of protein glycosylation. Coupled
with new bioorthogonal reactions (e.g., tetrazene clycoadditions) that enable
more diverse functional groups to be incorporated into MCRs (48-50), we predict
that a diverse library of MCRs can be created to enable the specific visualization
and identification of the several types of glycosylation in mammals and other or-
ganisms.
Materials and Methods
All reagents used for chemical synthesis were purchased from Sigma-Aldrich,
Alfa Aesar or EMD Millipore unless otherwise specified and used without further
purification. DBCO-biotin was purchased from Click Chemistry Tools. FITC-
avidin was purchased from Sigma. All anhydrous reactions were performed un-
der argon or nitrogen atmosphere. Analytical thin-layer chromatography (TLC)
225
was conducted on EMD Silica Gel 60 Å F254 plates with detection by ceric ammo-
nium molybdate (CAM), anisaldehyde or UV. For flash chromatography, 60 Å sil-
ica gel (EMD) was utilized.
1
H spectra were obtained at 400, 500, or 600 MHz on
a Varian spectrometers Mercury 400, VNMRS-500, or -600. Chemical shifts are
recorded in ppm (δ) relative to solvent. Coupling constants (J) are reported in
Hz.
13
C spectra were obtained at 100, 125 or 150 MHz on the same instruments.
Chemical Synthesis.
Known chemical reporters Ac4GlcNAz(40) and Ac36AzGlcNAc (40), were synthe-
sized according to literature procedures. The fluorescent detection tag alk-rho(51)
and the OGA inhibitor Thiamet-G(39) were also synthesized in lab according to
literature procedures. Ac36AzGlcNAc and alkyne-azo-biotin were synthesized ac-
cording to literature procedures as described below.
Compound 6.1 1,3,4-Tri-O-acetyl-2-acetamido-6-azido-2,6-dideoxy-D-
glucopyranose (6AzGlcNAc). Commercially available 2-
deoxy-2-N-acetyl-glucopyranose (1.00 g, 4.52 mmol) was dis-
solved in anhydrous pyridine under nitrogen and cooled to
-20 °C. p-Toluenesulfonyl chloride (1.04 g, 5.43 mmol) was
dissolved in anhydrous pyridine (3 mL) and the solution was added dropwise
over 20 minutes to the above reaction mixture. Upon completion of the addition,
the reaction was warmed to rt and stirred for 18 hours. The mixture was concen-
O
AcO
NH
O
N
3
AcO
OAc
226
trated by vacuum and used without further purification. The crude was then re-
suspended in N, N-dimethylformamide (20 mL) under a nitrogen atmosphere.
Sodium azide (1.47 g, 22.6 mmol) was added and the reaction was stirred for 3 d
at 50 °C. The reaction mixture was then concentrated by vacuum and resus-
pended in pyridine (30 mL). Acetic anhydride (10.0 mL, 66.0 mmol) was added
and the mixture was stirred for 16 h at rt. Upon completion, solvent was removed
under reduced pressure and the resulting mixture was redissolved in CH2Cl2 (200
mL) and washed with 1 M HCl (2 × 100 mL), saturated aqueous sodium bicar-
bonate (2 × 100 mL) and water (2 × 100 mL). Organic layer was dried over so-
dium sulfate. The resulting crude mixture was purified by column chromatogra-
phy (65% ethyl acetate in hexanes) to afford 1.01 g of the product in 60% yield
over three steps.
1
H NMR (400 MHz, CDCl3): δ (ppm) 6.18 (d, J = 3.7 Hz, 1H),
5.61 (d, J = 8.9 Hz, 1H), 5.22 (ddd, J = 10.8, 9.5, 0.4 Hz, 1H), 5.13 (dd, J = 9.9,
9.4 Hz, 1H), 4.46 (ddd, J = 10.9, 9.0, 3.7 Hz, 1H), 4.14 – 4.02 (m, 1H), 3.94
(dddd, J = 10.0, 5.6, 3.1, 0.6 Hz, 1H), 3.38 – 3.26 (m, 2H), 2.19 (s, 3H), 2.06 –
2.01 (m, 6H), 1.92 (d, J = 2.6 Hz, 3H).
Compound 6.2 4-(prop-2-yn-1-yl)phenol (52). Hydroquinone (11.0 g, 0.910
mmol) and propargyl chloride (7.50 g, 0.101 mmol) were
dissolved in ethanol (20 mL) under an argon atmosphere in
a three-neck flask equipped with an addition funnel. The re-
action was heated to reflux to dissolve all solids. KOH (0.10 M in water) was
OH
227
added dropwise through the addition funnel. The mixture was then stirred for 20
h upon which time the reaction was cooled and solvent was removed by vacuum.
The resulting crude mixture was dissolved in CH2Cl2 and extracted with dilute,
aqueous KOH. The aqueous layer was then brought to a neutral pH by the addi-
tion of 1 M HCl and subsequently extracted with CH2Cl2. The organic layer was
washed with water and dried over sodium sulfate, filtered and concentrated. The
crude mixture was purified by column chromatography (10% ethyl
acetate:hexanes) to afford the pure product (4.07 g, 28%).
1
H NMR (400 MHz,
CDCl3): δ (ppm) 6.87 (dd, J = 9.1, 1.1 Hz, 2H), 6.79 (d, J = 9.0 Hz, 2H), 4.62 (dd,
J = 2.4, 1.0 Hz, 2H), 2.51 (t, J = 2.4 Hz, 1H).
Compound 6.3 (E)-methyl 4-((2-hydroxy-5-(prop-2-yn-1-
yl)phenyl)diazenyl)benzoate. To a suspension of
methyl-4-amino-benzoate in 6 M HCl was added so-
dium nitrite at 0 °C. The reaction was let stir for 30
mins. The crude reaction mixture was used in the fol-
lowing reaction. Compound 6.2 (1.30 g, 18.9 mmol) was dissolved in water:THF
(2:1) and cooled to 0 °C. Potassium carbonate (52.0 g, 376 mmol) was added and
re ac t ion l e t s t ir f or 30 mins u p on w hic h t ime 4-
(methoxycarbonyl)benzenediazonium chloride was added dropwise. The reaction
was allowed to warm to rt and was stirred for 18 h. The reaction was poured over
water and extracted with ethyl acetate (3 x 200 mL). The organic layer was dried
OH
N
N
O
O
228
over sodium sulfate, filtered, and concentrated. The resulting crude mixture was
purified by column chromatography by first starting at 10% ethyl acetate:hexanes
and increasing to 20% ethyl acetate:hexanes to elute the product. Concentration
under decreased pressure affords the product as a yellow oil (2.00 g, 90%).
1
H
NMR (400 MHz, CDCl3): δ (ppm) 8.21 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 8.6 Hz,
2H), 7.56 (d, J = 3.1 Hz, 1H), 7.12 (dd, J = 9.1, 3.1 Hz, 1H), 7.01 (d, J = 9.1 Hz,
1H), 4.76 (d, J = 2.3 Hz, 2H), 3.97 (s, 3H), 2.57 (t, J = 2.4 Hz, 1H).
13
C NMR (125
MHz, CDCl3): δ (ppm) 166.25, 153.08, 150.83, 148.27, 137.01, 131.92, 130.80,
130.17, 128.36, 123.68, 122.02, 119.17, 111.60, 78.41, 75.86, 56.77, 52.38. MALDI-
MS calculated for C17H15N2O3 [M+H]
+
: 295.1083, found 293.9045.
Compound 6.4 (E)-4-((2-hydroxy-5-(prop-2-yn-1-yl)phenyl)diazenyl)benzoic
acid. Compound 6.3 (0.124 g, 0.421 mmol) was dis-
solved in tetrahydrofuran (2 mL). NaOH (0.758 mg,
1.90 mmol) dissolved in water was added and reac-
tion let stir 18 h. Upon completion, a color change
from purple to orange is seen. The reaction was neutralized by the dropwise addi-
tion of acetic acid and subsequently concentrated under reduced pressure to re-
move solvent. The resulting crude mixture was dissolved in CH2Cl2 and washed
with water (2 x 50 mL). The organic layer was dried over sodium sulfate, filtered,
and concentrated. The crude mixture was column purified (8:1.5:0.5 ethyl
acetate:methanol:water) to afford the pure product as a bright orange solid
OH
N
N
OH
O
229
(0.825 g, 70%).
1
H NMR (600 MHz, D6-DMSO): δ (ppm) 8.05 (d, J = 8.5 Hz,
2H), 7.85 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 3.0 Hz, 1H), 7.01 (dd, J = 9.2, 3.0 Hz,
1H), 6.88 (d, J = 8.9 Hz, 1H), 4.68 (d, J = 2.3 Hz, 2H), 2.87 (t, J = 2.4 Hz, 1H).
13
C
NMR (125 MHz, D6-DMSO): δ (ppm) 216.29, 178.12, 162.96, 160.46, 160.22,
148.17, 146.80, 140.14, 132.53, 132.07, 129.05, 115.05, 89.05, 88.03, 65.94, 40.41.
MALDI-MS calculated for C16H12N2O3Na [M+Na]
+
: 303.0740, found 302.9541.
Compound 6.5 (E)-2,5-dioxopyrrolidin-1-yl-4-((2-hydroxy-5-(prop-2-yn-1-yl)
phenyl)diazenyl)benzoate. To a solution of 6.4
(0.180 g, 0.642 mmol) in THF under argon was
added N-hydroxysuccinimide (0.177 g, 1.54 mmol)
and N,N’-Dicyclohexylcarbodiimide (0.317 g, 1.54
mmol). The reaction was let stir for 18 h at rt at which time the reaction was con-
centrated by vacuum. The mixture was dissolved in ethyl acetate and filtered to
remove solids. The flow-through was concentrated and the crude product was pu-
rified by column chromatography (1:10 ethyl acetate:CH2Cl2) to afford the prod-
uct as a dark red solid that was used in the subsequent reaction without further
purification.
OH
N
N
O
O
N
O
O
230
Compound 6.6 (E)-4-((2-hydroxy-5-(prop-2-yn-1-yl)phenyl)diazenyl)-N-(13-
oxo-17-(2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl)-3,6,9-
trioxa-12-azaheptadecyl)benzamide
(Alk-azo-biotin). Compound 6.5 (0.040 g, 0.101 mmol) was dissolved in anhy-
drous N,N’-dimethylformamide (1 mL) under argon. EZ-Link Amine PEG3-Biotin
(0.460 mg, 0.111 mmol) (Thermo Scientific) was added and reaction let stir for 18
h upon which time solvent was removed by vacuum. The resulting crude mixture
was purified by RP-HPLC over a C18 semi-preparative column (The Nest Group)
using a 5.5-44% B linear gradient over 10 min before switching to a 44-100% B
linear gradient over 40 mins,
t
R = 18 min (buffer A: 0.1% TFA in water, buffer B:
0.1% TFA, 90% ACN in water) and lyophilized to afford the pure product as an
orange solid (0.022 g, 31%). ESI-MS calculated for C34H40N6O8S (oxidized at the
biotin cysteine) [M+Na]
+
: 719.28, found 719.20.
Compound 6.7 6-O-p-methylbenzenesulfonate-N-acetyl-glucosamine. Com-
mercially available 2-deoxy-2-N-acetyl-glucopyranose (2.50
g, 11.3 mmol) was co-evaporated from toluene and dis-
solved in anhydrous pyridine (20 mL). The reaction mix-
ture was cooled to -20 °C. p-Toluenesulfonyl chloride (2.59 g, 13.6 mmol) was
then dissolved is anhydrous pyridine (5 mL) and added drop-wise to the stirring
mixture. Upon completion of addition, the reaction was allowed to warm to room
OH
N
N
O
H
N
O
3
H
N
O
S
NH
HN
O
O
AcHN
OTs
HO
HO
OH
231
temperature and stirred for 16 h under an argon atmosphere. To purify, the reac-
tion was concentrated under reduced pressure and the crude mixture purified by
column chromatography (7:1:0.5 ethyl acetate:methanol:water) to afford the
product as a yellow oil (1.78 g).
1
H NMR (500 MHz, CD3OD) α-anomer: δ 7.74 (d,
J = 8.3 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 5.13 (d, J = 3.6 Hz, 1H), 3.99 (m, 1H),
3.90 (dd, J = 2.2, 10.6 Hz, 1H), 3.73 (m, 1H), 3.55 (dd, J = 3.2, 13.3 Hz, 1H), 3.44
(dd, J = 5.4, 12.6 Hz, 1H), 3.38 (m, 1H), 2.39 (s, 3H), 2.01 (s, 3H). The product
was used in the subsequent reaction with no further characterization.
Compound 6.8 6-azido-6-deoxy-N-acetyl-glucosamine
(6AzGlcNAc, 9) (40). Compound 6.7 (1.78 g, 4.73 mmol)
was coevaporated from toluene and dissolved in anhydrous
N,N’-dimethylformamide (20 mL). Sodium azide (1.54 g, 23.7 mmol) was then
added and the reaction warmed to 50 °C. The reaction was stirred for 3 d after
which time the reaction was cooled and concentrated under reduced pressure.
The crude mixture was purified by silica gel chromatography (9:1:0.5 ethyl
acetate:methanol:water) to afford the product as a white solid (402 mg, 14% yield
over 2 steps). The sugar was further purified by RP-HPLC over a C18 semi-
preparative column (The Nest Group) using a 5-15% B linear gradient over 10
min,
t
R = 2.5 min (buffer A: 0.1% TFA in water, buffer B: 0.1% TFA, 90% ACN in
water).
1
H NMR (500 MHz, (CD3)2SO) α-anomer: δ 7.69 (d, J = 8.3 Hz, 1H), 4.94
(app s, 1H), 3.78 (m, 1H), 3.62 (m, 1H), 3.50 (m, 2H), 3.37 (m, 1H), 3.10 (app t, J
O
AcHN
N
3
HO
HO
OH
232
= 9.2 Hz, 1H), 1.83 (s, 3H).
13
C NMR (125 MHz,(CD3)2SO) β-anomer: δ 169.39,
90.77, 71.85, 70.52, 70.17, 54.21, 51.60, 22.67.
Compound 6.9 α-1-O-benzyl-N-acetyl-glucosamine (33). The procedure was
adapted from literature.(53) Commercially available 2-deoxy-
2-N-acetyl-glucopyranose (5.00 g, 22.6 mmol) was sus-
pended in benzyl alcohol (50 mL) and concentrated HCl was
added (1 mL). The solution was warmed to 75 °C and stirred for 4 h after which
time the reaction was cooled and poured into diethyl ether (400 mL) with vigor-
ous stirring. A white precipitate was observed and the mixture left at 4 °C for 16
h. The precipitate was then filtered and washed with diethyl ether (50 mL) to re-
move remaining benzyl alcohol. The filtrated was dried and recrystallized in a
minimal amount of isopropanol to afford the product as white solid (2.48 g, 7.98
mmol, 35% yield).
1
H NMR (500 MHz, (CD3)2SO): δ 7.82 (d, J = 8.2 Hz, 1H),
7.38-7.28 (m, 5H), 4.71 (d, J = 3.5 Hz, 1H), 4.68 (d, J = 12.5 Hz, 1H), 4.31 (d, J =
12.5 Hz, 1H), 3.80 (q, J = 6.1 Hz, 1H), 3.70-3.64 (m, 2H), 3.55-3.47 (m, 2H), 3.18
(t, J = 9.02 Hz, 1H), 1.83 (s, 3H).
Compound 6.10 α-1-O-benzyl-6-O-p-methylbenzenesulfonate-N-acetyl-
glucosamine (33). Compound 6.9 (2.36 g, 7.58 mmol) was co-
evaporated from toluene and dissolved in anhydrous pyridine
(20 mL) under an argon atmosphere. The mixture was then
O
AcHN
OH
HO
HO
OBn
O
AcHN
OTs
HO
HO
OBn
233
cooled to -20 °C. p-toluenesulfonyl chloride (1.74 g, 9.10 mmol), freshly rescrys-
tallized from CH2Cl2, was dissolved in pyridine (7 mL) and added dropwise over
20 min. The reaction was stirred at -20 °C for 1 h and the dry ice bath replaced
with an ice bath. The reaction was allowed to warm to room temperature over 16
h. Upon completion, the mixture was concentrated to remove pyridine and puri-
fied over silica gel (9:1:0.5 EtOAc:methanol:water) to afford product (1.85 g, 3.97
mmol, 52% yield).
1
H NMR (500 MHz, CD3OD): δ 7.85 (d, J = 10.4 Hz, 2H), 7.46
(d, J = 1.1 Hz, 2H), 7.36 (m, 5H), 4.76 (d, J = 4.5 Hz, 1H), 4.65 (d, J = 15 Hz, 1H),
4.45 (d, J = 14.8 Hz, 1H), 4.36 (dd, J = 2.6, 13.6 Hz, 1H), 4.27 (dd, J = 7.3, 13.6
Hz, 1H), 3.88 (dd, J = 4.6, 13.5 Hz, 1H), 3.81-3.78 (m, 1H), 3.71-3.66 (m, 1H),
3.35-3.31 (m, 1H), 2.46 (s, 3H), 1.97 (s, 3H).
Compound 6.11 3,4-di-O-acetyl-α-1-O-benzyl-6-O-p-methylbenzenesulfonate-
N-acetyl-glucosamine (33). 6.10 (1.85 g, 3.97 mmol) was
resuspended in pyridine (20 mL) and acetic anhydride (1.12
mL, 11.01 mmol). The reaction was stirred for 3 h at room
temperature after which time the reaction mixture was con-
centrated and purified over silica gel (75% EtOAc in hexanes) to afford product in
quantitative yield (2.18 g, 3.97 mmol).
1
H NMR (500 MHz, CDCl3): δ 7.79 (d, J =
10.3 Hz, 2H), 7.37-7.29 (m, 7H), 5.63 (d, J = 11.9 Hz, 1H), 5.20 (dd J = 11.7, 13.5
Hz, 1H), 4.96 (t, J = 12.1 Hz, 1H), 4.82 (d, J = 4.6 Hz, 1H), 4.67 (d, J = 14.7 Hz,
O
AcHN
OTs
AcO
AcO
OBn
234
1H), 4.44 (d, J = 14.8 Hz, 1H), 4.26 (td, J = 4.6, 12.6 Hz, 1H), 4.07 (d, J = 5.0 Hz,
2H), 4.04-4.00 (m, 1H), 2.44 (s, 3H), 1.98 (d, J = 2.6 Hz, 6H), 1.86 (s, 3H).
Compound 6.12 3,4-di-O-acetyl-6-O-p-methylbenzenesulfonate-N-acetyl-
glucosamine. Procedure adapted from published literature
(33). 6.11 (969 mg, 1.76 mmol) was resuspended in metha-
nol. Pd(OH)2/C (10% Pd) was added and a balloon of H2 was
attached. The reaction was monitored by TLC (75% EtOAc in
hexanes) and stirred for 48 h to completion. The mixture was then filtered over a
pad of celite and the flow-through evaporated to yield the product (710 mg, 1.55
mmol) that was used in subsequent reactions with no further characterization.
Compound 6.13 Diallyl(3,4-di-O-acetyl-6-O-p-methylbenzenesulfonate-N-
acetyl-glucosamine)-α-1-phosphate. 6.12 (629 mg,
1.37 mmol) was coevaporated with toluene and resus-
pended in CH2Cl2 (10 mL) under an argon atmos-
phere. 5-(Ethylthio)-1H-tetrazole (1.07 g, 8.22 mmol)
was added and the reaction stirred for 15 min. Diallyl-
N,N’-diisopropylphosphoramidite (1.00 g, 4.11 mmol) was added dropwise, and
the reaction stirred for 2 h until completed as determined by TLC (5% methanol
in CH2Cl2). At this time, the reaction was cooled to -78 °C and freshly recrystal-
lized m-chloroperoxybenzoic acid was added (1.18 g, 6.85 mmol). The reaction
O
AcHN
OTs
AcO
AcO
OH
O
AcHN
OTs
AcO
AcO
O
P
O
O
O
235
was allowed to proceed for 10 min after which time the dry ice bath was replaced
with an ice bath, and the reaction was slowly warmed to room temperature over 1
h. Upon completion, the reaction was diluted with CH2Cl2 (50 mL) and washed
2x each with saturated sodium thiosulfate, saturated sodium bicarbonate, water
and brine. The organic layer was then concentrated and purified over silica gel
(35%-45% acetone in hexanes) to afford the product (717 mg,74% yield over 2
steps).
1
H NMR (500 MHz, CDCl3): δ 7.70 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 7.8
Hz, 2H), 6.06 (d, J = 9.4 Hz, 1H), 5.93-5.82 (m, 2H), 5.54 (dd, J = 3.3, 6.3 Hz,
1H), 5.32 (ddd, J = 17.1, 12.3, 1.4 Hz, 2H), 5.23 (ddd, J = 10.6, 9.6, 1.2 Hz, 1H),
5.12 (dd, J = 10.9, 9.4 Hz, 1H), 4.96 (dd, J = 10.3, 9.5 Hz, 1H), 4.54-4.49 (m, 3H),
4.25-4.21 (m, 1H), 4.19-4.15 (m, 1H), 4.04 (dd, J = 11.1, 2.6 Hz, 1H), 3.98 (dd, J =
11.1, 5.1 Hz, 1H), 2.39 (s, 3H), 1.94 (s, 3H), 1.93 (s, 3H), 1.86 (s, 3H);
13
C NMR
(125 MHz, CDCl3): δ 171.08, 170.31, 169.06, 145.21, 132.23, 132.12, 132.07, 131.91,
131.86, 129.86, 128.05, 119.06, 118.93, 95.65, 95.60, 69.73, 69.37, 68.79, 67.67,
66.99, 51.69, 51.63, 22.87, 21.63, 20.58, 20.45;
31
P NMR (500 MHz, CDCl3): δ
-2.77; APCI-HRMS calculated for C25H34NO13PSNa [M+Na]
+
: 642.1488, found
642.1398.
236
Compound 6.14 Diallyl(6-O-p-methylbenzenesulfonate-N-acetyl-
glucosamine)-α-1-phosphate. 6.13 (669 mg, 1.08
mmol) was resuspended is methanol (10 mL). Freshly
made NaOMe was added dropwise until pH 9-10 was
reached. The reaction was monitored by TLC (10%
methanol in CH2Cl2) and was determined complete af-
ter 1.5 h. Upon completion, the reaction was quenched with acetic acid and con-
centrated to afford the crude. Silica gel chromatography (7% methanol in CH2Cl2)
yielded the product (282 mg, 57% yield).
1
H NMR (500 MHz, CD3OD): 7.79 (d,
2H, J = 8.3 Hz), 7.44 (d, J = 8.5 Hz, 2H), 6.02-5.92 (m, 2H), 5.59 (dd, J = 8.5, 8.5
Hz, 1H), 5.42-5.37 (m, 2H), 5.29-5.26 (m, 2H), 4.60-4.55 (m, 4H), 4.32 (dd, J =
11.0, 1.9 Hz, 1H), 4.21 (dd, J = 11.0, 5.7 Hz, 1H), 3.93-3.89 (m, 1H), 3.86-3.82 (m,
1H), 3.64 (dd, J = 10.8, 8.8 Hz, 1H), 3.36 (t, J = 8.7 Hz, 1H), 2.46 (s, 3H), 1.98 (s,
3H);
13
C NMR (125 MHz, CDCl3): δ 173.75, 146.59, 134.05, 133.63, 133.58, 133.57,
133.53, 131.02, 129.09, 118.89, 118.87, 97.39, 97.34, 73.35, 71.15, 71.12, 70.22,
69.89, 69.84, 69.79, 69.75, 55.08, 55.01, 22.51, 21.62;
31
P NMR (500 MHz,
CDCl3): δ -2.51; APCI-HRMS calculated for C21H30NO11PSNa [M+Na]
+
: 558.1169,
found 558.1154.
O
AcHN
OTs
HO
HO
O
P
O
O
O
237
Compound 6.15 Diallyl(6-azido-6-deoxy-N-acetyl-glucosamine)-α-1-
phosphate. 6.14 (282 mg, 0.527 mmol) was coevapo-
rated from toluene and resuspended in N,N’-
dimethylformamide (20 mL) under an argon atmos-
phere. Sodium azide (172 mg, 2.63 mmol) was added,
and the reaction reaction warmed to 60 °C. The reaction proceeded for 48 h after
which time the reaction was concentrated and purified over silica gel (7:2:1
EtOAc:methanol:water) to afford the product (151 mg, 71% yield).
1
H NMR (500
MHz, CD3OD): 6.07-5.73 (m, 2H), 5.40 (dd, J = 7.4, 3.3 Hz, 1H), 5.27-5.20 (m,
2H), 5.06-5.01 (m, 2H), 4.34-4.31 (m, 2H), 4.27-4.24 (m, 2H), 3.89-3.81 (m, 2H),
3.59 (dd, J = 10.6, 8.9 Hz, 1H), 3.49 (dd, J = 13.2, 2.5 Hz, 1H), 3.36-3.31 (m, 2H),
3.22-3.21 (m, 1H), 1.91 (s, 3H);
13
C NMR (125 MHz, CDCl3): δ 173.81, 135.99,
135.92, 116.18, 116.15, 95.59, 95.54, 73.63, 72.48, 72.37, 67.33, 67.29, 67.17, 67.13,
55.33, 55.27, 52.63, 22.85;
31
P NMR (500 MHz, CDCl3): δ 0.66, -1.37.
Compound 6.16 6-azido-6-deoxy-N-acetyl-glucosamine-1-phosphate (54).
6.15 (50 mg, 0.123 mmol) was resuspended in 4 mL
methanol:THF (1:1) under an argon atmosphere. p-
Toluenesulfinic acid sodium salt (44 mg, 0.246 mmol) and
Tetrakis(triphenylphosphine)-Palladium(0) (11 mg, 0.095 mmol) were added.
The the reaction was monitored by TLC (3:2:1 N-propanol:acetic acid:water) and
determined complete. The reaction was then evaporated under reduced pressure
O
AcHN
N
3
HO
HO
O
P
O
O
O
O
AcHN
N
3
HO
HO
OPO
3
-2
238
and purified by silica gel chromatography (3:2:1 N-propanol:acetic acid:water).
The sugar was further purified by RP-HPLC over a C18 semi-preparative column
(The Nest Group) using a 0% B isocratic flush over 10 min followed by a 0-50% B
linear gradient from 10-20 min and a second linear gradient 50-0% B 20-30 min,
t
R = 2.5-4 min (buffer A: 0.1% TFA in water, buffer B: 0.1% TFA, 90% ACN in wa-
ter).
1
H NMR (500 MHz, D2O): δ 5.33 (dd, J = 7.3, 3.4 Hz, 1H), 3.94–3.83 (m,
2H), 3.68 (dd, J = 10.5, 9.1 Hz, 1H), 3.64-3.56 (m, 2H), 3.53-3.43 (m, 2H), 1.95
(s, 3H);
13
C NMR (125 MHz, D2O): δ 174.63, 163.38, 163.10, 162.82, 162.53,
119.78, 117.46, 115.14, 112.82, 93.43, 93.38, 71.34, 70.42, 70.35, 58.59, 53.73,
53.66, 50.67, 33.71, 21.92;
31
P NMR (500 MHz, D2O): δ -1.65; ESI-MS calculated
for C8H14N4O8P [M-H]
-
: 323.04, found 325.00.
Cell Culture.
COS-7, HEK293, HeLa and MCF7 cells were cultured in DMEM media (Corning)
enriched with 10% fetal bovine serum (HyClone, ThermoScientific). AmphoPack-
293 retroviral packaging cells (Clontech) were cultured in DMEM media (Corn-
ing) enriched with 10% fetal bovine serum (HyClone, ThermoScientific). NIH3T3
and MEF cells were cultured in high-glucose DMEM media (Corning) enriched
with 10% fetal calf serum (HyClone, ThermoScientific). H1299 cells were cultured
in RPMI media enriched with 10% fetal bovine serum (HyClone, ThermoScien-
tific). SH-SY5Y cells were cultured in a 1:1 mixture of DMEM:F12 Medium (Corn-
239
ing) enriched with 10% fetal bovine serum (HyClone, ThermoScientific). All cell
lines were maintained in a humidified incubator at 37 ℃ and 5.0% CO2.
Metabolic Labeling.
To cells at 80-85% confluency, media containing Ac4GlcNAc, Ac4GlcNAz,
Ac4GlcNAlk, Ac36AzGlcNAc, Ac36AlkGlcNAc (1,000 x stock in DMSO), or DMSO
vehicle was added as indicated. For chase experiments, existing media was re-
placed with media supplemented with 200 µM Ac4GlcNAc (Sigma) or 200 µM
Ac4GlcNAc (Sigma) plus 10 µM Thiamet-G (1,000 x stock in DMSO) as indicated.
Preparation of Nonidet P-40 (NP-40)-Soluble Lysates.
The cells were collected by trypsinization and pelleted by centrifugation at for 4
min at 2,000 x g, followed by washing 2x with PBS (1 mL). Cell pellets were then
resuspended in 100 µL of 1% NP-40 lysis buffer [1% NP-40, 150 mM NaCl, 50
mM triethanolamine (TEA) pH 7.4] with Complete, Mini, EDTA-free Protease
Inhibitor Cocktail Tablets (Roche) for 20 min and then centrifuged for 10 min at
10,000 x g at 4 ℃. The supernatant (soluble cell lysate) was collected and the
protein concentration was determined by BCA assay (Pierce, ThermoScientific).
Cu(I)-Catalyzed [3 þ 2] Azide-Alkyne Cycloaddition.
Cell lysate (200 µg) was diluted with cold 1% NP-40 lysis buffer to obtain a de-
sired concentration of 1 µg/µL. Newly-made click chemistry cocktail (12 µL) was
240
added to each sample [alkynyl-rhodamine tag (100 µM, 10 mM stock solution in
DMSO); tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (1 mM, 50 mM
freshly prepared stock solution in water); tris[(1-benzyl-1-H-1,2,3-triazol-4-
yl)methyl]amine (TBTA) (100 µM, 10 mM stock solution in DMSO); CuSO4•5H2O
(1 mM, 50 mM freshly prepared stock solution in water) for a total reaction vol-
ume of 200 µL. The reaction was gently vortexed and allowed to sit at room tem-
perature for 1 h. Upon completion, 1 mL of ice cold methanol was added to the
reaction, and it was placed at -20 ℃ for 2 h to precipitate proteins. The reactions
were then centrifuged at 10,000 x g for 10 min at 4 ℃. The supernatant was re-
moved, the pellet was allowed to air dry for 15 min, and then 50 µL 4% SDS
buffer (4% SDS, 150 mM NaCl, 50 mM TEA pH 7.4) was added to each sample.
The mixture was sonicated in a bath sonicator to ensure complete dissolution,
and 50 µL of 2x SDS-free loading buffer (20% glycerol, 0.2% bromophenol blue,
1.4% β-mercaptoethanol, pH 6.8) was then added. The samples were boiled for 5
min at 97 ℃, and 40 µg of protein was then loaded per lane for SDS-PAGE sepa-
ration (Any Kd, Criterion Gel, Bio-Rad).
In-Gel Fluorescence Scanning.
Following SDS-PAGE separation, gels were scanned on a Typhoon 9400 Variable
Mode Imager (GE Healthcare) using a 532 nm for excitation and 30 nm bandpass
filter centered at 610 nm for detection.
241
Reverse Transcriptase-PCR.
Information about primers used for GALK2 and GAPDH available upon request.
RNA from NIH3T3 cells was isolated using the RNAeasy Kit (Qiagen). Concentra-
tions of RNA were obtained by UV-Vis. PCR was conducted in an Eppendorf
Mastercycler thermocycler. To a 0.2 mL thermo-walled PCR tube was added 2X
Reaction Mix (SuperScript™ One-Step RT-PCR with Platinum® Taq, Invitro-
gen), template RNA from NIH3T3 cells (1,000 ng), sense and anti-sense primers
for GALK2 (10 µM), sense and anti-sense primers for GAPDH (10 µM), water,
and Taq enzyme (SuperScript™ One-Step RT-PCR with Platinum
®
Taq, Invitro-
gen). The provided PCR cycle was used according to SuperScript™ One-Step RT-
PCR with Platinum® Taq (Invitrogen ) with an extension time of 32 sec (1 min/
kbp). Products were diluted with 6X sample loading dye (Bio-Rad) and analyzed
by electrophoresis on a 5% agarose gel (500 mg agarose in 1X TAE buffer, tris/
acetic acid/EDTA, Bio-Rad). The gel was subsequently visualized using a Chemi-
Doc XRS+ molecular imager (Bio-Rad).
GalNAc Kinase 2 (GalK2) Assay.
Recombinant human GalK2 was prepared as previously described.(55) Recombi-
nant GalK2 (8 µg mL
−1
) was incubated in triplicate with GalNAc, GlcNAc or
6AzGlcNAc (40 mM) in 25 µL reaction buffer (10 mM MgCl2, 50 mM Tris HCl pH
8.0) containing 1 mg/mL BSA and 5 mM [
32
P]ɣATP (1000 cpm/nmol) for 60 min
242
at 37 °C. After this time, reactions were terminated by the addition of water (0.75
mL) and applied to a Dowex 1 x 8 (Cl-) column (0.7 cm x 3.0 cm). Unreacted
starting material was eluted by washing with 2 mL of 25 mM NH4HCO3 before
eluting the sugar-1-phosphates with 100 mM NH4HCO3. The fractions (2 mL)
were counted in a liquid scintillation counter (µBeta, Perkin-Elmer). Controls
without acceptor substrates were treated in the same way. Column profiles were
compared to detect the presence of overlapping radioactive peaks corresponding
to degradation products. If present, these peaks were subtracted from the assay
chromatogram.
Expression of phosphoacetylglucosamine mutase (AGM1).
Homo sapiens phosphoacetylglucosamine mutase 3 (AGM1/PGM3) cDNA was
obtained from Biovalley (Marne-la-Vallée, France), amplified by PCR, sequenced
and cloned in pTrcHis A (Invitrogen)). The 6His-tagged PGM3 protein was ex-
pressed in Escherichia coli DH5α (Invitrogen) cultured for 24 h at 18 °C in 2 TY
medium supplemented with 1 mM IPTG and 2 mM MgCl2. Bacteria were lysed in
Y-Per (ThermoScientific) and the lysate diluted with 5 volumes of 50 mM phos-
phate buffer pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.1 mM PMSF and 0.1
mM TCEP. After application of the lysate on a HisTrap FF 5mL column (GE
Healthcare), AGM1 was eluted with 250 mM imidazole in 25 mM phosphate
buffer pH 8.0, 150 mM NaCl, 0.1 mM TCEP. AGM1 activity was checked in a cou-
pled assay by 2 h incubation at 37 °C with GlcNAc-6-phosphate (2 mM), in 75
243
mM Tris HCl pH 8.8, 5 mM MgCl2, containing 0.1 mg BSA and 2 µL of the PGM3
enzyme solution, and coupling with AGX1 (0.6−6.3 µg mL
−1
, 2.2−22 mU mL
−1
)
and yeast inorganic pyrophosphatase (1.6 µg mL
−1
, 3 U mL
−1
) in the presence of
[
3
H]UTP (Perkin-Elmer, 2 mM, 260 cpm nmol
−1
). After AGM1/AGX1/yeast inor-
ganic phosphatase heat denaturation, calf intestinal alkaline phosphatase (NEB,
80 U mL
−1
) was added in order to hydrolyze all the UTP and UDP present, and
the products were monitored as described below for AGX1 enzymatic tests (sepa-
ration on paper chromatography). Finally, the production of UDP-GlcNAc was
estimated by scintillation counting. Under these conditions, the recombinant
AGM1 activity was estimated to 20 nmol h
−1
µg
−1
of the AGM1 enzyme solution.
Phosphoacetylglucosamine mutase (AGM1) Assay.
6AzGlcNAc (2.25 mM final concentration) was incubated in 100 µL of 75 mM
Tris HCl pH 8.8, 5 mM MgCl2, containing 0.1 mg BSA and 10 µg of the PGM3 en-
zyme solution. Incubations were run at 37°C for 1 h 30 min either without cofac-
tor or in the presence of Glc-6P, Glc1,6-diP or GlcNAc-6P (1.00 mM final concen-
tration). Reactions were stopped by freezing at –20 °C. After thawing, the reac-
tion mixtur es wer e incubated for 1 h at 37 °C with
bicyclo[6.1.0]nonyne-(POE)3-NH-Dye495 conjugate (Synaffix, Oss, Netherlands).
The reaction mixtures which contain the azido sugars coupled through their azido
moiety to the BCN fluorescent Dye495, were then laid onto a 46 x 57 cm sheet of
Whatman 3MM Paper (GE Healthcare) and run for descending chromatography
244
in ethyl acetate/formic acid/water (70:20:10) for 5 h. After drying, fluorescent
spots (Rf ~ 0.8) were cut out and the products eluted from the paper in 50%
methanol. They were further concentrated under vacuum before MS analysis.
UDP-GalNAc pyrophorylase (AGX1) Assay.
Recombinant hAGX1 was prepared as previously described.(55) AGX1 (0.6-6.3 µg
mL
−1
, 2.2-22 mU mL
−1
) was incubated with GlcNAc-1P or 6AzGlcNAc-1P in 25 µL
reaction buffer (1 mM MgCl2, 75 mM Tris HCl pH 8.8) containing 1 mg mL
−1
BSA
and 2 mM [
3
H]UTP (260 cpm/nmol) for 10 min at 37 °C. Yeast inorganic pyro-
phosphatase (1.6 µg mL
−1
, 3 mU mL
−1
) was added to inhibit the reverse reaction.
Reactions were terminated by heating for 6 min at 80 °C. To hydrolyze excess
UTP and UMP, calf intestinal alkaline phosphatase (New England Biolabs, 80 U/
mL) was added to the reaction and incubated for 2 h at 37 °C. The reaction was
spotted onto Whatman 3MM chromatography paper and submitted to descend-
ing chromatography in ethyl acetate/formic acid/water (70:20:10) to remove
[
3
H]Uridine (Rf = 0.25). Spots with [
3
H]UDP-sugars (Rf = 0.01) were cut out of
the paper and counted in a liquid scintillation counter. Control samples without
sugar-1P received the same treatment in order to deduct background radioactiv-
ity. Tests were run in triplicates and each experiment was repeated three times.
Km and Kcat were calculated using the Enzyme Kinetic Module 1.3 of SigmaPlot
10, from plots obtained with different concentrations of sugar-1P and different
amounts of AGX1.
245
MTS Assay
NIH3T3 cells were pretreated with 200 µM Ac36AzGlcNAc or DMSO for 72 hrs
prior to plating. NIH-3T3 cells (1 x 10
4
cells) were plated per well in a 96-well,
white bottom dish 24 hours before treatment with 200 µM Ac4GlcNAc or
Ac36AzGlcNAc for 16 hours in triplicate. CellTiter 96
®
AQueous Non-Radioactive
Cell Proliferation Assay (Promega, Madison, WI) was used according to the pro-
vided protocol. Absorbance at 490 nm was read using a BioTek Synergy H4
Multi-Mode Microplate reader.
Flow Cytometry of Cell-Surface Labeling with DBCO-Biotin.
NIH3T3 cells grown in 6-well plates at 80-85% confluency were treated with 200
µM Ac4GlcNAc, Ac4GlcNAz, Ac4GalNAz or Ac36AzGlcNAc in triplicate for 16
hours at which time media was removed and cells were gently washed with PBS
before being detached from the plate with 1 mM EDTA in PBS. Cells were col-
lected by centrifugation (5 min, 300 x g at 4 ℃) and were washed three times
with PBS (5 min, 300 x g at 4 ℃). Cells were then resuspended in 200 µL PBS
containing DBCO-biotin (Click Chemistry Tools, 60 µM) for 1 h, after which time
they were washed three times with PBS (5 min, 300 x g at 4 ℃) before being re-
suspended in ice-cold PBS containing fluorescein isothiocynate (FITC) conju-
gated avidin (Sigma, 5 µg/mL, 30 mins at 4 ℃). Cells were then washed three
times in PBS (5 min, 300 x g at 4 ℃) before being resuspended in 400 µL PBS for
246
flow-cytometry analysis. A total of 10,000 cells [dead cells were excluded by
treatment with propdium iodide (2.5 µg/mL in water, 30 mins)] were analyzed
on a BD SORP LSRII Flow Cytometer using the 488 nm argon laser.
FoxO1 Labeling.
NIH3T3 cells stably expressing FLAG-tagged FoxO1 were treated with 200 µM
Ac4GlcNAz, Ac36AzGlcNAc (1,000x stock in DMSO) or DMSO and allowed to in-
cubate overnight. After 16 h, cells were washed with PBS, trypsinized and pel-
leted. Cell pellets were resuspended in 100 µl of 1% NP-40 lysis buffer [1% NP-
40, 150 mM NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete Mini,
EDTA-free Protease Inhibitor Cocktail Tablets (Thermo Scientific) for 20 min
and then centrifuged at 4 ℃ for 10 min at 10,000 x g. The supernatant was col-
lected and the protein concentration was determined by BCA assay (Pierce,
ThermoScientific). Total cell lysate (1.5 mg) was diluted as necessary to a final
volume of 1 mL with 1% NP-40 buffer with Complete Mini, EDTA-free Protease
Inhibitor Cocktail Tablets (Thermo Scientific). EZview Red ANTI-FLAG M2 affin-
ity beads (30 µL, Sigma), pre-washed with cold NP-40 buffer 2X followed by cold
PBS 2x, were added to each sample. The samples were placed on a rotator for 2 h
at 4 ℃. Beads were collected by centrifugation at 2,000 x g for 2 min at 4 ℃, and
the supernatant was carefully removed. Beads were then washed with cold PBS
by rotating for 5 mins before centrifuging 2 mins at 2,000 x g. The final PBS wash
was carefully removed, and the beads were suspended in 40 µL 4% SDS buffer
247
and boiled for 5 min at 97 ℃. The appropriate amount of click chemistry cocktail
was added, and the reaction was allowed to proceed for 1 h after which time 30
µL of 2x loading buffer was added. Samples were boiled for 5 minutes at 97 ℃.
Protein samples (40 µg) were then loaded per lane for SDS-PAGE separation
(Any Kd Criterion Gel, Bio-Rad) and imaged by in-gel fluorescence scanning.
GlyCAM-IgG Labeling.
NIH3T3 cells stably expressing GlyCAM-IgG in 6-well dishes at 80-85% conflu-
ency were treated in DMEM with 10% FCS and 200 µM Ac4GlcNAz, Ac36AzGlc-
NAc (1,000x stock in DMSO) or DMSO for 24 hours. The media from each sam-
ple was collected by centrifugation at 3,000 x g for 10 min at 4 ˚C to remove cell
debris. The supernatant (1mL) was incubated with 50 µL of recombinant protein
G sepharose beads (Invitrogen) in 100 mM TEA pH 8 overnight. Beads were col-
lected by centrifugation at 2,000 x g for 2 min at 4 ℃. Beads were washed 3x
with 1 mL 100 mM TEA pH 8. GlyCAM-Ig was eluted by addition of 50 µL 4%
SDS buffer (4% SDS, 150 mM NaCl, 50 mM TEA pH 7.4) and boiling for 5 min at
97 ˚C. Protein concentration was determined by BCA assay (ThermoScientific).
Final SDS concentration was diluted to 0.5% by addition of 50 mM TEA pH 7.4.
The appropriate amount of click chemistry cocktail was added and the reaction
was allowed to proceed for 1 h after which time 4x loading buffer (200 mM Tris
HCl, 4% SDS, 40% glycerol, 0.4% bromophenol blue, 1.4% β-mercaptoethanol,
248
pH 6.8) was added. Samples were boiled for 5 min at 97 ˚C and 50 µg were
loaded for SDS-PAGE separation (Any Kd Criterion Gel, Bio-Rad).
Western Blotting.
Proteins were separated by SDS-PAGE before being transferred to PVDF mem-
brane (Bio-Rad) using standard western blotting procedures. All western blots
were blocked in TBST (0.1% Tween-20, 150 mM NaCl, 10mM Tris pH 8.0) con-
taining 5% non-fat milk for 1 h at rt. The blots were then incubated with the ap-
propriate primary antibody in blocking buffer for 1 h at rt. The anti-FLAG anti-
body (Thermo) and anti-MAb414 antibody (Covance) were used at a 1:5,000 dilu-
tion and 1:1,000 for detection of Foxo1A and p62, respectively. The anti-Nedd4
antibody (Millipore) was used at a 1:10,000 dilution to detect Nedd-4 and the
anti-Pyruvate kinase antibody (Abcam) was used at 1:1,000. The blots were then
washed three times in TBST for 10 min and incubated with the horseradish per-
oxidase (HRP)-conjugated secondary antibody for 1 h in blocking buffer at rt.
HRP-conjugated anti-mouse, anti-rabbit, anti-goat and anti-human antibodies
(Jackson ImmunoResearch) were used at 1:10,000 dilutions. After being washed
three more times with TBST for 10 min, the blots were developed using ECL rea-
gents (Bio-Rad) and the ChemiDoc XRS+ molecular imager (Bio-Rad).
249
Biotin Enrichment and On-bead Trypsinolysis.
NIH3T3 cell-pellets labeled with Ac36AzGlcNAc, Ac3GlcNAz, Ac3GalNAz or
Ac4GlcNAc for 16 hours were resuspended in 200 µL H2O, 60 µL PMSF in H2O
(250 mM), and 500 µL 0.05% SDS buffer (0.05% SDS, 10 mM TEA pH 7.4, 150
mM NaCl) with Complete Mini protease inhibitor cocktail (Roche Biosciences).
To this was added 8 µL Benzonase (Sigma), and the cells were incubated on ice
for 30 min. Then, 4% SDS buffer (2000 µL) was added, and the cells were briefly
sonicated in a bath sonicator followed by centrifugation (20,000 x g for 10 min at
15 °C). Soluble protein concentration was normalized by BCA assay (Pierce,
ThermoScientific) to 1 mg/mL, and 10 mg of total protein was subjected to the
appropriate amount of click chemistry cocktail containing alkyne-PEG3-biotin (5
mM, Click Chemistry Tools) for 1 h, after which time 10 volumes of ice-cold
MeOH were added. Precipitation proceeded 2 hours at -20 °C. Precipitated pro-
teins were centrifuged at 5,200 x g for 30 min at 0 ℃ and washed 3 times with 40
mL ice-cold MeOH, with resuspension of the pellet each time. The pellet was then
air-dried for 1 h. To capture the biotinylated proteins by streptavidin beads, the
air-dried protein pellet was resuspended in 2 mL of resuspension buffer (6 M
urea, 2 M thiourea, 10 mM HEPES pH 8.0) by bath sonication. To cap cysteine
residues, 100 µl of freshly-made TCEP (200 mM stock solution, Thermo) was
then added and the mixture incubated for 30 min, followed by 40 µl of freshly
prepared iodoacetamide (1 M stock solution, Sigma) and incubation for a further
30 min in the dark. Steptavadin beads (250 µL of a 50% slurry per sample,
250
Thermo) were washed 2x with 1 mL PBS and 1x with 1 mL resuspension buffer
and resuspended in resuspension buffer (200 µL). Each sample was combined
with streptavadin beads and incubated on a rotator for 2 h. These mixtures were
then transferred to Mini Bio-Spin
®
columns (Bio-Rad) and placed on a vacuum
manifold. Captured proteins were then washed with agitation 5x with resuspen-
sion buffer (10 mL), 5x PBS (10 mL), 5x with 1% SDS in PBS (10 mL), 30x with
PBS (1 mL per wash, vacuum applied between each wash), and 5x 2M urea in PBS
(1 mL per wash, vacuum applied between each wash). Beads were then resus-
pended in 2 M urea in PBS (1 mL), transferred to screw-top tubes, and pelleted by
centrifugation (2000 x g for 2 min). At this time, 800 µL of the supernatant was
removed, leaving a volume of 200 µL. To this bead-mixture was added 2 µL of
CaCl2 (200 mM stock, 1 mM final concentration) and 2 µL of 1 mg/mL sequence
grade trypsin (Promega) and incubated at 37 ℃ for 18 hours. The resulting mix-
tures of tryptic peptides and beads were transferred to Mini Bio-Spin
®
columns
(Bio-Rad) and the eluent was collected by centrifugation (1,000 x g for 2 min).
Any remaining peptides were eluted by addition of 100 µL of 2 M urea in PBS fol-
lowed by centrifugation as immediately above. The tryptic peptides were then ap-
plied to C18 spin columns (Pierce) according to manufacturer's instructions,
eluted with 70% acetonitrile in H2O, and concentrated to dryness on a speedvac.
251
LC-MS Proteomics Analysis.
Peptides were desalted on a trap column following separation on a 12cm/75um
reversed phase C18 column (Nikkyo Technos Co., Ltd. Japan). A 3 hour gradient
increasing from 10% B to% 45% B in 3 hours (A: 0.1% Formic Acid, B:
Acetonitrile/0.1% Formic Acid) was delivered at 150 nL/min. The liquid chroma-
tography setup (Dionex, Boston, MA, USA) was connected to an Orbitrap XL
(Thermo, San Jose, CA, USA) operated in top-5-mode. Acquired tandem MS
spectra (CID) were extracted using ProteomeDiscoverer v. 1.3 (Thermo, Bremen,
Germany) and queried against the human Uniprot protein database using MAS-
COT 2.3.02 (Matrixscience, London, UK). Peptides fulfilling a Percolator calcu-
lated 1% false discovery rate threshold were reported. All LC-MS/MS analysis
were carried out at the Proteomics Resource Center at The Rockefeller Univer-
sity, New York, NY, USA.
252
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262
Chapter Seven. A Chemical Reporter for Visualizing Meta-
bolic Cross-Talk between Carbohydrate Metabolism and
Protein Modification
*
Introduction
An increasing number of posttranslational modifications (PTMs) have been dis-
covered that can have dramatic effects on the function (i.e., activity, localization,
stability, etc.) of substrate proteins. To catalog and investigate these important
modifications, a variety of chemical approaches have been developed to visualize
and identify PTMs in cell lysates, living cells and in vivo (1,2). One of the most
successful chemical technologies involves the biosynthetic incorporation of syn-
thetic analogs of endogenous PTMs onto proteins in living cells or animals (3).
Typically, these metabolic chemical reporters (MCRs) contain unique chemical-
functionalities that can undergo bioorthogonal reactions to install visualization or
affinity tags. Until recently, research using MCRs has primarily focused on the
end-point of their biosynthetic incorporation, namely the specific PTM of inter-
est. However, because MCRs must be metabolically transformed, typically into
high-energy donor substrates [e.g., uridine-diphosphate (UDP) monosaccharides
or acetyl-CoA], they provide a direct opportunity to chemically track cellular me-
tabolism. For example, we and others demonstrated that after the azide-
263
*
Kelly N. Chuh (University of Southern California) contributed to the work presented in this
chapter.
containing MCR N-azidoacetyl glucosamine (GlcNAz) is metabolized into UDP-
GlcNAz, it can be enzymatically converted to UDP N-azidoacetyl galactosamine
(UDP-GalNAz), resulting in the incorporation into at least three classes of glyco-
proteins (4,5). While this “metabolic crosstalk” is less than ideal for the analysis
of a single type of glycosylation, it raises the possibility that MCRs could be used
to isolate, analyze and potentially discover different branching biosynthetic-
pathways from common metabolic intermediates (Figure 7-1). For example, one
recently-discovered branching pathway involves metabolism from the N-acetyl
glucosamine (GlcNAc) salvage pathway (6) to protein acetylation (7). Specifically,
Varki and co-workers demonstrated that the previously uncharacterized enzyme
amidohydrolase-domain-containing 2 (AMDHD2) converts GlcNAc-6-phosphate
into glucosamine-6-phosphate and acetate (8). This acetate might then be acti-
vated on CoA and subsequently used for protein acetylation. While it had been
previously demonstrated that acetyl-CoA was required for de novo synthesis of
UDP-GlcNAc from glucose through the hexosamine biosynthetic pathway (9,10),
these data reveal that under certain nutrient or metabolic conditions, cells may
utilize scavenged GlcNAc for not only for the biosynthesis of glycans but also for
other posttranslational modifications.
264
Figure 7-1. Using metabolic chemical reporters (MCRs) to detect cellular metabolism.
A) Salvaged N-acetyl glucosamine (GlcNAc) can enter a linear biosynthetic pathway that
yields UDP-GlcNAc that can be directly incorporated onto glycoproteins. Additionally,
GlcNAc metabolic intermediates can enter branching pathways to generate acetate and
other monosaccharides. B) MCRs have the potential to isolate branching metabolic
pathways, like the transformation of GlcNAc into acetate and subsequent acetylation of
proteins.
Here, we report the development of a MCR that isolates the metabolism of
GlcNAc into posttranslational modifications that are not glycosylation. This
MCR, termed 1-deoxy-GlcNAlk, builds upon our published chemical reporter for
glycosylation, N-pentynyl glucosamine (GlcNAlk) (5), but structurally lacks the 1-
hydroxyl group that is absolutely required for biosynthesis into the correspond-
ing UDP-monosaccharide and subsequent incorporation into glycans. Treatment
of a variety of cells with 1-deoxy-GlcNAlk, followed by copper-catalyzed azide-
alkyne cycloaddition (CuAAC) with a fluorescent tag, resulted in differential la-
beling that is detectable in a majority of cell-lines. Notably, the intensity of this
265
signal was inhibited by the addition of the acetyl-transferase inhibitor curcumin
and competition with sodium acetate, suggesting that some of the protein label-
ing is a result of lysine acetylation. Furthermore, proteomic analysis using 1-
deoxy-GlcNAlk identified 60 known acetylated proteins. Finally, labeling of the
acetylated-proteins histones H1.1 and H2B was confirmed using in-gel fluores-
cence scanning. These data demonstrate that 1-deoxy-GlcNAlk is a MCR of pro-
tein modification and more importantly suggest that MCRs can be used to char-
acterize and potentially discover branching metabolic-pathways in living cells.
Results
Fluorescent detection of 1-DeoxyGlcNAlk labeling
To create a MCR capable of isolating the cellular metabolism of GlcNAc into pro-
tein modifications that are not glycosylation, we synthesized a structural analog
of our previously-published glycoprotein MCR, GlcNAlk (Scheme 7-1) (5). This
analog, 1-deoxy-GlcNAlk, lacks the 1-hydroxyl group and therefore cannot enter
any glycosylation metabolic-pathways. Additionally, we generated the per-
acetylated derivative, 1-deoxy-Ac3GlcNAlk, as the acetates allow for passive diffu-
sion of the MCR into living cells where they are subsequently removed by es-
terases (11). NIH3T3 cells were treated with either 1-deoxy-Ac3GlcNAlk (200
µM), 1-deoxy-GlcNAlk (10 mM) or Ac4GlcNAlk (200 µM) as a positive control.
After 16 hours, the corresponding cell-lysates were subjected to the bioorthogonal
reaction copper-catalyzed azide-alkyne cycloaddition (CuAAC) with a fluorescent
266
tag, azido-rhodamine (az-rho). In-gel fluorescence scanning revealed that both
versions of the 1-deoxy MCR were robustly incorporated onto proteins (Figure 7-
2A), albeit at a lower level than the highly-efficient GlcNAlk. The per-acetylated
MCR, 1-deoxy-Ac3GlcNAlk, was incorporated more efficiently than 1-deoxy-
GlcNAlk, consistent with other MCRs (11), and was therefore used in all our sub-
sequent experiments.
Scheme 7-1. Synthesis of Ac3-1-DeoxyGlcNAc, Ac3-1-DeoxyGlcNAlk, and 1-
DeoxyGlcNAlk. (a) Acetyl Chloride 16 h, RT, 38% yield; (b) Tributyltin Hydride, AIBN,
Toluene, 1.5 h, Reflux, 98% yield; (c) 2.5 M HCl, 2 h, Reflux, 75% yield; (d) i. DCC, 4-
Pentynoic Acid, TEA, cat. DMAP, DMF, 16 h, RT ii. Acetic Anhydride, Pyridine, 16 h, RT,
33% yield; (e) NaOMe, MeOH, pH 9, 1 h, RT, 84% yield.
We and others have previously demonstrated that protein labeling by certain
MCRs can be competed by the availability of specific nutrients in cell culture. For
example, MCRs that largely read out on the intracellular glycosylation O-GlcNAc
modification can be competed by increasing glucose concentrations (5), and in-
creasing the amount of serum can inhibit the incorporation of radio-labelled glu-
267
cosamine (12). To investigate the sensitivity of 1-deoxy-GlcNAlk to different cell-
culture conditions, NIH3T3 cells were treated with 1-deoxy-Ac3GlcNAlk (200
µM) in the presence of low or high glucose concentrations (1.0 vs. 4.5 g/mL) or
three different amounts of serum (0, 2 or 10% v/v). In-gel fluorescence scanning,
following lysis and CuAAC with az-rho, demonstrated that 1-deoxy-GlcNAlk la-
beling is largely insensitive to these different culture conditions (Figure 7-3A).
268
Figure 7-2. Characterization of proteins that are labeled by the MCR 1-deoxy-GlcNAlk.
A) NIH3T3 cells were treated with the indicated MCRs for 16 hours before the corre-
sponding lysates were subjected to CuAAC with az-rho and analyzed by in-gel fluores-
cence scanning. B) NIH3T3 cells were treated with 1-deoxy-Ac3GlcNAlk with or without
sodium acetate for 6 hours before CuAAC az-rho and in-gel fluorescence scanning. C)
NIH3T3 cells were pretreated with the protein acetyltransferase inhibitor curcumin for
30 min before addition of 1-deoxy-Ac3GlcNAlk for an additional 5.5 hours. Labeled pro-
teins were then visualized using in-gel fluorescence scanning following CuAAC with az-
rho. Coomassie blue staining shows equal loading.
269
1-deoxy-GlcNAlk is metabolized and incorporated onto acetylated proteins
To determine if any 1-deoxy-GlcNAlk labeling could be attributable to protein
acetylation, we used sodium acetate and the p300-specific acetyltransferase in-
hibitor curcumin (13). NIH3T3 cells were treated with or without sodium acetate
(10 mM) and 1-deoxy-Ac3GlcNAlk (200 µM) for 6 hours. In-gel fluorescence
showed that sodium acetate was able to compete 1-deoxy-GlcNAlk labeling (Fig-
ure 7-2B). To investigate whether any observed protein acetylation by 1-deoxy-
GlcNAlk is enzymatic in nature, NIH3T3 cells were pretreated with curcumin (60
µM) for 30 minutes prior to treatment with 1-deoxy-Ac3GlcNAlk (200 µM) for 5.5
hours. Cell lysates were then subjected to CuAAC with az-rho and analyzed by in-
gel fluorescence scanning (Figure 7-2C). Curcumin-treated cells also showed re-
duced 1-deoxy-GlcNAlk labeling compared to controls. Notably, in both of these
experiments, the effect on the labeling of different proteins was not uniform. For
example, labeled proteins in the region of histones and other small proteins (~15
kDa) are more sensitive to both competition by sodium acetate and curcumin
treatment. Together, these data suggest that pentynoic acid is likely removed
from 1-deoxy-GlcNAlk, where it is know to be enzymatically incorporated into
protein acetylation (14) and potentially other protein modifications. We next di-
rectly compared 1-deoxy-Ac3GlcNAlk to the known acetylation reporter sodium
pentynoate (14). Specifically, NIH3T3 cells were treated with 1-deoxy-
Ac3GlcNAlk (200 µM) or sodium pentynoate (200 or 5000 µM) for 8 hours.
Visualization of the labeled proteins by in-gel fluorescence showed that sodium
270
pentynoate is a more efficient MCR, even at equal concentrations (Figure 7-3B).
Notably, the pattern of proteins that are labeled by 1-deoxy-GlcNAlk and penty-
noate are also different. Together, these data suggest that while at least some of
the proteins that become modified by 1-deoxy-Ac3GlcNAlk treatment are acety-
lated, its metabolism and/or distribution into different types of posttranslational
modification (e.g., acetylation vs. long-chain fatty acylation) are different than
sodium pentynoate.
Figure 7-3. Characterization of 1-deoxy-GlcNAlk labeling. A) 1-deoxy-GlcNAlk labeling
is not affected by glucose or serum levels in media. NIH-3T3 cells were treated with 1-
deoxy-Ac3GlcNAlk (200 µM) for 16 h under high- or low-glucose conditions, 4.5 g/L or 1
g/L respectively, supplemented with 10%, 2% or 0% fetal calf serum. Lysate from treated
cells was subjected to CuAAC and in-gel fluorescence scanning. B) Comparison of 1-
deoxy-Ac3GlcNAlk and sodium 4-pentynoate labeling. MEFs were treated with or with-
out 1-deoxy-Ac3GlcNAlk (200 µM), sodium 4-pentynoate (200 µM or 5 mM) or DMSO
vehicle for 8 h. Lysate from treated cells was subjected to CuAAC following by SDS-
PAGE separation and in-gel fluorescence scanning.
To further characterize this MCR, NIH3T3 cells were treated with various con-
centrations of 1-deoxy-Ac3GlcNAlk for 16 hours prior to lysis and CuAAC with az-
271
Type to enter text
rho. In-gel fluorescence scanning showed that proteins are dose-dependently la-
belled by 50-200 µM 1-deoxy-Ac3GlcNAlk treatment (Figure 7-4A). To determine
the kinetics and dynamics of 1-deoxy-GlcNAlk labeling, we next performed pulse
and pulse-chase experiments. We first treated NIH3T3 cells with 1-deoxy-
Ac3GlcNAlk (200 µM) for different lengths of time. After lysis and CuAAC with
az-rho, in-gel fluorescence scanning revealed protein labeling in as little as 2
hours, with similar kinetics to other direct MCRs of protein acetylation (Figure 7-
4B) (14). NIH3T3 cells were then treated with 1-deoxy-Ac3GlcNAlk (200 µM) for
16 hours, after which time the growth medium was replaced with fresh media
containing 1-deoxy-N-acetyl glucosamine (1-deoxy-Ac3GlcNAc, 200 µM). In-gel
fluorescence scanning after CuAAC revealed a time-dependent loss of signal (Fig-
ure 7-4C). To ascertain the generality of 1-deoxy-GlcNAlk as a MCR, a small panel
of cell-lines were treated with 1-deoxy-Ac3GlcNAlk (200 µM) for 16 hours before
lysis and reaction with az-rho using CuAAC. In-gel fluorescence scanning showed
labeling of proteins in each of the cell-lines tested (Figure 7-5). To determine if
treatment of cells with the MCR resulted in any toxicity, NIH3T3 cells were
treated with either 1-deoxy-Ac3GlcNAc (200 µM), 1-deoxy-Ac3GlcNAlk (200 µM)
or DMSO vehicle. After 24 or 48 hours of treatment, the viability of the cells was
measured using a commercially available MTS assay (Figure 7-6). No toxicity was
observed with 1-deoxy-Ac3GlcNAlk treatment, despite some toxicity with the con-
trol compound 1-deoxy-Ac3GlcNAc.
272
Figure 7-4. Dose-dependence and dynamics of 1-deoxy-GlcNAlk protein labeling. A)
NIH3T3 cells were treated with the indicated concentrations of 1-deoxy-Ac3GlcNAlk for
16 hours before CuAAC with az-rho and analysis by in-gel fluorescence scanning. B)
NIH3T3 cells were treated with 1-deoxy-Ac3GlcNAlk (200 µM) for the indicated lengths
of time. Cell lysates were reacted with az-rho and visualized with in-gel fluorescence
scanning. C) NIH-3T3 cells were treated with 1-deoxy-Ac3GlcNAlk (200 µM) for 16 h af-
ter which time media was exchanged with fresh media containing 1-deoxy-Ac3GlcNAc
(200 µM) for the indicated lengths of time. Time dependent loss of protein labeling was
visualized using in-gel fluorescence scanning. Coomassie blue staining shows equal load-
ing.
273
Figure 7-5. Generality of 1-deoxy-GlcNAlk labeling. The indicated cell lines were
treated with 200 µM 1-deoxy-Ac3GlcNAlk for 16 hours before modified proteins were
subjected to CuAAC with az-rho and in-gel fluorescent scanning.
Figure 7-6. Toxicity of 1-deoxy-Ac3GlcNAlk. Cell viability following treatment for 24-48
h with 1-deoxy-Ac3GlcNAc, 1-deoxy-Ac3GlcNAlk or DMSO vehicle was measured using a
commercially available MTS assay (Promega). Quantitation is from three individual ex-
periments normalized to DMSO treated cells; error bars indicate ±s.e.m.
274
Identification of 1-deoxy-GlcNAlk labelled proteins
Finally, we performed a large-scale mass spectroscopy experiment to identify
proteins labeled by 1-deoxy-GlcNAlk and directly compare them to those modi-
fied by our published MCR GlcNAlk. NIH3T3 cells were treated in triplicate with
1-deoxy-GlcNAlk (200 µM), GlcNAlk (200 µM) or GlcNAc (200 µM) as a nega-
tive control for 16 hours. Treated cells were pelleted and lysed with a denaturing
buffer (4% SDS). Protein concentration was normalized, and 10 mg of protein
was subjected to CuAAC with an azide-functionalized biotin affinity-tag. The
biotinylated samples were enriched with streptavidin beads, washed extensively
and subjected to on-bead trypsin digestion, and the recovered peptides were sub-
jected to LC-MS/MS analysis. Proteins were identified using Proteome Discover
and Mascot and curated using the following criteria to identify “hits”: (1) Pro-
teins must be identified in all 3 runs (at least 1 spectral count per run) with a sum
of at least 4 spectral-counts overall; (2) The sum of the spectral counts must be
4-fold greater in the 1-deoxy-GlcNAlk or GlcNAlk samples than the GlcNAc-
treated samples; (3) The number of spectra counts in the MCR-treated sample
compared to the control must be statistically significant (p-value < 0.05, t test).
Following these requirements, we identified 99 proteins modified by 1-deoxy-
GlcNAlk (Figure 7-7A and Table 7-1) and 433 proteins modified by GlcNAlk (Fig-
ure 7-8 and Table 7-2). Of the 1-deoxy-GlcNAlk labeled proteins, 60 have been
previously identified as acetylated proteins, including those annotated in Figure
7-7A, supporting this MCR as a reporter of acetylation. Forty-six proteins identi-
275
fied using 1-deoxy-GlcNAlk were also present in the GlcNAlk treated samples
(Figure 7-7B and Tables 7-1 and 7-2), suggesting that these proteins are either
simultaneously modified by O-GlcNAc glycosylation or that GlcNAlk can also be
metabolized through an “off-target” pathway. Notably, 16 of these overlapping
proteins are known acetylated proteins, suggesting that both MCRs may be me-
tabolized into the protein acetylation pathway. The proteins identified using 1-
deoxy-GlcNAlk also contained 39 previously uncharacterized substrates (Table 7-
1), suggesting that this MCR can be used to find new modification (e.g., acetyla-
tion) events. To confirm 1-deoxy-GlcNAlk labeling of known acetylated proteins,
Histones H1.1 and H2B.(7) Histones were enriched from NIH-3T3 cells treated
with 1-deoxy-Ac3GlcNAlk (200 µM) or 1-deoxy-Ac3GlcNAc (200 µM) as negative
control for 16 h using acid precipitation (15). Purified histones were then sub-
jected to CuAAC with az-rho and in-gel fluorescence scanning confirmed labeling
of Histones 1.1 and H2B (Figure 7-7C).
276
Figure 7-7. Identification of posttranslationally modified proteins using 1-deoxy-
GlcNAlk. (A) NIH3T3 cells were treated with 1-deoxy-Ac3GlcNAlk, Ac4GlcNAlk or
Ac4GlcNAc (all at 200 µM concentration) for 16 hours. At this time, the corresponding
cell-lysates were subjected to CuAAC with azide-biotin, enrichment with streptavidin-
coated beads, and on-bead trypsinolysis. Proteins identified by LC-MS/MS are graphi-
cally presented as total number of positive minus total number of control spectral
counts. Three known acetylated proteins are annotated in black. (B) Overlap between
proteins identified using 1-deoxy-Ac3GlcNAlk and Ac4GlcNAlk. (C) Enriched histones
from NIH3T3 cells labeled with 1-deoxyAc3GlcNAlk or DMSO vehicle were subjected to
CuAAC with az-rho. In-gel fluorescence reveals modification of Histones 1.1 and 2B.
Discussion
Bioorthogonal chemistries have enabled the creation of MCRs for the visualiza-
tion and enrichment of a wide array of PTMs (3) including glycosylation (5,9,16),
lipidation (17), methylation (18), and different forms of acetylation (14,19). Be-
cause MCRs must be metabolized by living cells, they provide unique opportuni-
ties to simultaneously interrogate a certain PTM and the upstream metabolic and
biosynthetic pathways. We have demonstrated that alterations in the chemical
structure of a MCR can impact its acceptance into different glycosylation path-
ways (5). Building upon those results, we synthesized and characterized a MCR
(1-deoxy-GlcNAlk) that reports on the metabolic crosstalk between the GlcNAc
salvage pathway and non-glycosylation modifications on proteins. Using a fluo-
277
rescent azide-tag, in combination with CuAAC, we demonstrated that 1-deoxy-
GlcNAlk treatment results in labeling of a range of proteins in different cell lines.
The labeling intensities in these cell-lines varies dramatically, raising the possi-
bility that MCRs could be used to classify metabolic flux in different cells. Co-
treatment with different nutrient sources and an inhibitor of protein acetyltrans-
ferases showed that 1-deoxy-GlcNAlk labeling is competed by exogenous acetate
and acetyltransferase inhibition. This demonstrates that at least some 1-deoxy-
GlcNAlk enters the protein acetylation pathway. This is further supported by our
proteomic identification of 60 previously identified, acetylated proteins, which
account for ~60% of the total proteins identified. The most likely pathway re-
sponsible for these observations is the one identified by Varki and co-workers
mentioned above (8). In this case, 1-deoxy-GlcNAlk would be phosphorylated and
then deacetylated by the enzyme AMDHD2 to generate pentynoic acid, although
this remains to be experimentally confirmed.
278
Figure 7-8. Identification of proteins labelled by GlcNAlk. NIH3T3 cells were treated
with Ac4GlcNAlk or Ac4GlcNAc (both at 200 µM concentration) for 16 hours. At this
time, the corresponding cell-lysates were subjected to CuAAC with azide-biotin, enrich-
ment with streptavidin-coated beads, and on-bead trypsinolysis. Proteins identified by
LC-MS/MS are graphically presented as total number of positive minus total number of
control spectral counts. Three known O-GlcNAcylated proteins are annotated in black.
However, not all of the labeled proteins were equally susceptible to competition
by sodium acetate or inhibition of the p300 acetyltransferase (Figures 7-2B and
7-2C). In the case of sodium acetate competition, the intensity of all of the labeled
proteins is reduced; however, the proteins at ~15 kDa molecular weight display a
more dramatic effect. This difference could be attributable to acetylation dynam-
ics. Rapidly cycling acetylation marks, like those on the core histones that are
found around 15 kDa (20,21), could be more sensitive to competition by excess
sodium acetate. In contrast, any long-lived pentynyl-modification events could
persist throughout the experiment. Likewise, treatment with curcumin resulted
in dramatic reduction of the labeling of proteins at low molecular weights but
less-so for other proteins. Since curcumin is a specific inhibitor of the p300 ace-
279
tyltransferase (13), the proteins that show no change in labeling intensity might
be modified by other acetyltransferases. We next directly compared 1-deoxy-
GlcNAlk with pentynoic acid. At equal concentrations, 1-deoxy-GlcNAlk is signifi-
cantly less efficient at labeling proteins, and pentynoate-labeling can be per-
formed at higher concentrations to maximize incorporation (Figure 7-3B). Inter-
estingly, 1-deoxy-GlcNAlk and pentynoate treatment resulted in the visualization
of different patterns of proteins. This demonstrates that 1-deoxy-GlcNAlk is not a
simple replacement of a known MCR of protein acetylation (14). The differences
between the two MCRs could simply arise from changes in their metabolism. For
example, if the two MCRs are metabolized at different rates, a different subset of
proteins could be modified after the same length of labeling. It is also possible
that the two MCRs are incorporated into different types of posttranslational
modifications. For example, short-chain fatty acid reporter could be metabolized
into the corresponding lipid-reporter (e.g., palmitoylation) (22). It is also possi-
ble that either pentynoate or 1-deoxy-GlcNAlk is metabolized into an unknown,
non-acetylation pathway that contributes to some of the signal, or results in non-
enzymatic modification of proteins (23,24).
Finally, to compare 1-deoxy-GlcNAlk to a glycoprotein MCR, we performed a pro-
teomics experiment using 1-deoxy-GlcNAlk and GlcNAlk. Enrichment with 1-
deoxy-GlcNAlk resulted in the identification of 99 proteins. Treatment with
GlcNAlk resulted in the identification of a large number of O-GlcNAc modified
280
proteins and 64 proteins that overlapped with the 1-deoxy-GlcNAlk sample. No-
tably, 16 of these proteins were also previously identified as being acetylated. This
raises the likely possibility that any glycoprotein MCR bearing its chemical func-
tionality at the N-acetyl position will read-out on some acetylated proteins.
Therefore, care should be taken to confirm the glycosylation of candidate pro-
teins identified using these reporters.
Conclusion
In summary, our competition, inhibition and proteomics experiments support
the conclusion that a large fraction of 1-deoxy-GlcNAlk is metabolized into the
protein acetylation pathway. We cannot definitively rule out the incorporation of
our MCR into other types of protein modifications, but believe that our data
demonstrates the unique utility of chemical synthesis to develop new MCRs that
can be used to visualize cellular metabolism in addition to their traditional roles
as probes of posttranslational modifications. Given the resurgent importance of
cellular metabolism in human disease (e.g., diabetes and cancer), we believe that
these tools can provide important and exact information on the transformation of
metabolites to PTMs where they can directly effect protein function.
Materials and Methods
All reagents used for chemical synthesis were purchased from Sigma-Aldrich,
Alfa Aesar or EMD Millipore unless otherwise specified and used without further
281
purification. All anhydrous reactions were performed under argon or nitrogen
atmosphere. Analytical thin-layer chromatography (TLC) was conducted on EMD
Silica Gel 60 F
254
plates with detection by ceric ammonium molybdate (CAM),
anisaldehyde or UV. For flash chromatography, 60 Å silica gel (EMD) was util-
ized. Electrospray (ESI) and Atmospheric Pressure Chemical Ionization (APCI)
was preformed on an Agilent LCTOF (2006) by University of California Riverside
Mass Spectrometry Facility.
1
H spectra were obtained at 400, 500, or 600 MHz
on a Varian spectrometers Mercury 400, VNMRS-500, or -600. Chemical shifts
are recorded in ppm (δ) relative to solvent.
13
C spectra were obtained at 100, 125
or 150 MHz on the same instruments.
Chemical Synthesis.
Known compounds Ac4GlcNAlk (5), azido-rhodamine (22) and sodium 4-
pentynoate (14) were synthesized according to literature procedures.
Compound 7.1 3,4,6-Tri-O-Acetyl-1-Chloro-1-Deoxy-N-Acetylglucosamine
(25). Commercially-available N-acetylglucosamine (10.00 g, 45.21
mmol) under Argon atmosphere was stirred vigorously in Acetyl
Chloride (17.68 mL, 248.63 mmol) for 16 h at room temperature.
Upon completion, CH2Cl2 was added and the resulting mixture
was extracted with ice-water (1x), saturated NaHCO3 (1x), H2O (1x), and Brine
(1x). The organic layer was dried over Na2SO4, filtered and concentrated. The
O
AcO
OAc
AcO
Cl
NH
O
282
crude mixture was then purified over silica-gel chromatography (33% CH2Cl2 in
EtOAc) to yield the purified product (6.33 g, 38% yield).
1
H NMR (500 MHz,
CDCl3) δ 6.18 (d, J = 3.7 Hz, 1H), 5.83 (d, J = 8.7 Hz, 1H), 5.35(t, J = 9.3 Hz, 1H),
5.23 (t, J = 9.5 Hz, 1H), 4.53 (ddd, J = 10.7, 8.7, 3.7 Hz, 1H), 4.32 – 4.23 (m, 2H),
4.13 (d, J = 10.4 Hz, 1H), 2.10 (d, J = 0.7 Hz, 3H), 2.05 (t, J = 1.0 Hz, 6H), 1.98
(d, J = 0.6 Hz, 3H).
Compound 7.2 3,4,6-Tri-O-Acetyl-1-Deoxy-N-acetylglucosamine (1-deoxy-
Ac3GlcNAc) (26). 7.1(6.30 g, 17.22 mmol) was dissolved in tolu-
ene (100 mL) and purged with Argon for 30 min. At this time,
tributyltin hydride (5.56 mL, 20.66 mmol) and azobisisobutyroni-
trile (560 mg, 3.44 mmol) were added and the reaction stirred at
reflux (~110 ℃) for 1.5 h. Upon completion, the reaction was concentrated and
purified by silica gel chromatography (50% CH2Cl2 in EtOAc for 2 column vol-
umes, 25% CH2Cl2 for 1 volume, 100% EtOAc for 1 volume, 10% MeOH in EtOAc
for 2 volumes) to afford the product (5.58 g, 98% yield).
Compound 7.3 1-Deoxyglucosamine hydrochloride (26). 7.2 (2.50 g, 7.55
mmol) was dissolved in 2.5 M HCl (20 mL) and refluxed for 2
h. Upon completion the mixture was concentrated. The re-
sulting oil was dissolved in minimal EtOH and Et2O was
added under stirring until the product precipitated and was filtered and washed
O
AcO
OAc
AcO
NH
O
O
HO
OH
HO
NH
3
+
Cl
-
283
with isopropanol to yield the product as an off-white powder (1.14 g, 75% yield).
1
H NMR (500 MHz, D2O) δ 3.99 – 3.80 (m, 3H), 3.72 (dd, J = 12.3, 5.8 Hz, 1H),
3.53 (dd, J = 10.1, 8.7 Hz, 1H), 3.43 (dd, J = 9.8, 8.7 Hz, 1H), 3.39-3.35 (m, 1H),
3.27 (t, J = 11.2 Hz, 1H).
Compound 7.4 3,4,6-Tri-O-Acetyl-1-Deoxy-N-4-pentynylglucosamine (1-
deoxy-Ac3GlcNAlk). 7.3 (250 mg, 1.25 mmol) was concen-
trated from toulene 3x and resuspended in DMF under Argon.
To the starting material was added TEA (183 µL, 1.31 mmol)
and the reaction proceeded for 10 min. 4-Pentynoic acid (147
mg, 1.50 mmol) followed by N,N’-Dicyclohexylcarbodiimide (309 mg, 1.50 mmol)
were then added followed by a catalytic amount of DMAP (~5 mg). The starting
material slowly went in to solution and the reaction proceeded for 16 h. Upon
completion, the reaction mixture was concentrated under reduced pressure and
purified by column chromatography (10% MeOH in CH2Cl2). The purified prod-
uct was then resuspended in pyridine (5 mL) and acetic anhydride (1.5 mL) and
allowed to stir for 16 h. Upon completion the reaction was concentrated, resus-
pended in CH2Cl2, washed with 1M HCl (1x), saturated NaHCO3 (1x), H2O (1x),
and Brine (1x). The organic layer was dried over Na2SO4, filtered and concen-
trated. The crude mixture was then purified over silica-gel chromatography 50-
60% EtOAc in Hexanes to afford crude (154 mg, 33% yield over 2 steps).
1
H NMR
(500 MHz, CDCl3) δ 5.78 (d, J = 7.2 Hz, 1H), 5.07 (t, J = 9.6 Hz, 1H), 4.97 (t, J =
O
AcO
NH
O
OAc
AcO
284
9.7 Hz, 1H), 4.26 – 4.16 (m, 3H), 4.13 (dd, J = 12.3, 2.4 Hz, 1H), 3.55 (ddd, J =
9.8, 5.0, 2.4 Hz, 1H), 3.20 (t, J = 12.5 Hz, 1H), 2.48 (td, J = 7.1, 2.7 Hz, 2H), 2.34
(t, J = 7.1 Hz, 2H), 2.09 (s, 2H), 2.06 (s, 2H), 2.04 (s, 3H), 1.98 (t, J = 2.7 Hz,
1H).
13
C NMR (126 MHz, CDCl3) δ 172.29, 171.24, 170.94, 169.52, 82.73, 76.84,
74.33, 69.66, 68.44, 68.33, 62.56, 50.80, 35.49, 21.05, 20.98, 20.84, 14.95. ESI-
MS calculated for C17H23NNaO8 [M+Na]
+
392.13, found 392.00.
Compound 7.5 1-Deoxy-N-4-pentynylglucosamine (1-deoxy-GlcNAlk). 1.4
(154 mg, 0.417 mmol) was dissolved in MeOH (10 mL). Freshly
prepared NaOMe (Na in MeOH) was added dropwise to reach
pH 9-10. The reaction was monitored TLC and determined fin-
ished in 1 h. The reaction was quenched with dilute acetic acid
in MeOH until pH 7 reached. The mixture was then concentrated and column
chromatograpy (10% MeOH in CH2Cl2) yielded the pure product (85 mg, 84%
yield).
1
H NMR (500 MHz, CD3OD) δ 3.91 (dd, J = 10.9, 5.2 Hz, 1H), 3.87 – 3.77
(m, 2H), 3.62 (dd, J = 11.9, 6.0 Hz, 1H), 3.38 (dd, J = 10.1, 8.6 Hz, 1H), 3.29 –
3.24 (m, 2H), 3.20 – 3.14 (m, 1H), 2.51 – 2.35 (m, 4H), 2.25 (t, J = 2.5 Hz, 1H).
13
C NMR (125 MHz, CD3OD) δ 173.05, 82.12, 81.02, 75.49, 71.04, 68.87, 67.69,
61.73, 51.64, 34.67, 14.36. ESI-MS calculated for C11H18NO5 [M]
+
244.12, found
244.20.
O
HO
NH
O
OH
HO
285
Cell Culture.
COS-7, HEK293, HeLa and MCF7 cells were cultured in high-glucose DMEM
media (Corning) enriched with 10% fetal bovine serum (HyClone, ThermoScien-
tific). NIH3T3 and MEF cells were cultured in high-glucose DMEM media (Corn-
ing) enriched with 10% fetal calf serum (HyClone, ThermoScientific). H1299 cells
were cultured in RPMI media enriched with 10% fetal bovine serum (HyClone,
ThermoScientific). All cell lines were maintained in a humidified incubator at 37
°C and 5.0% CO2.
Metabolic Labeling.
To cells at 80-85% confluency, media containing 1-deoxy-Ac3GlcNAlk,
Ac4GlcNAlk (1,000 x stock in DMSO) or 1-deoxy-GlcNAlk (dissolved directly in
media) or DMSO vehicle was added as indicated. For chase experiments, media
was replaced with media supplemented with 1-deoxy-Ac3GlcNAc (200 µM, 1,000
x stock in DMSO).
Preparation of Nonidet P-40 (NP-40)-Soluble Lysates.
The cells were then collected by trypsinization and pelleted by centrifugation at
for 4 min at 500 x g, followed by washing 2x with PBS (1 mL). Cell pellets were
then resuspended in 100 µl of 1% NP-40 lysis buffer [1% NP-40, 150 mM NaCl,
50 mM triethanolamine (TEA) pH 7.4] with Complete, Mini, EDTA-free Protease
Inhibitor Cocktail Tablets (Roche Biosciences) for 20 min and then centrifuged
286
for 10 min at 10,000 x g at 4 °C. The supernatant (soluble cell lysate) was col-
lected and the protein concentration was determined by BCA assay (Pierce,
ThermoScientific).
Preparation of 4% SDS-soluble lysates
Cells were collected by trypsinization and pelleted by centrifugation for 4 min at
500 x g, followed by washing 2x with PBS (1 mL). Cell pellets were then resus-
pended in 75 µL 0.05% SDS buffer (0.05% SDS, 10 mM TEA pH 7.4, 150 mM
MgCl2) with Complete Mini protease inhibitor cocktail (Roche Biosciences). To
this was added 1 µL Benzonase (Sigma), and the cells were incubated on ice for
30 min. Then, 200 µL 4% SDS buffer (4% SDS, 150 mM NaCl, 50 mM TEA pH
7.4) was added, and the cells were briefly sonicated in a bath sonicator followed
by centrifugation (20,000 x g for 10 min at 15 °C). Soluble protein concentration
was normalized by BCA assay (Pierce, ThermoScientific) to 1 mg/mL.
Cu(I)-Catalyzed [3 + 2] Azide-Alkyne Cycloaddition.
Cell lysate (200 µg) was diluted with cold 1% NP-40 lysis buffer to obtain a de-
sired concentration of 1 µg/µL. Newly made click chemistry cocktail (12 µL) was
added to each sample [azido- or alkynyl-rhodamine tag (100 µM, 10 mM stock
solution in DMSO); tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (1 mM,
50 mM freshly prepared stock solution in water); tris[(1-benzyl-1-H-1,2,3-triazol-
4-yl)methyl]amine (TBTA) (100 µM, 10 mM stock solution in DMSO); Cu-
287
SO4•5H2O (1 mM, 50 mM freshly prepared stock solution in water) for a total re-
action volume of 200 µL. The reaction was gently vortexed and allowed to sit at
room temperature for 1 h. Upon completion, 1 mL of ice cold methanol was added
to the reaction, and it was placed at -20 °C for 2 h to precipitate proteins. The re-
actions were then centrifuged at 10,000 x g for 10 min at 4 . The supernatant was
removed, the pellet was allowed to air dry for 15 min, and then 50 µL 4% SDS
buffer (4% SDS, 150 mM NaCl, 50 mM TEA pH 7.4) was added to each sample.
The mixture was sonicated in a bath sonicator to ensure complete dissolution,
and 50 µL of 2x SDS-free loading buffer (20% glycerol, 0.2% bromophenol blue,
1.4% β-mercaptoethanol, pH 6.8) was then added. The samples were boiled for 5
min at 97 °C, and 40 µg of protein was then loaded per lane for SDS-PAGE sepa-
ration (Any Kd, Criterion Gel, Bio-Rad).
In-Gel Fluorescence Scanning.
Following SDS-PAGE separation, gels were scanned on a Typhoon 9400 Variable
Mode Imager (GE Healthcare) using a 532 nm for excitation and 30 nm bandpass
filter centered at 610 nm for detection.
Metabolic labelling comparison of Sodium Pentynoate and 1-deoxy-Ac3GlcNAlk.
MEFs were treated with 0.2 mM, 5 mM or DMSO vehicle for 6 h. Upon treatment
completion, cells were washed, trypsinized and pelleted. Cell pellets were washed
288
3x with PBS, lysed with 1% NP-40 buffer and soluble lysate subjected CuAAC
with azido-rhodamine as described in the methods section above.
Competition of 1-deoxy-Ac3GlcNAlk labelling with NaOAc.
NIH-3T3 cells were treated with or without NaOAc (10 mM) and 1-deoxy-
Ac3GlcNAlk (200 µM) for 6 h. Cells were then lysed by 4% SDS buffer with ben-
zonase as outlined in the methods section above .
Inhibition of histone acetyl transferases by curcumin.
NIH-3T3 cells were treated with or without curcumin (60 µM) for 30 min prior to
treatment with 1-deoxy-Ac3GlcNAlk (200 µM) for 5.5 h. Cells were then lysed by
4% SDS buffer with benzonase as outlined in the methods section above.
MTS Assay
MEFs (10
4
) plated 4 h prior in a 96-well plate were treated for 24 or 48 h with ei-
ther 1-deoxy-Ac3GlcNAlk (200 µM) or DMSO vehicle in triplicate. Upon treat-
ment completion, cell viability was determined by MTS assay according to litera-
ture procedure (CellTiter 96 AQeous Non-Radioactive Cell Proliferation Assay,
Promega, Madison, WI) with the following minor change. After sufficient color
development, a reaction-quenching formazan-solubilization solution (100 µL)
was added to each well (50% DMF, 20% SDS in H2O). Absorbance at 490 nm was
read using a BioTek Synergy H4 Multi-Mode Microplate reader.
289
Biotin enrichment and On-bead trypsinolysis.
NIH-3T3 cell pellets labeled with 1-deoxy-Ac3GlcNAlk, Ac4GlcNAlk or 1-deoxy-
Ac3GlcNAc for 16 hours were resuspended in 200 µL H2O, 60 µL PMSF in H2O
(250 mM), and 500 µL 0.05% SDS buffer (0.05% SDS, 10 mM TEA pH 7.4, 150
mM MgCl2) with Complete Mini protease inhibitor cocktail (Roche Biosciences).
To this was added 8 µL Benzonase (Sigma), and the cells were incubated on ice
for 30 min. Then, 2000 µL 4% SDS buffer (4% SDS, 150 mM NaCl, 50 mM TEA
pH 7.4) was added, and the cells were briefly sonicated in a bath sonicator fol-
lowed by centrifugation (20,000 x g for 10 min at 15 °C). Soluble protein concen-
tration was normalized by BCA assay (Pierce, ThermoScientific) to 1 mg/mL, and
10 mg of total protein was subjected to the appropriate amount of click chemistry
cocktail containing azido-PEG3-biotin (5 mM, Click Chemistry Tools) for 1 h, af-
ter which time 10 volumes of ice-cold MeOH were added. Precipitation proceeded
2 hours at -20 °C. Precipitated proteins were centrifuged at 5,200 x g for 30 min
at 0 °C and washed 3 times with 40 mL ice-cold MeOH, with resuspension of the
pellet each time. The pellet was then air-dried for 1 h. To capture the biotinylated
proteins by streptavidin beads, the air-dried protein pellet was resuspended in 2
mL of resuspension buffer (6 M urea, 2 M thiourea, 10 mM HEPES pH 8.0) by
bath sonication. To cap cysteine residues, 100 µl of freshly-made TCEP (200 mM
stock solution, Thermo) was then added and the mixture incubated for 30 min,
followed by 40 µl of freshly prepared iodoacetamide (1 M stock solution, Sigma)
290
and incubation for a further 30 min in the dark. Steptavadin beads (250 µL of a
50% slurry per sample, Thermo) were washed 2x with 1 mL PBS and 1x with 1 mL
resuspension buffer and resuspended in resuspension buffer (200 µL). Each
sample was combined with streptavidin beads and incubated on a rotator for 2 h.
These mixtures were then transferred to Mini Bio-Spin® columns (Bio-Rad) and
placed on a vacuum manifold. Captured proteins were then washed with agitation
5x with resuspension buffer (10 mL), 5x PBS (10 mL), 5x with 1% SDS in PBS (10
mL), 30x with PBS (1 mL per wash, vacuum applied between each wash), and 5x
2M urea in PBS (1 mL per wash, vacuum applied between each wash). Beads were
then resuspended in 2 M urea in PBS (1 mL), transferred to screw-top tubes, and
pelleted by centrifugation (2000 x g for 2 min). At this time, 800 µL of the super-
natant was removed, leaving a volume of 200 µL. To this bead-mixture was
added 2 µL of CaCl2 (200 mM stock, 1 mM final concentration) and 2 µL of 1 mg/
mL sequence grade trypsin (Promega) and incubated at 37 °C for 18 hours. The
resulting mixtures of tryptic peptides and beads were transferred to Mini Bio-
Spin® columns (Bio-Rad) and the eluent was collected by centrifugation (1,000 x
g for 2 min). Any remaining peptides were eluted by addition of 100 µL of 2 M
urea in PBS followed by centrifugation as immediately above. The tryptic pep-
tides were then applied to C
18
spin columns (Pierce) according to manufacturer's
instructions, eluted with 70% acetonitrile in H2O, and concentrated to dryness on
a speedvac.
291
LC-MS Analysis.
Peptides were desalted on a trap column following separation on a 12cm/75µm
reversed phase C18 column (Nikkyo Technos Co., Ltd. Japan). A 3 hour gradient
increasing from 10% B to% 45% B in 3 hours (A: 0.1% Formic Acid, B:
Acetonitrile/0.1% Formic Acid) was delivered at 150 nL/min. The liquid chroma-
tography setup (Dionex, Boston, MA, USA) was connected to an Orbitrap XL
(Thermo, San Jose, CA, USA) operated in top-5- mode. Acquired tandem MS
spectra (CID) were extracted using ProteomeDiscoverer v. 1.3 (Thermo, Bremen,
Germany) and queried against the human Uniprot protein database using MAS-
COT 2.3.02 (Matrixscience, London, UK). Peptides fulfilling a Percolator calcu-
lated 1% false discovery rate threshold were reported. All LC-MS/MS analysis
were carried out at the Proteomics Resource Center at The Rockefeller Univer-
sity, New York, NY, USA.
Acid extraction of histones.
NIH-3T3 cells were treated with 1-deoxy-Ac3GlcNAlk (200 µM) or 1-deoxy-
Ac3GlcNAc (200 µM) for 16 hr. Cells were collected by trypsinization and pelleted
by centrifugation at 4 ℃ for 2 min at 2,000 x g, followed by washing with PBS (1
mL) two times. Cell pellets were then resuspended in ice-cold hypotonic lysis
buffer [10 mM triethanolamine (TEA), 1 mM KCl, 1.5 mM MgCl2, 1 mM PMSF,
pH 7.4 with Complete Mini protease inhibitor cocktail (Roche Biosciences)]. The
resuspended cells were homogenized by Dounce homogenizer and lysed in 3 cy-
292
cles of freeze-thaw. Intact nuclei were pelleted at 4 ℃ for 10 min at 10,000 x g
and washed 2x with ice-cold hypotonic lysis buffer. The nuclear pellet was resus-
pended in 0.4 N H2SO4 and agitated overnight on a rotator at 4 ℃. Nuclear de-
bris was pelleted at 4 ℃ for 10 min at 16,000 x g and the supernatant containing
histones was collected and precipitated in ice-cold MeOH in -80 ℃ overnight.
Precipitated histones were collected at 4 ℃ for 10 min at 16,000 x g and washed
2x with ice-cold MeOH. The resulting protein pellet was air dried and resus-
pended in water. Concentration was determined by BCA Assay and normalized
with 1% NP-40 lysis buffer [1% NP-40, 150 mM NaCl, 50 mM triethanolamine
(TEA) pH 7.4] with Complete Mini protease inhibitor cocktail] for CuAAC.
293
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298
Chapter Eight. Investigation of O-GlcNAc Glycosylation on
the E3 Ubiquitin Ligase NEDD4-1
*
Introduction
N-aceytylglucosamine (O-GlcNAc) modification is a dynamic, nutrient-sensitive
post-translational modification that plays a regulatory role in cell fate and func-
tion. In addition to O-GlcNAc directly regulating protein localization (1), stability
(2), activity (2-4) and protein-protein interactions (5-7), O-GlcNAcylation has
been shown to regulate the occupancy of other sites of post-translational modifi-
cation including acetylation, phosphorylation and ubiquitination(5,8-11). O-
GlcNAc is known to be upregulated during cell stress and in cancers including
breast cancer (12) and chronic lymphocytic leukemia (13), among others(14,15).
Conversely, O-GlcNAcylation levels have been shown to be decreased in neurode-
generation and increasing O-GlcNAc levels have been shown to rescue a neuro-
degenerative phenotype(16,17). More specifically, glycosylation of neurodegen-
erative proteins α-synuclein and Tau has been shown to directly prevent protein
aggregation associated with Parkinson’s and Alzheimer’s pathology, respective-
ly(17,18).
299
*
Kelly N. Chuh (University of Southern California) contributed to the work presented in this
chapter.
Ubiquitin is a small-protein modifier that is postranslationally transferred onto
proteins targeting them for degradation or changes in localization and has been
shown to be a regulator of a variety of cellular processes(19-22). The ubiquitina-
tion process is three steps that result in the recruitment of a target protein by its
corresponding E3 ligase for ubiquitination. NEDD4-1 (NEDD4) is one such E3
ligase with substrates that include members of the PI3K signaling pathway (Fig-
ure 8-1A)(23-27). More specifically, NEDD4 promotes cell-surface localization of
the receptor tyrosine kinase IGF1R (28), directly activates AKT kinase activity
and downregulates the opposing phosphatase PTEN(26,29). NEDD4 is a large
protein that consists of 7 major domains including 4 WW domains which deter-
mine protein-protein interactions, the catalytic HECT domain where Ubiquitin
transfer occurs and the C2 domain (Figure 8-1B). In order for NEDD4 to be ac-
tive towards some substrates, it must be anchored to a membrane(30). This is a
calcium-mediated mechanism in which the Ca
2+
binds to the C2 domain resulting
in C2 membrane recruitment and a release of the C2-bound HECT domain for
subsequent E3 ligase activation(30,31). Therefore, the C2 domain is indispensa-
ble in regulating NEDD4 activity. As a member of the ubiquitin proteasome sys-
tem, it is not surprising that NEDD4 is upregulated in response to cell stress in in
vivo models(32,33). Importantly, these models do not demonstrate an increase in
mRNA levels of NEDD4, suggesting a translational or post-translational mecha-
nism for upregulation.
300
Figure 8-1. NEDD4 and its substrates. (A) NEDD4 regulates members of the pro-
growth PI3K signaling pathway, including AKT, PTEN, IGF1R and EGFR. (B) The do-
mains of NEDD4.
NEDD4 has been shown to regulate the cell surface turnover of a handful of
transmembrane receptors, participate in proliferative signaling in cancer, and fa-
cilitate the budding of viral particles including HIV (34,35). The E3 ligase has
also been implicated in neurodegeneration. Tofaris et al reported that NEDD4
responsible for ubiquitinating α-synuclein, a protein that aggregates in the brains
of Parkinson’s disease victims(36). These protein aggregates, termed Lewy bodies
or Lewy neurites are a primary attribute of Parkinsonian pathology. NEDD4-
mediated ubiqutinated occurs at lysine 63 and was shown to target α-synuclein
for lysosomal degradation, suggesting a potential mechanism by which the pro-
tein can be cleared by cells and preventing accumulation of the protein.
301
Figure 8-2. The C2 domain of NEDD4 is O-GlcNAcylated. (A) HEK293 cells expressing
HA-tagged truncation mutants of NEDD4 were treated with GlcNAlk, lysed and sub-
jected to CuAAC with azido-azo-biotin. Following enrichment, Western blotting con-
firmed labeling of FL NEDD4 and the C2 trunctation. (B) Cells expressing either the HA-
tagged C2 domain or the C2 deficient NEDD4 were treated with GlcNAlk, lysed and sub-
jected to CuAAC with azido-azo-biotin. Following biotin enrichment, Western blot analy-
sis was conducted.
NEDD4 and O-GlcNAc have been independently implicated in biochemically
regulating signaling processes and pro-survival mechanisms during cells stress
and neurodegeneration. Both are upregulated in several breast, prostate and
bladder cancers as well as neuroblastomas and small lung cell carcino-
mas(12,14,37,38). Additionally, NEDD4 is the known E3 ligase responsible for
regulating expression and localization of several proteins in the pro-growth PI3K
signaling pathway, and glycosylation of members of the PI3K/AKT signaling cas-
302
cade has also been shown to be important for both cancer and well as diabetes
(see Chapter 1)(38,39). We have previously reported that NEDD4 itself is an O-
GlcNAc modified protein, suggesting a possible mechanism by which NEDD4
levels are positively regulated by O-GlcNAcylation (See Chapter 2, (40)).
Results
Site Identification of O-GlcNAcylation of NEDD4.
We next wanted to identify the site of modification, in hopes that the location of
the site would provide further insight into its biochemical role. Given the large
size of the NEDD4 protein, we chose to first identify which domain(s) contained
O-GlcNAc modification. To this end, I generated 5 truncation mutants in which I
systematically deleted domains from N-terminus to C-terminus (Figure 8-2A).
HEK293 cells expressing truncations of NEDD4 or full-length NEDD4, all of
which were C-terminally HA-tagged, were treated with GlcNAlk (200 µM, 2.3)
overnight, lysed and subjected to CuAAC with azido-azo-biotin (2.10). Following
streptavidin enrichment, the samples were subjected to Western blotting. The
anti-HA blot revealed that glycosylation is lost following deletion of the C2 do-
main (Figure 8-2A). This suggested that a majority of NEDD4 O-GlcNAc modifi-
cation occurs in the C2 domain. In order to confirm this result, I next generated
cells expressing either the C2 domain on its own or a C2-deficient mutant of
NEDD4 (NEDD4C2Δ). These cells were then subjected to the same GlcNAlk
treatment and biotin enrichment protocol. Western blotting revealed that the C2
303
domain on its own is indeed O-GlcNAc modified while the C2 deletion of NEDD4
is not (Figure 8-2B).
Figure 8-3. NEDD4 stability it response to O-GlcNAcylation and cell stress. (A) Cells
were treated with Thiamet-G for 24 h, lysed, reacted with a chemoenzymatic chemical
reporting strategy to transfer GalNAz onto O-GlcNAc residues and subjected to CuAAC
to alkyne-rhodamine. In-gel fluorescence reveals an increase in O-GlcNAc. (B) Cells ex-
pressing HA-tagged FL, C2-NEDD4 or WW1-NEDD4 truncations or RI332 were treated
with Thiamet-G and the lysate subjected to Western blotting. (C) Oxidative stress in-
creases O-GlcNAc levels in cancer cells. A549 and H1299 cells were treated with H2O2
(H, 1mM), diamide (D, 250 µM) or DMSO for 6 h. Following treatment, cells were lysed
and reacted with CCR. In-gel fluorescence after CuAAC reveals increases to O-GlcNAc
levels upon stress. Western blotting from the gel reveals increased levels of NEDD4.
304
NEDD4 Stability is affected by O-GlcNAc modification.
In order to investigate the role of O-GlcNAcylation on NEDD4 stability, we syn-
thesized Thiamet-G (8.3, Scheme 8.1), the highly-specific, small-molecule inhibi-
tor of O-GlcNAcase, the enzyme responsible for removing O-GlcNAc resi-
dues(41). We next treated HEK293 cells expressing either full-length NEDD4, the
C2 truncation or the WW1 truncation with DMSO vehicle, the proteasome inhibi-
tor MG132 (50 µM) or Thiamet-G (100 µM). We confirmed changes to O-GlcNAc
levels in response to both small-molecules by in-gel fluorescence scanning follow-
ing chemoenzymatic labeling of O-GlcNAcylated residues utilizing a post-lysis
chemical reporting strategy developed by the Hsieh-Wilson laboratory (Figure 8-
3A)(42). Treated cells were lysed and the soluble fraction subjected to SDS-PAGE
and Western blotting. As expected, cells treated with MG132 showed an increase
in stability of all three NEDD4 constructs (Figure 8-3B). However, treatment
with Thiamet-G revealed even higher expression levels of NEDD4 for both glyco-
sylated proteins, full-length and C2, while inhibitor treatment seemed to have no
effect on the WW1 truncation (Figure 8-3B). This suggests a mechanism by which
O-GlcNAc modification directly stabilizes NEDD4. Since O-GlcNAcylation of the
proteasome has been shown to inhibit proteasomal turnover, we employed a
rapidly-turned-over, truncation of the protein ribophorin, RI332, as a control for
non-specific protein stabilization(4). RI332 is an ER-resident protein, and there-
fore it cannot be O-GlcNAcylated, but it is a known substrate of the proteasome.
HEK293s expressing RI332 were treated with DMSO vehicle, MG132 or Thiamet-
305
G and lysed. The soluble fraction was subjected to SDS-PAGE following by West-
ern blotting (Figure 8-3B). MG132 treatment also increased stability of RI332 as
anticipated but treatment with Thiamet-G did not stabilize RI332.
Scheme 8.1. Synthesis of Thiamet-G. (a) Ac4Glucosamine HCl, ethyl-thioisocyanate,
TEA, MeCN, 16 h, 80 ℃, 66% yield (b) SnCl4, CH2Cl2, 16 h, RT, 84% yield (c) K2CO3,
MeOH, 3 h, RT, 94% yield.
O-GlcNAc and NEDD4 are upregulated under oxidative stress.
Global O-GlcNAcylation as well as NEDD4 are upregulated during cell stress and
tumorigenesis in some cancers. In order to demonstrate this, we treated lunch
cancer cells (A549 and H1299) with or without either H2O2 (H) or Diamide (D)
for 6 h. Global O-GlcNAc modification levels were visualized by in-gel fluores-
cence following chemoenzymatic transfer of GalNAz onto GlcNAc residues using
UDP-GalNAz and GalT1 Y289L and CuAAC with alkyne-rhodamine (Figure 8-
3C). O-GlcNAc levels were increased in both cell lines under both oxidative
stress/hypoxia conditions. Soluble lysates from treated cells were also subjected
to SDS-PAGE and Western blotting to reveal increased that NEDD4 levels in
A549 and H1299 cells under oxidative stress (Figure 8-3C).
306
The C2 domain is O-GlcNAcylated in vitro
We next employed a bacterial coexpression strategy to confirm O-GlcNAcylation
of the C2 domain of NEDD4 in vitro. E. coli do not have O-GlcNAc modification
or the corresponding O-GlcNAc Transferase (OGT) and O-GlcNAcase enzymes.
As such, Vocadlo and co-workers have developed a system in which OGT and a
protein of interest are coexpressed in E. coli, allowing for O-GlcNAcylation of
proteins in vitro(41). However, this system has been inconsistent in its ability to
glycosylate target substrates(18). Recent reports identified NagZ as the enzyme
responsible for cleaving O-GlcNAc residues installed during OGT coexpression
and generated a nagZ knockout E. coli strain to circumvent this challenge(43).
We obtained the nagZ strain as a gift from the Macnaughtan lab and expressed
the His-tagged GST-C2 domain protein and OGT. We confirmed O-
GlcNAcylation of the GST-C2 fusion protein by chemoenzymatic chemical report-
ing since no commercially available O-GlcNAc antibodies recognize glycosylation
on the C2 domain (Figure 8-4A). Purification of the GST-C2 by Ni affinity chro-
matography and subsequent cleavage of the GST tag upon treatment with en-
terokinase yielded the purified C2 domain. Glycosylation of the C2 domain on its
own was then confirmed and the protein sent for mass spectroscopy analysis/site
identification. We are currently awaiting results.
307
Type to enter text
Figure 8-4. O-GlcNAcylation of the C2 domain in vitro. C2-GST is O-GlcNAc modfied.
OGT and GST-C2 were coexpressed in nagZ knockout E. coli. Chemoenzymatic labelling
of O-GlcNAcylated proteins with GalNAz followed by CuAAC with alk-rho reveals O-
GlcNAcylation of GST-C2 by in-gel fluorescence.
Discussion and Conclusion
Despite its importance in a variety of biological processes, the regulation of
NEDD4 is not well understood. Our discovery of the O-GlcNAcylation of NEDD4
potentially uncovers an important regulatory mechanism. First, we demonstrated
that NEDD4 is O-GlcNAcylated in the C2 domain, which is required for mem-
brane binding and auto-activation. We went on to show that NEDD levels are
dramatically increased during treatment with an inhibitor of O-GlcNAcase and
that this affect is diminished upon loss of the C2 domain. Importantly, this affect
seems to be a direct result of O-GlcNAc regulation and not due to inhibition of
the proteasome. Finally, we coexpressed the C2 domain and OGT in a nagZ
knockout strain of E. coli and confirmed glycosylation in vitro. This O-
GlcNAcylated protein was enriched and submitted for mass spectroscopy analysis
for site identification.
308
C2 domains are evolutionarily conserved domains that target proteins to the cell
membrane following Ca
2+
binding. Proteins containing C2 domains include
NEDD4, PTEN and SMURF1/2, among others. Given that the NEDD4 C2 domain
itself is O-GlcNAcylated, both within cells as well as in vitro, suggests that per-
haps O-GlcNAc plays a regulatory role in NEDD4 membrane localization. To in-
vestigate this hypothesis, further work is necessary to identify specific site(s) of
modification and interrogate their biochemical role in NEDD4 activity. Upon site
identification, O-GlcNAcylated NEDD4’s effect on the stability of downstream
targets, including PTEN and α-Synuclein, can also be determined, further eluci-
dating the function of O-GlcNAcylation in human diseases including cancer and
neurodegeneration.
Materials and Methods
All reagents used for chemical synthesis were purchased from Sigma-Aldrich un-
less otherwise specified and used without further purification. All anhydrous re-
actions were performed under argon atmosphere. Analytical TLC was conducted
on Silica Gel 60 F
254
plates (EMD Chemicals) with detection by ceric ammonium
molybdate (CAM), anisaldehyde, or UV. For flash chromatography, 60 Å silica gel
(EMD Chemicals) was utilized. Electrospray ionization mass spectrometry (ESI-
MS) was performed using a Shimadzu liquid chromatography (LC)-MS 2020. 1H
spectra were obtained at 600 MHz on a Varian VNMRS-600. Chemical shifts are
309
recorded in ppm (δ) relative to CHCl3 (7.26 ppm) for spectra acquired in CDCl3 .
13
C spectra were obtained at 200 MHz on the same instrument.
Chemical Synthesis.
Known compounds 1,3,4,6-Tetra-O-Acetyl-N-4-pentynylglucosamine (2.3,
GlcNAlk; (44), N-(6-(diethylamino)-9-(2-(4-hept-6-ynoylpiperazine -1-
carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium (2.2, alk-rho;
(45)), azido-azo-biotin (2.10) (46), Thiamet-G (41) and UDP-GalNAz(47) were
synthesized according to literature procedures or as described below.
Compound 8.1 2-deoxy-2-ethylthioureido-1,3,4,6-tetra-O-acetyl-β-D-
glucopyranose. Synthesis carried out according to Yuzwa et
al(41). Ac4Glucosamine·HCl (3.46 g, 9.02 mmol) was synthesized
according to literature procedure (48) and resuspended in MeCN
(40 mL). TEA (2.26 mL, 16.2 mmol) and ethyl thioisocyanate (2.37 mL, 27.1
mmol) were then added and the mixture was heated to reflux (~80 ℃) and
stirred until determined complete by TLC (50% EtOAc in Hexanes, ~ 24 h). Upon
completion the reaction was concentrated and resuspended in CH2Cl2. The or-
ganic later was then washed with saturated sodium bicarbonate, water and brine.
The organic layer was then dried over Na2SO4 and concentrated to afford crude.
Purification by silica gel column chromatography (50% EtOAc in Hexanes)
yielded the product (2.59 g, 66% yield).
1
H NMR (400 MHz, CDCl3) δ 6.22 (dd, J
O
AcO
NH
OAc
OAc
AcO
S
HN
310
= 9.6, 5.3 Hz, 1H), 6.10 (s, 1H), 5.74 (d, J = 8.5 Hz, 1H), 5.26 – 5.10 (m, 1H), 4.97
– 4.88 (m, 0H), 4.33 – 4.21 (m, 1H), 4.16 – 4.05 (m, 2H), 3.87 – 3.78 (m, 1H),
3.53 – 3.23 (m, 2H), 2.10 (dd, J = 6.9, 1.6 Hz, 3H), 2.08 – 2.04 (m, 7H), 2.03 (d,
J = 1.7 Hz, 3H), 1.19 (dtd, J = 14.5, 7.3, 1.6 Hz, 3H).
Compound 8.2 3,4,6-Tri-O-acetyl-1,2-dideoxy-2’-ethylamino-a-D-
glucopyranoso-[2,1-d]-Δ2’-thiazoline. Synthesis carried out ac-
cording to Yuzwa et al(41). 8.2 (2.59 g, 5.96 mmol) was resuspended
in anhydrous CH2Cl2 and SnCl4 (2.80 mL, 23.8 mmol) was added
dropwise. The reaction was stirred overnight and quenched with saturated so-
dium bicarbonate. The aqueous layer was then extracted with CH2Cl2 (2x) and
the combined organics dried over Na2SO4 and concentrated. Purification by silica
gel column chromatography (70 - 80% EtOAc in Hexanes) yielded the product
(1.88 g, 84% yield).
1
H NMR (500 MHz, CDCl3) δ 6.18 (d, J = 6.5 Hz, 1H), 5.39
(dd, J = 4.1, 2.6 Hz, 1H), 4.91 (ddd, J = 9.5, 2.7, 1.1 Hz, 1H), 4.32 (ddd, J = 6.5,
4.1, 1.1 Hz, 1H), 4.14 – 4.06 (m, 2H), 3.83 – 3.77 (m, 1H), 3.28 (ddq, J = 43.5,
13.1, 7.2 Hz, 2H), 2.07 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.17 (t, J = 7.2 Hz, 3H).
Compound 8.3 1,2-dideoxy-2’-ethylamino-a-D-glucopyranoso-[2,1-d]-Δ2’-
thiazoline (Thiamet-G). Synthesis carried out according to Yuzwa et
al(41). 8.3 (841 mg, 2.25 mmol) was resuspended in MeOH (20 mL)
and K2CO3 was added until the reaction was basic (~5% w/v). The
O
AcO
OAc
AcO
S
N
NH
O
HO
OH
HO
S
N
NH
311
reaction was allowed to proceed until completion (~ 2 h) as determined by TLC.
Upon completion, the reaction was filtered, concentrated and purified by silica
gel column chromatography (7:2:1 EtOAc:MeOH:H2O) to yield the pure product
(521 mg, 94% yield).
1
H NMR (400 MHz, CD3OD) δ 6.39 (d, J = 6.4 Hz, 1H), 4.03
(t, J = 6.4 Hz, 1H), 3.84 (t, J = 6.2 Hz, 1H), 3.79 – 3.73 (m, 1H), 3.66 – 3.55 (m,
2H), 3.44 (dd, J = 8.9, 6.1 Hz, 1H), 3.28 – 3.23 (m, 2H), 1.16 (t, J = 7.2 Hz, 3H).
Cell Culture.
HEK293 or Tet-On HEK293 were cultured in high-glucose DMEM media (Hy-
Clone, Thermo-Scientific) enriched with 10% FBS (HyClone, Thermo-Scientific).
Cell lines were maintained in a humidified incubator at 37 °C and 5.0% CO2. Sta-
ble cell lines were generated using AmphoPack293 viral packaging cells (Clone-
tech) and standard CaCl2 transfection.
Metabolic Labeling.
To cells at 80–85% confluency, low-glucose media containing Ac4GlcNAc or
Ac4GlcNAlk (1,000x stock in DMSO) was added as indicated.
Cu(I)-Catalyzed [3 + 2] Azide-Alkyne Cycloaddition.
Cell lysate (200 µg) was diluted with cold 1% NP-40 lysis buffer to obtain a de-
sired concentration of 1 µg⁄µL. Newly-made click chemistry cocktail (12 µL) was
added to each sample [alkynyl-rhodamine tag (100 µM, 10 mM stock solution in
312
DMSO); tris (2-carboxyethyl)phosphine hydrochloride (1 mM, 50 mM freshly-
prepared stock solution in water); tris[(1-benzyl-1-H-1,2,3-triazol-4-
yl)methyl]amine (100 µM, 10 mM stock solution in DMSO); CuSO4 •5H2O (1 mM,
50 mM freshly prepared stock solution in water)] for a total reaction volume of
200 µL. The reaction was gently vortexed and allowed to sit at room tempera-
ture for 1 h. Upon completion, 1 mL of ice-cold methanol was added to the reac-
tion, and it was placed at −80 °C for 2 h to precipitate proteins. The reactions
were then centrifuged at 10;000 × g for 10 min at 4 °C. The supernatant was re-
moved, the pellet was allowed to air dry for 5 min, and then 50 µL 4% SDS buffer
(4% SDS, 150 mM NaCl, 50 mM TEA pH 7.4) was added to each sample. The
mixture was sonicated in a bath sonicator to ensure complete dissolution, and 50
µL of 2x loading buffer (20% glycerol, 0.2% bromophenol blue, 1.4% β-
mercaptoethanol) was then added. The samples were boiled for 5 min at 97 °C,
and 30 µg of protein was then loaded per lane for SDS-PAGE gel separation (4–
20% Tris • HCl Criterion Gel, Bio-Rad).
Biotin Enrichment.
Cell pellets from 1 150 × 25 mm plate of cells expressing their corresponding
NEDD4 mutant, labeled with Ac4GlcNAlk (200 µM) or DMSO were resuspended
in 150 µL 0.05% SDS buffer (0.05% SDS, 10 mM TEA, pH 7.4, 150 mM NaCl)
with Complete Mini protease inhibitor cocktail (Roche Biosciences) and 2 µL
MG-132 (50 µM, 1000x stock in DMSO, Cayman Chemical). To this was added 1
313
µL Benzonase (Sigma), and the cells were incubated on ice for 30 min. At this
time, 4% SDS buffer (400 µL) was added and the cells were briefly sonicated in a
bath sonicator and collected by centrifugation at 20;000 × g for 10 min at 15 °C.
Protein concentration was normalized by BCA assay (Pierce, Thermo-Scientific)
to 1 mg⁄mL (1.5 mg total cell lysate). The appropriate amount of click chemistry
cocktail (substituting 5mM azido-azo-biotin for 10 mM rhodamine) was added
and the reaction was allowed to proceed for 1 h, after which time 10 volumes of
ice-cold methanol were added. Precipitation proceeded overnight or 2 h at
−20°C. Precipitated proteins were centrifuged at 5,200 × g for 30 min at 0 °C and
washed three times with 5x volume ice-cold MeOH, with resuspension of the pel-
let each time. The pellet was then air dried for 1 h. To capture the biotinylated
proteins by streptavidin beads, the air-dried protein pellet was resuspended in
600 µL of HEPES buffer (6 M urea, 2 M thiourea, 10 mM HEPES, pH 8.0) by
bath sonication.
Streptavidin beads were then washed twice with PBS (37.5 µL) and once with
HEPES buffer. Beads resuspended in HEPES buffer were then incubated on a ro-
tator for 2 h, washed twice with HEPES buffer, twice with PBS, and twice with 1%
SDS in PBS vortexing at each wash (1 mL per wash, 2;000 × g, 2 min). Beads
were then incubated in 37.5 µL of sodium dithionite solution (1% SDS, 25 mM
sodium dithionite) for 30 min at RT to elute captured proteins. The beads were
centrifugated for 2 min at 2;000 × g and the eluent collected. Collected eluent
314
was then precipitated with 5x methanol for 2 h at -20 ℃. Precipitated proteins
were centrifuged at 10,000 × g for 10 min at 0 °C and air-dried. Dried pellets
were resuspended in 20 µL 4% SDS buffer by sonication. 2x SDS-free loading
buffer (20 µL, 20% glycerol, 0.2% bromophenol blue, 1.4% β-mercaptoethanol)
was added and the samples boiled for 5 min.
Preparation of Nonidet P-40 (NP-40)-Soluble Lysates.
Cells were collected by trypsinization and pelleted by centrifugation 4 °C for 3
min at 2;000 × g, followed by washing with PBS (1 mL) two times. Cell pellets
were then resuspended in 200 µL of 1% NP-40 lysis buffer [1% NP-40, 150 mM
NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete EDTA-free Mini pro-
tease inhibitor cocktail (Roche Biosciences) and 1 µL MG-132 (50 µM, 1000x
stock in DMSO, Cayman Chemical), for 10 min and then centrifuged at 4 °C for
10 min at 10;000 × g. The supernatant (soluble cell lysate) was collected and the
protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce,
Thermo-Scientific).
In-Gel Fluorescence Scanning.
Following SDS-PAGE gel separation, the gel was incubated in destaining solution
(50% methanol, 40% H2O, 10% glacial acetic acid) for 5 min followed by H2O for
an additional 5 min prior to scanning. The gel was scanned on a Molecular Im-
315
ager FX (Bio-Rad) using a 580-nm laser for excitation and a 620-nm bandpass
filter for detection.
Western Blotting.
Proteins were separated by SDS-PAGE before being transferred to PVDF mem-
brane (Bio-Rad) using standard Western blotting procedures. All Western blots
were blocked in TBST (0.1% Tween-20, 150 mM NaCl, 10 mM Tris, pH 8.0) con-
taining 5% nonfat milk for 1 h at room temperature (RT), then incubated with the
appropriate primary antibody in blocking buffer overnight at 4 °C. The anti-HA
antibody (Covance) was used at a 1∶1;000 dilution for detection of NEDD4 mu-
tant constructs and RI332. The anti-actin antibody (Sigma) was used at 1:2,000
dilution for detection of actin. The anti-WW2 antibody (Millipore) was used at
1:10,000 dilution for detection of NEDD4. The blots were then washed three
times in TBST and incubated with the HRP-conjugated secondary antibody for 1
h in blocking buffer at RT. HRP-conjugated anti-mouse and anti-rabbit antibod-
ies (Jackson ImmunoResearch) were used at 1∶10;000 dilutions. After being
washed three more times with TBST, the blots were developed using ECL rea-
gents (Bio-Rad) and the ChemiDoc XRS+ molecular imager (Bio-Rad).
Chemoenzymatic transfer of GalNAz onto O-GlcNAcylated proteins.
Soluble cell lysate was diluted to 1 mg/mL (4x total desired protein amount) in
1% SDS lysis buffer to a final volume of 800 µL (1% SDS, 150 mM MgCl2, 50 mM
316
TEA pH 7.4). To this was added 3 x volume methanol, and the reaction vortexed
briefly. Chloroform (0.75 x volume) was added and vortexed briefly. Water (2 x
volume) was added and vortexed briefly. The mixture was centrifuged (maximum
speed, 5 min, RT) and upper aqueous phase discard while leaving interface intact.
Additional methanol (2.25 x) was added to tube and vortex briefly. The mixture
was centrifuged (maximum speed, 5 min, RT) and supernatant discarded. The
pellet was air-dried and resuspended in 200 µL 1% SDS/HEPES buffer (1% SDS,
20 mM HEPES pH 8.0). Protein concentration was determined by BCA Assay
(Pierce, Thermo-Scientific). Protein was diluted to 2.5 mg/mL using 1% SDS/
HEPES buffer and cooled on ice (100 µg reaction, 40 µL protein). The enzymatic
reagents were added in the following order: MilliQ water (49 µL), 2.5 x labeling
buffer (80 µL, 5% NP-40, 125 mM NaCl, 50 mM HEPES pH 7.9), 100 mM in H2O
MnCl2 (11 µL), vortexed and centrifuged briefly, UDP-GalNAz(47) (10 µL, 0.5 mM
in 10 mM HEPES pH 7.9) and gently mixed, and GalT1 Y289L enzyme (11.25 µL,
10 mM Tris pH 8). Incubated at 4 ℃ for 16 to 20 h.
Upon reaction completion, 3 x volume methanol was added, and the reaction vor-
texed briefly. Chloroform (0.75 x volume) was added and vortexed briefly. Water
(2 x volume) was added and vortexed briefly. The mixture was centrifuged
(maximum speed, 5 min, RT) and upper aqueous phase discard while leaving in-
terface intact. Additional methanol (2.25 x) was added to tube and vortex briefly.
The mixture was centrifuged (maximum speed, 5 min, RT) and supernatant dis-
317
carded. The pellet was air-dried and resuspended in 4% SDS buffer (100 µL, 4%
SDS, 150 mM MgCl2, 50 mM TEA pH 7.4) and subjected to CuAAC as described
above.
Bacterial coexpression of GST-C2 and OGT
NagZ D3 Rosetta cells, a gift from Megan Macnaughtan, contransformed with
OGT and His-tagged GST-C2 were grown in Luria broth at 37 ℃ until an OD
0.600 nm. Expression was induced with IPTG (0.5 mM, 250 mM stock in H2O),
and the culture was allowed to grow for 4 h at 37 ℃. Upon completion, the cells
were pelleted and washed with PBS 2 x. Cells were then resuspended in 1% SDS
buffer (1% SDS, 150 mM MgCl2, 50 mM TEA pH 7.4) on ice and sonicated using a
tip sonicator to break up cells (30% intensity, 6 min, 20 sec on, 40 sec off). Lysed
cells were then centrifuged at 4 °C for 10 min at 10;000 × g. The supernatant
(soluble cell lysate) was collected and the protein concentration was determined
by bicinchoninic acid (BCA) assay (Pierce, Thermo-Scientific).
318
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Chapter Nine. Synthesis of O-GlcNAc-Related Compounds
and Additional Small Molecules
Introduction
Chemical tools have proven invaluable in discovering and characterizing the bio-
chemical roles of proteins and their post-translational modifications. Some func-
tion as small-molecule inhibitors or activators while others are used as enrich-
ment probes or visualization tags. The following compounds have been synthe-
sized in support of projects not discussed in this thesis.
Ac5SGlcNAc
Scheme 9-1. Synthesis of Ac5SGlcNAc. (9.8). (a) 2,2’-dimethoxypropane, p-
toluenesulfonic acid, 1,4-dioxane, 24 h, 80 ℃, 67% yield; (b) 80% acetic acid, 2 h, 40 ℃,
77% yield; (c) benzyl chloride, pyridine, 6 h, -10 ℃, 74% yield; (d) mesyl chloride, pyri-
dine, 16 h, RT, 74% yield; (e) NaOMe, CH2Cl2, 5 h, -10 ℃, 95% yield; (f) SC(NH2)2,
MeOH, 1.5 h, 60 ℃, 47% yield; (g) KOAc, AcOH, acetic anhydride, 20 h, 140 ℃, 79%
yield; (h) i. AcOH:2M HCl (10:1), 48 h, 40 ℃ ii. acetic anhydride, pyridine, 18 h, RT,
68% yield.
328
N-acetylglucosamine (O-GlcNAc) modification is a post-translational glycosyla-
tion event that is required for cell survival is implicated in a variety of human dis-
eases including diabetes, neurodegeneration and cancer. In order to interrogate
the biological role of O-GlcNAc modification small molecule modulators of O-
GlcNAc have been developed. Since there is a single enzyme O-GlcNAc Transfe-
rase (OGT) that transfers the residue onto target proteins and a single enzyme
that removes the modification, O-GlcNAcase (OGA), OGT and OGA are desirable
targets for inhibitor development. Notably, the Vocadlo lab has developed a
highly-specific and potent inhibitor of each enzyme, Thiamet-G in the case of
OGA (1) and Ac5SGlcNAc in the case of OGT (Scheme 9.1, Chapter 1) (2).
Ac5SGlcNAc is accepted by enzymes of the Hexosamine Biosynthetic pathways to
generate the UDP donor sugar UDP-5SGlcNAc. However, the carbohydrate is a
poor substrate for OGT, preventing enzymatic transfer of endogenous GlcNAc.
1,3-Dibromoacetone
Scheme 9-2. Synthesis of 1,3-Dibromoacetone. (9.9). (a) Br2, MeOH, 16 h, RT, 12%
yield.
1,3-Dibromoacetone (Scheme 9-2, 9.9) is being used to activate ubiquitin and
subsequently conjugate the activated protein to target substrates, generating a
non-hydrolyzable linkage.
329
3-(2-(bromomethyl)-1,3-dioxolan-2-yl)prop-2-en-1-amine
The electrophilic trap, 3-(2-(bromomethyl)-1,3-dioxolan-2-yl)prop-2-en-1-amine
(Scheme 9-3, 9.15), has been used to enrich and characterize deubiquitylases
(3).Our lab aims to employ the same probe to identify similar enzymes involved
in the removal of post-translational modifications.
Scheme 9-3. Synthesis of 3-(2-(bromomethyl)-1,3-dioxolan-2-yl)prop-2-en-1-amine
(9.15). (a) TFA, CH2Cl2, 5 h, 0 ℃ → RT, quantitative yield; (b) NaH, diethyl (2-
oxopropyl)phosphonate, THF, 4 h, RT, 88% yield; (c) TMSOTf, TEA, toluene, 16 h, RT;
(d) NBS, NaHCO3, THF, 4 h, 0 ℃, 75% yield over 2 steps; (e) p-TsOH, ethylene glycol,
benzene, 8-16 h, reflux, 80% yield; (f) CH3NH2, MeOH, 48 h, RT, 87% yield.
UDP-GalNAlk
UDP-GalNAz has been employed by the Hsieh-Wilson laboratory in a chemoen-
zymatic chemical reporting (CCR) strategy to label O-GlcNAcylated proteins
(Chapter 1) (4). In the CCR reaction, cell lysate is incubated with UDP-GalNAz in
the presence of the mutant galatosyltransferase Y289L GalT1. GalNAz residues
330
are transferred onto O-GlcNAc residues to generate an azide-modified disaccha-
ride that be subsequently reacted with alkyne-bearing fluorescence or affinity
tags for visualization/identification. Since an alkyne chemical reporter is more
desirable, we synthesized UDP-GalNAlk (9.18) and attempted to chemoenzy-
matically transfer GalNAlk residues onto O-GlcNAc modifications using Y289L
GalT1 but with no luck. We thought that perhaps the larger N-acetate side chain
was preventing the UDP sugar from entering the active site, so we expressed
Y289I GalT1, a different mutant of the enzyme that has a larger binding pocket.
Unfortunately, no appreciable transfer was seen.
Scheme 9-4. Synthesis of UDP-GalNAlk. (9.18). (a) benzylamine, THF, 16 h, 50 ℃,
90% yield; (b) N-N’-diisopropyldiallylphosphoramidite, 5-(ethylthio)-1-H-tetrazole, m-
CPBA, CH2Cl2, 4 h, RT→-40 ℃→RT, 76% yield; (c) i. Pd(PPh3)4, p-toulenesulfinic acid
sodium salt, THF:MeOH 1:1, 3 h, RT; ii. UMP-N-methylimidazolide, TEA, MeCN, 3 h, 0
℃; iii. MeOH: H2O:TEA 5:2:1, 16 h, RT 61% yield.
Materials and Methods
All reagents used for chemical synthesis were purchased from Sigma-Aldrich un-
less otherwise specified and used without further purification. All anhydrous re-
actions were performed under argon atmosphere. Analytical TLC was conducted
on Silica Gel 60 Å F
254
plates (EMD Chemicals) with detection by ceric ammo-
nium molybdate (CAM), anisaldehyde, ninhydrin or UV. For flash chromatogra-
phy, 60 Å silica gel (EMD Chemicals) was utilized. Electrospray ionization mass
331
spectrometry (ESI-MS) was performed using a Shimadzu liquid chromatography
(LC)-MS 2020.
1
H spectra were obtained at 600 MHz on a Varian VNMRS-600
unless otherwise specified. Chemical shifts are recorded in ppm (δ) relative to
CHCl3 (7.26 ppm) for spectra acquired in CDCl3.
13
C spectra were obtained at 200
MHz on the same instrument.
Compound 9.1 2-Acetamido-2-deoxy-3,4,5,6-di-O-isopropylidine-aldehydo
-D-glucose dimethyl acetal. Synthesis carried out according to
Gloster et al (2). N-acetylglucosamine (5.00 g, 22.6 mmol) was
resuspended in 1,4 dioxane (50 mL) and warmed to 80 ℃. As the
reaction warmed, the sugar went in to solution. Dimethoxypro-
pane (20 mL) and p-toluene sulfonic acid monohydrate (748 mg, 3.93 mmol)
were added and reaction proceeded for 24 h. Upon completion, the reaction was
allowed to cool, neutralized with sodium bicarbonate, filtered and concentrated
to afford crude. The product was then purified using column chromatography
(2% MeOH in CH2Cl2) to afford 5.277 g in 67% yield.
1
H NMR (600 MHz, CDCl3)
δ 5.84 (d, J = 9.8 Hz, 1H), 4.46 – 4.40 (m, 1H), 4.35 – 4.31 (m, 1H), 4.22 (dd, J =
8.2, 1.3 Hz, 1H), 4.11 – 4.07 (m, 1H), 4.06 – 4.00 (m, 1H), 3.95 – 3.91 (m, 1H),
3.63 – 3.57 (m, 1H), 3.38 (s, 3H), 3.34 (s, 3H), 1.99 (s, 3H), 1.44 (s, 3H), 1.36 (s,
3H), 1.34 (s, 3H), 1.31 (s, 3H).
O
O
O
O
NHAc
OMe MeO
332
Compound 9.2 2-Acetamido-2-deoxy-3,4-O-isopropylidine-aldehydo-D-
glucose dimethyl acetal. Synthesis carried out according to
Gloster et al (2). 9.1 (393 mg, 1.13 mmol) was dissolved in 80%
acetic acid in water (10 mL) and stirred at 40 ℃ for 2 h. The re-
action was monitored by TLC (40 - 60% Acetone in Hexanes, us-
ing Ninhydrin (CAM stains the product well but not the starting material). Upon
completion, the reaction was concentrated and coevaporated with toluene to re-
move excess acid. The product was afforded following column chromatography
(40 - 60% Acetone in Hexanes; 237 mg, 77% yield).
1
H NMR (400 MHz, CDCl3) δ
6.07 (d, J = 9.5 Hz, 1H), 4.45 (d, J = 6.7 Hz, 1H), 4.36 (ddd, J = 9.5, 6.7, 1.4 Hz,
1H), 4.24 (dd, J = 8.4, 1.4 Hz, 1H), 3.75 (dd, J = 10.7, 3.3 Hz, 1H), 3.71 – 3.54 (m,
3H), 3.39 (s, 3H), 3.30 (s, 3H), 2.04 (s, 3H), 1.35 (s, 3H).
Compound 9.3 2-Acetamido-6-O-benzoyl-2-deoxy-3,4-O-isopropylidene-
aldehydo-D-glucose dimethyl acetal. Synthesis carried out ac-
cording to Gloster et al (2). 9.2 (2.00 g, 6.51 mmol) was coevapo-
rated from toluene, dissolved in pyridine and the reaction was
cooled to -20 ℃ using a dry-ice and acetone bath. Benzoyl chlo-
ride (794 µL, 6.51 mmol) was added and the mixture was stirred for 6 h at -10 ℃
using a ice-water and salt bath. Upon completion, the mixture was diluted with
CH2Cl2, washed with 1 M HCl, saturated sodium bicarbonate, water and brine.
The organic layer was then dried over Na2SO4 and concentrated to afford crude.
OH
OH
O
O
NHAc
OMe MeO
OH
OBz
O
O
NHAc
OMe MeO
333
Purification by silica gel column chromatography (20-40% Acetone in Hexanes)
yielded the product (1.92 g, 72% yield).
1
H NMR (500 MHz, CDCl3) δ 8.12 (dd, J
= 8.3, 1.3 Hz, 2H), 7.58 – 7.52 (m, 1H), 7.43 (dd, J = 8.4, 7.1 Hz, 2H), 4.51 – 4.40
(m, 5H), 4.24 (dd, J = 8.5, 1.6 Hz, 1H), 3.90 (ddd, J = 8.8, 4.4, 2.8 Hz, 1H), 3.70
(t, J = 8.7 Hz, 1H), 3.40 (s, 4H), 3.30 (s, 4H), 2.05 (s, 4H), 1.37 (d, J = 0.8 Hz,
4H), 1.33 (d, J = 0.8 Hz, 4H).
Compound 9.4 2-Acetamido-6-O-benzoyl-2-deoxy-3,4-O-isopropylidine-5-O
-mesyl-aldehydo-D-glucose dimethyl acetal. Synthesis carried
out according to Gloster et al (2). 9.3(1.92 g, 4.66 mmol) was
coevaporated with toluene, resuspended in anhydrous pyridine
(4 mL) and the reaction wass cooled to 0 ℃. Mesyl chloride (457
µL, 5.91 mmol) was added and the reaction stirred at 0 ℃ for 2 h and allowed to
warm to room temperature overnight. Upon completion the reaction was concen-
trated, diluted with CH2Cl2, washed with 1 M HCl, saturated sodium bicarbonate,
water and brine. The organic layer was then dried over Na2SO4 and concentrated
to afford crude. Purification by silica gel column chromatography (30-40% Ace-
tone in Hexanes) yielded the product (1.69 g, 74% yield). Note: the starting mate-
rial runs at the same Rf as the product.
1
H NMR (600 MHz, CDCl3) δ 8.10 – 8.06
(m, 2H), 7.57 – 7.52 (m, 1H), 7.43 (dd, J = 8.2, 7.4 Hz, 2H), 5.87 (d, J = 9.6 Hz,
1H), 5.10 (ddd, J = 7.0, 6.0, 2.6 Hz, 1H), 4.76 (dd, J = 12.5, 2.7 Hz, 1H), 4.45 (dd,
J = 12.5, 7.1 Hz, 1H), 4.42 – 4.38 (m, 2H), 4.35 (ddd, J = 9.6, 6.3, 1.8 Hz, 1H),
OMs
OBz
O
O
NHAc
OMe MeO
334
3.96 (dd, J = 7.9, 6.1 Hz, 1H), 3.40 (s, 3H), 3.30 (s, 3H), 3.11 (s, 3H), 2.03 (s, 3H),
1.41 (s, 3H), 1.39 (s, 3H).
Compound 9.5 2-Acetamido-5,6-anhydro-2-deoxy-3,4-O-isopropylidene-
aldehydo-L-idose dimethyl acetal. Synthesis carried out accord-
ing to Gloster et al (2). 9.4(1.54 g, 3.15 mmol) was resuspended
in anhydrous chloroform (15 mL) and reaction cooled to -20 ℃.
Freshly prepared NaOMe (120 mg Na in 4.8 mL MeOH, 5.23
mmol) was added and was added and the mixture was stirred for 5 h at -10 ℃ us-
ing a ice-water and salt bath. Upon completion, the reaction was quenched with
MeOH (5 mL), neutralized with H
+
anion exchange resin until pH 7 was reached.
The mixture was then filtered and dried to afford crude. Material was then titu-
rated with hexanes and diethyl ether to afford pure solid with no further purifica-
tion necessary (870 mg, 95% yield).
Compound 9.6 2-Acetamido-2,5,6-trideoxy-5,6-epithio-3,4-O-isopropylidine
-aldehydo-D-glucose dimethyl acetal. Synthesis carried out ac-
cording to Gloster et al (2). 9.5 (148 mg, 0.511 mmol) was resus-
pended in dry MeOH (5 mL) and thiourea (117 mg, 1.53 mmol)
was added.The mixture was warmed to 60 ℃ and stirred for 1.5
h. Completion was determined by TLC (30% Acetone in Hexanes) and the mix-
ture was cooled and concentrated. Purification by silica gel column chromatogra-
O
O
NHAc
OMe MeO
O
O
O
NHAc
OMe MeO
S
335
phy (30% Acetone in Hexanes) yielded the product (78.3 mg, 50% yield).
1
H
NMR (400 MHz, CDCl3) δ 5.81 (d, J = 9.7 Hz, 1H), 4.43 – 4.25 (m, 3H), 3.42 (s,
3H), 3.38 (s, 3H), 3.26 (t, J = 7.9 Hz, 1H), 2.93 (ddd, J = 7.9, 6.1, 5.2 Hz, 1H),
2.56 – 2.47 (m, 1H), 2.27 (dd, J = 5.2, 1.4 Hz, 1H), 2.01 (s, 3H), 1.45 (s, 3H), 1.41
(s, 3H).
Compound 9.7 2-Acetamido-6-O-acetyl-5-S-acetyl-2-deoxy-3,4-O-
isopropylidine-5-thio-aldehydo-D-glucose dimethyl acetal.
Synthesis carried out according to Gloster et al (2). 9.6 (78.3 mg,
0.255 mmol) was resuspended in acetic anhydride (2.4 mL) and
acetic acid (382 µL). To this was added potassium acetate (117
mg, 1.19 mmol) and the reaction was warmed to 140 ℃. The reaction proceeded
under reflux for 20 h. Upon completion, the reaction was cooled and concen-
trated. The crude mixture was then resuspended in CH2Cl2, washed with 1 M HCl,
saturated sodium bicarbonate, water and brine. The organic layer was then dried
over Na2SO4 and concentrated to afford crude. Purification by silica gel column
chromatography (25% Acetone in Hexanes) yielded the product (82 mg, 79%
yield).
1
H NMR (400 MHz, CDCl3) δ 5.76 (d, J = 9.5 Hz, 1H), 4.44 – 4.35 (m, 2H),
4.33 (d, J = 6.8 Hz, 1H), 4.27 (dd, J = 11.5, 5.1 Hz, 1H), 4.17 (dd, J = 7.5, 1.3 Hz,
1H), 3.97 (ddd, J = 8.8, 5.1, 4.3 Hz, 1H), 3.83 (dd, J = 8.6, 7.5 Hz, 1H), 3.38 (s,
3H), 3.31 (s, 3H), 2.37 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H).
O
O
NHAc
OMe MeO
SAc
OAc
336
Compound 9.8 2-Acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-α-D-
glucopyranose (Ac5SGlcNAc). Synthesis carried out according to
Gloster et al (2). 9.7 (380 mg, 0.930 mmol) was resuspended in
a 10:1 solution of acetic acid and 2M HCl (11 mL). The mixture
was warmed to 40 ℃ and allowed to stir for 48 h. Upon completion, the reaction
mixture was concentrated, resuspended in pyridine (7.5 mL) and acetic anhy-
dride (2 mL) and allowed to stir and additional 16 h. Upon completion the reac-
tion was concentrated and column chromatography (30% Acetone in Hexanes)
afforded the product (255 mg, 68% yield). Note: the reaction works better when
using over 100 mg reactant.
1
H NMR (400 MHz, CDCl3) δ 5.93 (d, J = 3.1 Hz,
1H), 5.77 (d, J = 8.9 Hz, 1H), 5.37 (dd, J = 10.9, 9.5 Hz, 1H), 5.17 (dd, J = 11.0, 9.5
Hz, 1H), 4.64 (ddd, J = 10.9, 8.9, 3.1 Hz, 1H), 4.34 (dd, J = 12.1, 4.9 Hz, 1H), 4.04
(dd, J = 12.1, 3.2 Hz, 1H), 3.48 (ddd, J = 10.8, 4.9, 3.2 Hz, 1H), 2.18 (d, J = 0.4
Hz, 3H), 2.06 (s, 3H), 2.05 – 2.02 (s, 6H), 1.91 (s, 3H).
Compound 9.9 1,3-Dibromoacetone. Synthesis was carried out according to
Huang et al (5). Br2 (10 mL) was added dropwise to a round-
bottom flask containing MeOH (72.9 mL) and Acetone (6.08 mL)
stirring in a H2O bath at room temperature. Upon addition com-
pletion, the reaction stirred an additional 2 h and then was transferred to 4 ℃
and then -20 ℃ overnight to ensure precipitation. Precipitation was collected by
filtration. The precipitate (2.218 g) was added to a 1 L flask followed by H2O (92
S
AcO
HN
OAc
AcO
OAc
O
O
Br Br
337
mL) and 95% sulfuric acid (1 mL) was added slowly. The reaction was warmed to
60 ℃ and allowed to stir for 48 h. Upon completion the reaction was cooled to
room temperature, and CH2Cl2 was used to extract the product (3 x 100 mL). The
combined organics were then washed with Brine (300 mL) and dried over
MgSO4. The organic layer was then filtered and concentrated at 30 ℃ to afford
the pure product (2.218 g, 12% yield).
1
H NMR (600 MHz, CDCl3) δ 4.16 (s, 4H).
Compound 9.10 2-(1,3-dioxoisoindolin-2-yl)acetaldehyde. Synthesis carried
out according to Zhuang and coworkers (3). Phthalimido-
acetaldehyde diethylacetal. (8.00 g, 30.38 mmol) in CH2Cl2 (92
mL) under nitrogen atmosphere was cooled in an ice/water bath.
Trifluoroacetic Acid (61.5 mL) was added and the resulting mixture stirred for 1 h
after which time the reaction was allowed to warm to room temperature and stir
for an additonal 5 h. Upon completion, the reaction was concentrated and co-
evaporated with toulene and CH2Cl2 to remove traces of acid. No further purifica-
tion was necessary to yield the pure product (5.747 g, quantitative yield).
1
H NMR
(500 MHz, CDCl3) δ 9.67 (s, 1H), 7.91 (dd, J = 5.4, 3.1 Hz, 2H), 7.77 (dd, J = 5.5,
3.0 Hz, 2H), 4.57 (s, 2H).
N
O
O
O
338
Compound 9.11 2-(4-oxopent-2-enyl)isoindoline-1,3-dione. Synthesis carried
out according to Zhuang and coworkers (3). NaH (486 mg,
20.25 mmol) and THF (82 mL) were stirred under nitrogen
atmosphere. To this was added diethyl(2-
oxopropyl)phosphonate (3.893 mL, 20.25 mmol) in THF (55
mL) dropwise over 10 min. The reaction was allowed to stir for 1 h after which
time 9.10 (5.747 g, 30.38 mmol) in THF (28 mL) was added dropwise. The reac-
tion stirred for 3 h. Upon completion the reaction was quenched with H2O and
concentrated. The crude was then extracted with CH2Cl2 (3 x 50 mL) and the
combined organics washed with 1 M HCl, saturated sodium bicarbonate, water
and brine. The organic layer was then dried over Na2SO4 and concentrated to af-
ford crude. Purification by silica gel column chromatography (20-35% EtOAc in
Hexanes) yielded the product (4.070 g, 88% yield).
1
H NMR (500 MHz,
Chloroform-d) δ 7.89 (dd, J = 5.5, 3.0 Hz, 2H), 7.76 (dd, J = 5.5, 3.0 Hz, 2H),
6.76 (dt, J = 16.0, 5.2 Hz, 1H), 6.12 (d, J = 16.1 Hz, 1H), 4.48 (dd, J = 5.2, 1.7 Hz,
2H), 2.26 (s, 3H).
Compound 9.12 2-(4-trimethylsilyloxopent-2-enyl)isoindoline-1,3-dione. Syn-
thesis carried out according to Zhuang and coworkers (3).
Under nitrogen, toluene (2 mL) and TEA (103 µL, 0.742
mmol) were added to 9.11 (100 mg, 0.436 mmol).
Trimethylsilyl-trifluoreoacetate (118 µL, 0.654 mmol) was
N
O
O
O
N
O
O
TMSO
339
added dropwise and the reaction stirred for 16 h. Upon completion, the reaction
was diluted with diethylether (25 mL) and quenched with saturated sodium bi-
carbonate. The organic later was extracted with diethylether (2 x 25 mL) and the
combined organics washed with H2O and dried over Na2SO4. The concentrated
crude was then used with no further purification or characterization.
Compound 9.13 2-(5-bromo-4-oxopent-2-enyl)isoindoline-1,3-dione. Synthe-
sis carried out according to Zhuang and coworkers (3). To
9.12 (0.436 mmol) was added THF (5 mL) and the solution
was cooled in an ice bath. Once cool, NaHCO3 (55 mg, 0.6543
mmol) was added followed by N-bromosuccinimide (85 mg, 0.4798 mmol). The
reaction stirred for 4 h after which time the reaction was diluted with diethylether
(25 mL) and quenched with saturated sodium bicarbonate. The organic later was
extracted with diethylether (2 x 25 mL) and the combined organics washed with
H2O and dried over Na2SO4. The concentrated crude was then purified by column
chromatography (20-25% EtOAc in Hexanes) to afford the pure product (101 mg,
75% yield over 2 steps).
1
H NMR (500 MHz, CDCl3) δ 7.94 – 7.84 (m, 2H), 7.82 –
7.72 (m, 2H), 6.94 (ddd, J = 15.8, 5.1 Hz, 1H), 6.37 (d, J = 15.8 Hz, 1H), 4.51 (dd,
J = 5.3, 1.7 Hz, 2H), 3.99 (s, 2H).
N
O
O
O Br
340
Compound 9.14 2-(3-(2-(bromomethyl)-1,3-dioxolan-2-yl)allyl)isoindoline
-1,3-dione.Synthesis carried out according to Zhuang and co-
workers (3). Under nitrogen, 9.13 (824 mg, 2.67 mmol) was
resuspended in benzene (10 mL) To this was added ethylene
glycol (1.50 mL, 26.7 mmol) and p-toluenesulfonic acid (46 mg, 0.267 mmol).
The mixture was refluxed for 8 h, during which time a Dean-Stark trap was used
to remove water from the reaction. Upon completion, the mixture was cooled,
and the reaction was diluted with diethylether (25 mL) and quenched with satu-
rated sodium bicarbonate. The organic later was extracted with diethylether (2 x
25 mL) and the combined organics washed with H2O and dried over Na2SO4. The
concentrated crude was then purified by column chromatography (20% EtOAc in
Hexanes) to afford the pure product (749 mg, 80% yield).
1
H NMR (500 MHz,
CDCl3) δ 7.87 (dd, J = 5.4, 3.1 Hz, 2H), 7.77 – 7.71 (m, 2H), 6.02 (dt, J = 15.4, 5.7
Hz, 1H), 5.70 (dt, J = 15.4, 1.5 Hz, 1H), 4.34 (dd, J = 5.7, 1.6 Hz, 2H), 4.11 – 4.02
(m, 2H), 3.99 – 3.88 (m, 2H), 3.45 (s, 2H).
Compound 9.15 3-(2-(bromomethyl)-1,3-dioxolan-2-yl)prop-2-en-1-amine.
Synthesis carried out according to Zhuang and coworkers (3).
Under nitrogen, 9.14 (200 mg, 0.568 mmol) was dissolved in
methanol (22 mL) and 40% methylamine and allowed to stir
for 48-72 h. Upon completion (as determined by TLC), the reaction was concen-
trated and purified by column chromatography (5-10% MeOH in CH2Cl2 with 1%
N
O
O
Br
O
O
H
2
N
O
O
Br
341
Ammonium Hyroxide) to afford 9.15 (33 mg, 43% yield).
1
H NMR (500 MHz,
CD3OD) δ 6.09 (dt, J = 15.5, 5.7 Hz, 1H), 5.60 (dt, J = 15.5, 1.8 Hz, 1H), 4.08 –
4.04 (m, 2H), 3.97 – 3.93 (m, 2H), 3.52 (s, 2H), 2.08 – 2.06 (m, 1H), 1.94 – 1.92
(m, 1H).
Compound 9.16 3,4,6-Tri-O-Acetyl-N-4-pentynylgalactosamine
(Ac3GalNAlk). This anomeric deprotection procedure was taken from Hang et al.
for the synthesis of 3,4,6-Tri-O-acetyl-2-azidoacetamido-2-deoxy-D-
galactopyranose (6). To a 50 mL, flame-dried round-bottom flask was added 2.6
(300 mg, 0.702 mmol). The material was coevaporated with 5 mL toluene three
times. Upon dryness, the product was stirred under Argon, resuspended in THF
(10 mL) and benzylamine added (92 µL. 0.843 mmol). The reaction was warmed
to 50 ℃ and allowed to stir overnight. Upon completion, the reaction was con-
centrated to afford the crude. Silica gel column chromatography (1 liter flush 10%
Acetone/90% CH2Cl2 and then 5% MeOH/95% CH2Cl2) afforded 9.16 (243 mg,
90% yield).
1
H NMR (500 MHz, CDCl3) α-anomer: δ 6.07 (d, J = 9.6 Hz, 1H),
5.40 – 5.36 (m, 1H), 5.31 (d, J = 3.6 Hz, 1H), 5.24 (dd, J = 11.4, 3.2 Hz, 1H), 4.57
(ddd, J = 11.3, 9.6, 3.6 Hz, 1H), 4.46 – 4.40 (m, 1H), 4.18 – 4.02 (m, 3H), 2.52 –
2.46 (m, 2H), 2.39 (ddd, J = 7.9, 6.4, 4.4 Hz, 2H), 2.16 (s, J = 0.7 Hz, 3H), 2.06 –
2.01 (s, 4H), 2.01 – 1.97 (s, 3H).
13
C NMR (125 MHz, CDCl3) α-anomer: δ 171.42,
171.04, 170.83, 170.40, 92.18, 82.61, 76.77, 69.48, 67.50, 66.44, 62.10, 47.88,
35.33, 20.84, 20.75, 20.73, 14.89.
342
Compound 9.17 3,4,6-Tri-O-Acetyl-N-4-pentynylgalactosamine-1-Phosphate.
This procedure for the phosphorylation and subsequent
oxidation of 9.16 was taken from Hang et al (6). To a 50
mL, flame-dried round-bottom flask was added 9.16 (243
mg, 0.631 mmol). The compound was rinsed and coevapo-
rated from toluene (3 x 5 mL). Under Ar was added CH2Cl2 (7 mL) and 5-
ethylthio-1H-tetrazole (411 mg, 3.16 mmol). The reaction stirred for 10 min or
until the tetrazole was completely dissolved. Diallyl-N,N-
diisopropylphosphoramidite (500 µL. 1.89 mmol) was then added dropwise, and
the reaction stirred for 3 h. After 3 h, the reaction was cooled to -40 ℃ and
freshly recrystallized m-CPBA (545 mg, 3.16 mmol) was added. The reaction pro-
ceeded for 10 min at -40 ℃, and then was placed in an ice bath to warm to room
temperature over 1 h. Upon completion, the reaction was diluted with CH2Cl2 (40
mL), washed with 10% aq solution of Na2SO3 (2 x 50 mL), saturated NaHCO3 (4 x
50 mL) and H2O (2 x 50 mL). The organic layer was then dried over Na2SO4, fil-
tered and concentrated. The crude was further purified via silica gel column
chromatography (10-25% Acetone/90-75% Toluene) to afford product 9.17 (254
mg, 76% yield).
1
H NMR (500 MHz, CDCl3): δ 6.00 - 5.88 (m, 2H), 5.72 (dd, J =
5.9, 3.3 Hz, 1H), 5.45 - 5.42 (m, 1H), 5.40 (dt, J = 9.2, 1.4 Hz, 1H), 5.36 (dt, J =
9.2, 1.4 Hz, 1H), 5.29 (ddt, J = 10.5, 8.4, 1.1 Hz, 2H), 5.21 - 5.15 (m, 1H), 4.73 -
4.65 (m, 1H), 4.58 (tdd, J = 8.6, 5.7, 1.5 Hz, 4H), 4.45 - 4.39 (m, 1H), 4.15 - 4.04
O
OAc
AcO
AcO
HN
OPO
3
-2
O
343
(m, 2H), 2.53 - 2.42 (m, 2H), 2.42 - 2.33 (m, 2H), 2.14 (d, J = 1.2 Hz, 3H), 2.01
(d, J = 1.1 Hz, 3H), 1.99 - 1.95 (m, 4H).
13
C NMR (125 MHz, CDCl3): 171.44,
170.77, 170.37, 170.22, 132.30, 132.24, 132.10, 132.05, 119.22, 119.18, 119.17,
97.04, 96.99, 82.75, 69.50, 68.82, 67.50, 66.92, 61.55, 47.66, 35.21, 20.77, 20.75,
14.76.
31
P NMR (400 MHz, CDCl3): -2.60.
Compound 9.18 Uridine-Diphosphate-N-4-pentynylgalactosamine (UDP-
GalNAlk). This procedure was taken from Hang
et al (6). UMP-N-methylimidazolide was syn-
thesized according to Marlow and Kiessling (7).
To 9.17 (200 mg, 0.377 mmol) was added 1:1
THF:MeOH (10 mL), p-toluenesulfinic acid sodium salt (134 mg, 0.754 mmol)
and Pd(PPh3)4 (21 mg, 0.018 mmol) in round bottom flask under Nitrogen at-
mosphere. The reaction stirred for 3 h at room temperature. Upon completion,
the reaction was concentrated and co-evaporated with toluene. The crude mix-
ture was then resuspended in MeCN (2 mL) and TEA (571 µL) added. The reac-
tion was cooled to 0 ℃ and UMP-N-methylimidazolide (168 mg, 0.452 mmol) in
MeCN (4 mL) was added dropwise. The reaction was stirred for 3 h at 0 ℃ and
concentrated upon completion. The reaction was then resuspended in
MeOH:H2O:TEA 5:2:1 (20 mL) and stirred for 16 h at room temperature. The re-
action was again concentrated and the crude resuspended in H2O (50 mL) and
washed with CH2Cl2 (3 x 50 mL) to remove any organics. The aqueous layer was
O
OH
HO
HO
HN
O
P
O
O
OH
P
O OH
O
O
O
HO OH
N
NH
O
O
344
then concentrated to afford a yellow foam. The foam was resuspended in 100 mM
NH4HCO3 (2 mL), loaded onto a P-2 Bio-Gel Econo-Column (Bio-Rad) and
eluted with 100 mM NH4HCO3 at 0.5 mL/min. To determine which fractions
contained the donor sugar, tubes were TLC’ed and checked by UV
(EtOH:NH4OH:H2O 5:3:1). Fractions containing the donor sugar were concen-
trated, resuspended in a minimal amount of H2O (2 mL) and subjected to ion-
exchange with AG 50W-X8 sodium resin (Bio-Rad). The resulting product was
lyophilized to afford the product (159 mg, 61% yield).
1
H NMR (500 MHz, D2O) δ
7.96 (d, J = 8.1 Hz, 1H), 6.04 – 5.93 (m, 2H), 5.57 (dd, J = 7.0, 3.5 Hz, 1H), 4.42 –
4.34 (m, 2H), 4.33 – 4.18 (m, 4H), 4.06 (dd, J = 3.1, 1.1 Hz, 1H), 3.98 (dd, J =
10.9, 3.1 Hz, 1H), 3.86 – 3.70 (m, 2H), 3.27 (q, J = 7.3 Hz, 1H), 2.66 – 2.55 (m,
2H), 2.52 (dd, J = 7.0, 2.6 Hz, 2H), 1.26 (t, J = 7.2 Hz, 1H).
13
C NMR (126 MHz,
D2O) δ 175.32, 166.93, 160.29, 152.30, 141.51, 129.15, 125.30, 102.64, 94.77,
94.72, 88.51, 83.65, 83.12, 83.04, 73.73, 72.00, 69.95, 69.58, 68.37, 67.53, 64.97,
64.93, 60.97, 58.92, 49.69, 49.62, 48.82, 34.27, 14.28, 7.42.
31
P NMR (202 MHz,
D2O) δ - 11.38, -13.00.
345
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Appendix A: Mass Spectroscopy Tables
Table 2-1 High Confidence GlcNAlk Labelled Proteins
Table 2-2 Medium Confidence GlcNAlk Labelled Proteins
Table 5-1 Asp-Alk Labelled Proteins
Table 6-1 6AzGlcNAc Labelled Proteins
Table 6-2 GlcNAz Labelled Proteins
Table 6-3 GalNAz Labelled Proteins
Table 7-1 1-DeoxyGlcNAlk Labelled Proteins
Table 7-2 GlcNAlk Labelled Proteins
398
Table 2-1. Proteins selectively identified with high confidence in GlcNAlk samples by mass spectrometry.
Data was considered high confidence if the number of assigned spectra was at least 10 fold greater for GlcNAlk samples than
DMSO control samples. Further, the protein must have been identified with spectral counts greater than or equal to 5. Data
was compared to proteomic data sets from Wells et al., Teo et al., Gurcel et al., Cieniewski-Bernard et al., Clark et al., Sprung
et al., Nandi et al., Wang et al., Vosseller et al., Khidekel et al. 2007, Hanover et al., Khidekel et al. 2004, Dehennaut et al., and
Boyce et al. Proteins were shaded blue if they have not been previously identified. Nuclear pore glycoprotein p62, Lamin-A/C,
HP1γ, and NEDD4-1 (confirmed by Western blotting) are highlighted in yellow.
Identified Proteins (142, 79 new)
Accession
Number
GlcNAlk DMSO Citation Function
Glyceraldehyde-3-phosphate dehydrogenase
IPI00273646
(+2)
447 15
Wells, Teo, Gurcel, Cienewski-
Bernard, Dehennaut, Wang
Metabolism
Cofilin-1 IPI00890117 131 0 Wells, Teo, Nandi Structural
Triosephosphate isomerase IPI00467833 129 8
Teo, Cieniewski-Bernard,
Sprung, Clark
Metabolism
Annexin A2 IPI00468203 127 3 Clark, Gurcel Structural
Elongation factor 2 IPI00466069 124 0 Clark, Teo Protein Synthesis, Quality Control, & Turnover
Isoform A of Lamin-A/C IPI00620256 67 3 Structural
Putative uncharacterized protein IPI00229080 62 6 Unknown
S-methyl-5'-thioadenosine phosphorylase IPI00132096 62 0 Nandi Metabolism
Annexin A1 IPI00230395 59 3 Wells, Teo, Nandi Signaling
E3 ubiquitin-protein ligase NEDD4 IPI00462445 59 0 Protein Synthesis, Quality Control, & Turnover
Vimentin IPI00227299 59 3 Wang, Teo Structural
L-lactate dehydrogenase A chain
IPI00319994
(+1)
58 0 Dehennaut, Wang, Metabolism
Isoform M1 of Pyruvate kinase isozymes M1/M2 IPI00407130 57 0
Clark, Teo, Gurcel, Sprung,
Wells, Dehennaut, Nandi
Metabolism
Isoform 2 of Tropomyosin alpha-3 chain IPI00230044 54 0 Structural
Heterogeneous nuclear ribonucleoprotein F IPI00226073 43 0 RNA Processing
Voltage-dependent anion-selective channel protein 3 IPI00876341 41 0 Intracellular Trafficking
Destrin IPI00127942 40 0 Structural
14-3-3 protein epsilon IPI00118384 34 2 Sprung, Clark Signaling
Isoform 1 of Heterogeneous nuclear ribonucleoprotein K
IPI00223253
(+3)
34 0 Wang, Clark, Nandi, Gurcel RNA Processing
Voltage-dependent anion-selective channel protein 2 IPI00122547 31 0 Clark Intracellular Trafficking
Isoform 1 of Heterogeneous nuclear ribonucleoprotein A3
IPI00269661
(+1)
30 0 Clark, Teo, Wang, Nandi RNA Processing
Poly(rC)-binding protein 1 IPI00128904 28 0 Nandi Protein Synthesis, Quality Control, & Turnover
Zyxin
IPI00387422
(+1)
28 0 Structural
Chromobox protein homolog 3, HP1γ
IPI00129468
(+1)
27 0 Gene Regulation & Transcription
399
Identified Proteins (142, 79 new)
Accession
Number
GlcNAlk DMSO Citation Function
Nucleolar protein 58 IPI00463468 27 0 Protein Synthesis, Quality Control, & Turnover
Host cell factor C1
IPI00828490
(+1)
26 0
Wells, Wang, Teo, Vosseller,
Khidekel (2007), Khidekel
(2004),
Gene Regulation & Transcription
Isoform 1 of 14-3-3 protein theta
IPI00408378
(+2)
25 0 Gurcel, Clark Signaling
AHNAK nucleoprotein isoform 1 IPI00553798 22 0 Signaling
caldesmon 1
IPI00122450
(+4)
22 0 Structural
Eukaryotic translation initiation factor 5A-1
IPI00108125
(+1)
22 0 Nandi Protein Synthesis, Quality Control, & Turnover
Adenylyl cyclase-associated protein 1
IPI00137331
(+1)
20 0 Structural
Alpha-actinin-1 IPI00380436 20 0 Nandi Structural
Asparagine synthetase [glutamine-hydrolyzing] IPI00116966 20 0 Metabolism
Bifunctional aminoacyl-tRNA synthetase IPI00339916 20 0 RNA Processing
Isoform 1 of Filamin-A
IPI00131138
(+3)
20 0 Nandi, Gurcel Structural
MICAL-like 2
IPI00280103
(+1)
20 0 Structural
Myosin-9 IPI00123181 20 0
Nandi, Teo, Cienewski-
Bernard
Structural
Protein S100-A4 IPI00124096 20 0 Signaling
Thioredoxin IPI00226993 20 0 Teo Signaling
Isoform 2 of F-actin-capping protein subunit beta
IPI00269481
(+2)
19 0 Clark Structural
caprin-1 isoform c
IPI00121515
(+2)
18 0 Teo Protein Synthesis, Quality Control, & Turnover
Cathepsin B IPI00113517 18 0 Signaling
Isoform Long of Delta-1-pyrroline-5-carboxylate synthetase
IPI00129350
(+2)
16 0 Wang Metabolism
T-complex protein 1 subunit beta IPI00320217 15 0 Clark Protein Synthesis, Quality Control, & Turnover
Tubulin beta-5 chain IPI00117352 15 4
Clark, Wang, Teo, Sprung,
Gurcel
Structural
Cadherin-3
IPI00109340
(+2)
14 0 Signaling
Far upstream element-binding protein 2 IPI00462934 14 0 Nandi RNA Processing
Eukaryotic translation initiation factor 4B IPI00221581 13 0 Protein Synthesis, Quality Control, & Turnover
Myb-binding protein 1A IPI00331361 13 0 Gene Regulation & Transcription
Putative uncharacterized protein IPI00132089 13 0 Unknown
similar to high-mobility group box 1 IPI00665601 13 0 Unknown
400
Identified Proteins (142, 79 new)
Accession
Number
GlcNAlk DMSO Citation Function
similar to Nucleophosmin IPI00849626 13 0 Unknown
ADP/ATP translocase 2 IPI00127841 12 0 Clark, Teo, Wang Metabolism
Elongation factor 1-gamma IPI00318841 12 0 Protein Synthesis, Quality Control, & Turnover
Glycyl-tRNA synthetase IPI00112555 12 0 Nandi RNA Processing
Heat shock protein HSP 90-alpha IPI00330804 12 0 Nandi, Teo, Wells Protein Synthesis, Quality Control, & Turnover
Inosine-5'-monophosphate dehydrogenase 2
IPI00323971
(+1)
12 0 Nandi, Teo Metabolism
Isoform 1 of Protein arginine N-methyltransferase 1
IPI00120495
(+3)
12 0 Signaling
LIM domain and actin-binding protein 1 isoform a IPI00112339 12 0 Structural
Peroxiredoxin-1
IPI00121788
(+2)
12 0 Metabolism
CTP synthase 1 IPI00111959 11 0 Metabolism
deoxyuridine triphosphatase isoform 1
IPI00187434
(+1)
11 0 Metabolism
Glutathione S-transferase omega-1 IPI00114285 11 0 Wang, Sprung Protein Synthesis, Quality Control, & Turnover
Homeobox protein engrailed-2 IPI00020031 11 0 Gene Regulation & Transcription
Isoform 1 of Nuclear autoantigenic sperm protein
IPI00130959
(+1)
11 2 Gene Regulation & Transcription
Isoform 5 of Ubiquitin-associated protein 2-like
IPI00407835
(+1)
11 0 Clark, Teo Signaling
Nucleolar RNA helicase 2 IPI00652987 11 0 RNA Processing
Src substrate cortactin
IPI00118143
(+1)
11 0 Teo Signaling
Cytochrome P450 4V3 IPI00120197 10 0 Metabolism
Isoform 2 of Fatty aldehyde dehydrogenase IPI00394758 10 0 Metabolism
Isoform CW17 of Splicing factor 1
IPI00116284
(+4)
10 0 Teo Gene Regulation & Transcription
Uncharacterized protein IPI00830960 10 0 Unknown
Actin, alpha skeletal muscle
IPI00110827
(+4)
9 0 Clark Structural
Calponin-2 IPI00116649 9 0 Signaling
Elongation factor 1-alpha 1 IPI00307837 9 0
Nandi, Teo, Wang, Gurcel,
Wells, Clark
Protein Synthesis, Quality Control, & Turnover
Isoform 1 of Lipoma-preferred partner homolog IPI00221494 9 0 Signaling
Isoform 1 of Poly(rC)-binding protein 2
IPI00127707
(+1)
9 0 Clark, Gurcel Protein Synthesis, Quality Control, & Turnover
Isoform 3 of A-kinase anchor protein 2
IPI00336504
(+5)
9 0 RNA Processing
PDZ and LIM domain protein 5 isoform ENH1
IPI00653381
(+1)
9 0 Structural
401
Identified Proteins (142, 79 new)
Accession
Number
GlcNAlk DMSO Citation Function
Putative uncharacterized protein
IPI00136883
(+1)
9 0 Unknown
Putative uncharacterized protein IPI00653643 9 0 Unknown
Tropomyosin alpha-4 chain IPI00421223 9 0 Structural
40S ribosomal protein S12
IPI00225634
(+2)
8 0 Protein Synthesis, Quality Control, & Turnover
D-3-phosphoglycerate dehydrogenase IPI00225961 8 0 Nandi, Clark Metabolism
Ewing sarcoma breakpoint region 1
IPI00322492
(+3)
8 0 Wells, Teo Unknown
Histone-binding protein RBBP7
IPI00122698
(+1)
8 0 Gene Regulation & Transcription
Isoform 1 of Plasminogen activator inhibitor 1 RNA-binding protein
IPI00471475
(+3)
8 0 Teo RNA Processing
Isoform Smooth muscle of Myosin light polypeptide 6
IPI00354819
(+3)
8 0 Nandi Structural
Matrin-3 IPI00453826 8 0 Gene Regulation & Transcription
Peroxiredoxin-4 IPI00116254 8 0 Nandi Metabolism
similar to LOC446231 protein
IPI00116718
(+1)
8 0 unknown
Vesicle-associated membrane protein-associated protein A IPI00125267 8 0 Intracellular Trafficking
Cytochrome b5 type B IPI00315794 7 0 Metabolism
Impa1 protein
IPI00473176
(+1)
7 0 Metabolism
Isoform 1 of Glyoxalase domain-containing protein 4
IPI00110721
(+1)
7 0 Metabolism
Multifunctional protein ADE2
IPI00322096
(+1)
7 0 Teo Metabolism
Protein PRRC1 IPI00130462 7 0 Teo Unknown
Putative pre-mRNA-splicing factor ATP-dependent RNA Processing helicase
DHX15
IPI00128818 7 0 RNA Processing
Putative uncharacterized protein IPI00126248 7 0 Unknown
Transmembrane protein 117 IPI00221687 7 0 Signaling
Xanthine dehydrogenase/oxidase
IPI00352984
(+1)
7 0 Metabolism
40S ribosomal protein S3 IPI00134599 6 0 Dehennaut, Clark, Wang Protein Synthesis, Quality Control, & Turnover
Anamorsin IPI00187301 6 0 Signaling
ATP-binding cassette sub-family E member 1 IPI00322869 6 0 Intracellular Trafficking
Calreticulin IPI00123639 6 0 Nandi, Sprung Protein Synthesis, Quality Control, & Turnover
eukaryotic peptide chain release factor GTP-binding subunit ERF3A isoform 1
IPI00230355
(+1)
6 0 Protein Synthesis, Quality Control, & Turnover
High mobility group protein HMGI-C IPI00331612 6 0 Gene Regulation & Transcription
402
Identified Proteins (142, 79 new)
Accession
Number
GlcNAlk DMSO Citation Function
Isoform 1 of Integrin beta-like protein 1 IPI00123829 6 0 Signaling
Isoform 1 of La-related protein 1 IPI00929786 6 0 RNA Processing
Isoform 3 of Ribosome-binding protein 1
IPI00121149
(+1)
6 0 Intracellular Trafficking
SUMO-activating enzyme subunit 2 IPI00023234 6 0 Protein Synthesis, Quality Control, & Turnover
Transgelin-2 IPI00125778 6 0 Nandi Structural
Tubby-related protein 1
IPI00130590
(+3)
6 0 Signaling
Ubiquitin thioesterase OTUB1
IPI00154004
(+1)
6 0 Clark Protein Synthesis, Quality Control, & Turnover
WW domain-binding protein 11 IPI00123333 6 0 RNA Processing
182 kDa tankyrase-1-binding protein IPI00459443 5 0 Signaling
60S acidic ribosomal protein P0 IPI00314950 5 0 Teo, Wang Protein Synthesis, Quality Control, & Turnover
60S ribosomal protein L10a
IPI00127085
(+1)
5 0 Teo Protein Synthesis, Quality Control, & Turnover
Aldose reductase-related protein 2 IPI00273096 5 0 Metabolism
BAT2 domain-containing protein 1
IPI00330171
(+2)
5 0 Wang Unknown
Branched-chain-amino-acid aminotransferase
IPI00653423
(+3)
5 0 Metabolism
BRI3-binding protein IPI00226771 5 0 Signaling
Calcyclin-binding protein IPI00115650 5 0 Protein Synthesis, Quality Control, & Turnover
Crk-like protein IPI00113362 5 0 Signaling
Elongation factor 1-beta IPI00320208 5 0 Sprung Protein Synthesis, Quality Control, & Turnover
Four and a half LIM domains 3 IPI00828338 5 0 Structural
Gamma-sarcoglycan IPI00110503 5 0 Structural
Glutamate--cysteine ligase regulatory subunit IPI00114329 5 0 Metabolism
Histone H1.2 IPI00223713 5 0 Teo Gene Regulation & Transcription
Isoform 1 of Eukaryotic translation initiation factor 4 gamma 1
IPI00421179
(+3)
5 0 Nandi Protein Synthesis, Quality Control, & Turnover
Isoform Alpha of Lamina-associated polypeptide 2, isoforms alpha/zeta
IPI00126338
(+1)
5 0 Gene Regulation & Transcription
KH domain-containing, RNA-binding, signal transduction-associated protein 1 IPI00458765 5 0 RNA Processing
neurogenic locus notch homolog protein 2 IPI00467908 5 0 Signaling
Nuclear pore glycoprotein p62 IPI00139994 5 0 Hanover, Teo, Wells Intracellular Trafficking
Nucleolin IPI00317794 5 0 Teo, Wang Gene Regulation & Transcription
403
Identified Proteins (142, 79 new)
Accession
Number
GlcNAlk DMSO Citation Function
Nucleoplasmin-3 IPI00131725 5 0 Teo RNA Processing
olfactory receptor 585
IPI00127945
(+2)
5 0 Signaling
Peptidyl-prolyl cis-trans isomerase FKBP4 IPI00230139 5 0 Clark, Teo Protein Synthesis, Quality Control, & Turnover
Ran-specific GTPase-activating protein (Fragment) IPI00321978 5 0 Teo Signaling
Ribonucleoside-diphosphate reductase large subunit
IPI00315127
(+1)
5 0 Nandi Metabolism
Ubiquitin-40S ribosomal protein S27a IPI00470152 5 0 Protein Synthesis, Quality Control, & Turnover
Isoform 1 of Nesprin-2 IPI00845851 5 0 Structural
404
Table 2-1 References
Boyce, M., Carrico, I.S., Ganguli, A.S., Yu, S.-H., Hangauer, M.J., Hubbard, S.C., Kohler, J.J., and Bertozzi, C.R. (2011). Meta-
bolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine sal-
vage pathway. Proc. Natl. Acad. Sci. U.S.a.
Cieniewski-Bernard, C., Bastide, B., Lefebvre, T., Lemoine, J., Mounier, Y., and Michalski, J.-C. (2004). Identification of O-
linked N-acetylglucosamine proteins in rat skeletal muscle using two-dimensional gel electrophoresis and mass spectrometry.
Mol Cell Proteomics 3, 577–585.
Clark, P.M., Dweck, J.F., Mason, D.E., Hart, C.R., Buck, S.B., Peters, E.C., Agnew, B.J., and Hsieh-Wilson, L.C. (2008). Direct
in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. J. Am. Chem. Soc. 130, 11576–11577.
Dehennaut, V., Slomianny, M.-C., Page, A., Vercoutter-Edouart, A.-S., Jessus, C., Michalski, J.-C., Vilain, J.-P., Bodart, J.-F.,
and Lefebvre, T. (2008). Identification of Structural and Functional O-Linked N-Acetylglucosamine-bearing Proteins in
Xenopus laevis Oocyte. Molecular & Cellular Proteomics 7, 2229–2245.
Gurcel, C., Vercoutter-Edouart, A., Fonbonne, C., Mortuaire, M., Salvador, A., Michalski, J., and Lemoine, J. (2008). Identifi-
cation of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Analytical and Bioanalytical Chemistry 390,
2089–2097.
Hanover, J.A., Cohen, C.K., Willingham, M.C., and Park, M.K. (1987). O-linked N-acetylglucosamine is attached to proteins of
the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. J. Biol. Chem. 262, 9887–9894.
Khidekel, N., Ficarro, S.B., Clark, P.M., Bryan, M.C., Swaney, D.L., Rexach, J.E., Sun, Y.E., Coon, J.J., Peters, E.C., and Hsieh-
Wilson, L.C. (2007). Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem
Biol 3, 339–348.
Khidekel, N., Ficarro, S.B., Peters, E.C., and Hsieh-Wilson, L.C. (2004). Exploring the O-GlcNAc proteome: direct identifica-
tion of O-GlcNAc-modified proteins from the brain. Proc Natl Acad Sci USA 101, 13132–13137.
Nandi, A., Sprung, R., Barma, D.K., Zhao, Y., Kim, S.C., Falck, J.R., and Zhao, Y. (2006). Global identification of O-GlcNAc-
modified proteins. Anal Chem 78, 452–458.
Sprung, R., Nandi, A., Chen, Y., Kim, S., Barma, D., Falck, J., and Zhao, Y. (2005). Tagging-via-substrate strategy for probing
O-GlcNAc modified proteins. J. Proteome Res. 4, 950–957.
405
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specific monoclonal antibodies suggest new roles for O-GlcNAc. Nat Chem Biol 6, 338–343.
Vosseller, K., Trinidad, J.C., Chalkley, R.J., Specht, C.G., Thalhammer, A., Lynn, A.J., Snedecor, J.O., Guan, S.,
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fication using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804.
406
Table 2-2. Proteins selectively identified with medium confidence in GlcNAlk samples by mass spectrometry.
Data was considered medium confidence if the number of assigned spectra was at least 2 fold greater for GlcNAlk samples
than DMSO control samples. Further, the protein must have been identified with spectral counts greater than or equal to 2.
Data was compared to proteomic data sets from Wells et al., Teo et al., Gurcel et al., Cieniewski-Bernard et al., Clark et al.,
Sprung et al., Nandi et al., Wang et al., Vosseller et al., Khidekel et al. 2007, Hanover et al., Khidekel et al. 2004, Dehennaut et
al., and Boyce et al. Proteins were shaded blue if they have not been previously identified. Nuclear pore glycoprotein p62,
Lamin-A/C, HP1γ, and NEDD4-1 (confirmed by Western blotting) are highlighted in yellow.
Identified Proteins (232, 200 new)
Accession
Number
GlcNAlk DMSO Citation Function
Actin, cytoplasmic 1
IPI00110850
(+2)
83 32 Nandi. Teo, Dehennaut Structural
microtubule-associated protein IPI00848818 54 12 Clark, Teo, Nandi Structural
Tubulin alpha-1C chain IPI00403810 13 4 Gurcel Structural
Transcriptional regulator ATRX
IPI00322707
(+2)
9 4 Gene Regulation & Transcription
Putative uncharacterized protein IPI00113294 6 2 Unknown
Receptor-type tyrosine-protein phosphatase mu
IPI00125753
(+1)
6 2 Signaling
MCG1046548 (Fragment) IPI00874855 5 2 Unknown
Neurogenic locus notch homolog protein 2 IPI00467908 5 0 Signaling
Very low-density lipoprotein receptor isoform b precursor
IPI00130648
(+1)
5 2 Intracellular Trafficking
60S ribosomal protein L14 IPI00133185 4 0 Protein Synthesis, Quality Control, & Turnover
BAG family molecular chaperone regulator 3
IPI00331334
(+1)
4 0 Protein Synthesis, Quality Control, & Turnover
Calnexin IPI00119618 4 0 Protein Synthesis, Quality Control, & Turnover
Cell division protein kinase 4
IPI00128326
(+2)
4 0 Signaling
ELAV-like protein 1 IPI00466032 4 0 Teo Unknown
Hephaestin-like protein 1 IPI00352073 4 0 Intracellular Trafficking
High mobility group protein B2 IPI00462291 4 0 Gene Regulation & Transcription
hypothetical protein isoform 1 IPI00458629 4 0 Unknown
hypothetical protein LOC329540 isoform 2
IPI00453784
(+1)
4 0 Unknown
Isocitrate dehydrogenase
IPI00135231
(+1)
4 0 Nandi Metabolism
Isoform 1 of Collagen alpha-1(I) chain IPI00329872 4 0 Structural
407
Identified Proteins (232, 200 new)
Accession
Number
GlcNAlk DMSO Citation Function
Isoform 1 of Complement regulatory protein Crry
IPI00138061
(+1)
4 0 Signaling
Isoform 1 of Dynein heavy chain 17, axonemal IPI00378509 4 2 Nandi Structural
Isoform 1 of Non-POU domain-containing octamer-binding protein IPI00320016 4 0 Teo, Wang Gene Regulation & Transcription
Isoform 1 of Protein phosphatase 1 regulatory subunit 12A
IPI00671847
(+1)
4 0 Clark, Teo Signaling
Isoform 2 of Ubiquitin carboxyl-terminal hydrolase 10
IPI00420601
(+1)
4 0 Protein Synthesis, Quality Control, & Turnover
keratin associated protein 4-2 IPI00654392 4 0 Structural
low affinity immunoglobulin gamma Fc region receptor II isoform 1
IPI00129485
(+2)
4 0 Signaling
Myosin-Ig IPI00387204 4 0 Intracellular Trafficking
Novel member of the keratin associated protein 4 (Krtap4) family IPI00605829 4 0 Structural
Nup98 protein IPI00474558 4 0 Intracellular Trafficking
Olfactory receptor Olfr570 IPI00127571 4 0 Signaling
Putative uncharacterized protein IPI00653426 4 0 Unknown
Putative uncharacterized protein
IPI00761759
(+2)
4 0 Unknown
RIKEN cDNA 2310007F04 IPI00132738 4 0 Unknown
Sodium channel protein type 11 subunit alpha IPI00126898 4 0 Signaling
Transient receptor potential cation channel subfamily M member 2
IPI00130116
(+1)
4 0 Signaling
Vesicle-associated membrane protein 4, isoform CRA_c IPI00404693 4 0 Intracellular Trafficking
Vomeronasal receptor V1RH8 IPI00153500 4 0 Unknown
Zinc finger protein-like 1 IPI00119045 4 0 Gene Regulation & Transcription
Actin-binding protein anillin IPI00172197 3 0 Structural
Apoptosis regulator BAX IPI00120684 3 0 Signaling
Attractin
IPI00224752
(+1)
3 0 Signaling
COP9 signalosome complex subunit 7b IPI00453769 3 0 Signaling
Coronin-1C IPI00124820 3 0 Nandi Structural
Cytosolic phospholipase A2
IPI00111169
(+1)
3 0 Signaling
Disintegrin and metalloproteinase domain-containing protein 25 IPI00128146 3 0 Signaling
Disintegrin and metalloproteinase domain-containing protein 9
IPI00626485
(+1)
3 0 Structural
408
Identified Proteins (232, 200 new)
Accession
Number
GlcNAlk DMSO Citation Function
embryonic stem cell- and germ cell-specific protein isoform 2 IPI00719878 3 0 Unknown
Eukaryotic translation initiation factor 2 subunit 3, X-linked IPI00230415 3 0 Gurcel, Nandi Protein Synthesis, Quality Control, & Turnover
Eukaryotic translation initiation factor 3 subunit G IPI00622371 3 0 Teo Protein Synthesis, Quality Control, & Turnover
Filamin-B IPI00663627 3 0 Structural
Gamma-aminobutyric acid receptor subunit gamma-3
IPI00118860
(+1)
3 0 Signaling
Hematological and neurological expressed 1-like protein IPI00107958 3 0 Unknown
Hepatoma-derived growth factor IPI00313817 3 0 Gene Regulation & Transcription
Hypoxia up-regulated protein 1 IPI00123342 3 0 Signaling
Importin subunit alpha-2 IPI00124973 3 0 Intracellular Trafficking
Isoform 1 of E3 ubiquitin-protein ligase UHRF2
IPI00169767
(+1)
3 0 Protein Synthesis, Quality Control, & Turnover
Isoform 1 of Heterogeneous nuclear ribonucleoprotein Q
IPI00406117
(+1)
3 0 Teo RNA Processing
Isoform 1 of Leucine-rich repeat flightless-interacting protein 1 IPI00654388 3 0 Signaling
Isoform 1 of Probable ATP-dependent RNA helicase DDX17
IPI00396797
(+1)
3 0 RNA Processing
Isoform 1 of Reticulon-3
IPI00470981
(+1)
3 0 Intracellular Trafficking
Isoform 1 of Reticulon-4 IPI00469392 3 0 Signaling
Isoform 1 of Septin-9
IPI00457611
(+2)
3 0 Structural
Isoform 1 of STE20-like serine/threonine-protein kinase
IPI00331076
(+1)
3 0 Signaling
Isoform 1 of Tenascin
IPI00403938
(+4)
3 0 Signaling
Isoform 2 of Calpastatin
IPI00230641
(+4)
3 0 Protein Synthesis, Quality Control, & Turnover
Isoform 2 of Thioredoxin reductase 1, cytoplasmic
IPI00469251
(+1)
3 0 Metabolism
Isoform 3 of Teneurin-4
IPI00157497
(+3)
3 0 Structural
Isoform C1 of Heterogeneous nuclear ribonucleoproteins C1/C2
IPI00223443
(+4)
3 0 Clark, Teo RNA Processing
isopentenyl-diphosphate Delta-isomerase 1 IPI00115850 3 0 Metabolism
killer cell lectin-like receptor subfamily B member 1A isoform 1 IPI00120881 3 0 Signaling
409
Identified Proteins (232, 200 new)
Accession
Number
GlcNAlk DMSO Citation Function
Kinesin-like protein KIF3B IPI00465809 3 0 Intracellular Trafficking
neuropeptide W preproprotein IPI00856498 3 0 Unknown
NF-kB2 splice variant 4
IPI00123474
(+1)
3 0 Khidekel (2004), Nandi Signaling
Niban-like protein 1 IPI00330695 3 0 Protein Synthesis, Quality Control, & Turnover
nuclear transcription factor, X-box binding-like 1 IPI00378780 3 0 Gene Regulation & Transcription
Nucleolar protein 56
IPI00318048
(+1)
3 0 Gene Regulation & Transcription
Olfactory receptor 1102 IPI00353195 3 0 Signaling
Peroxiredoxin-6
IPI00555059
(+2)
3 0 Metabolism
Phosphoserine phosphatase IPI00117146 3 0 Signaling
Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated
channel 2
IPI00133980 3 0 Signaling
Probable histone-lysine N-methyltransferase ASH1L IPI00553465 3 0 Gene Regulation & Transcription
Proteasome activator complex subunit 2
IPI00124225
(+2)
3 0 Nandi Protein Synthesis, Quality Control, & Turnover
Protein IMPACT IPI00319956 3 0 Protein Synthesis, Quality Control, & Turnover
Protein Noxp20 IPI00331605 3 0 Unknown
protein phosphatase 1 regulatory subunit 11 IPI00851027 3 0 Gene Regulation & Transcription
Protein-lysine 6-oxidase IPI00310056 3 0 Protein Synthesis, Quality Control, & Turnover
Putative uncharacterized protein IPI00224729 3 0 Unknown
RAS-related C3 botulinum substrate 1, isoform CRA_a
IPI00127408
(+1)
3 0 Signaling
Ras-related protein Rab-5C
IPI00224518
(+1)
3 0 Signaling
S-adenosylmethionine decarboxylase proenzyme 2
IPI00122482
(+2)
3 0 Metabolism
scaffold attachment factor B IPI00944159 3 0 Structural
Serpin B6
IPI00121471
(+1)
3 0 Protein Synthesis, Quality Control, & Turnover
Sperm-associated antigen 7 IPI00273232 3 0 Unknown
Splicing factor 3A subunit 1 IPI00408796 3 0 RNA Processing
410
Identified Proteins (232, 200 new)
Accession
Number
GlcNAlk DMSO Citation Function
TAR DNA-binding protein 43 IPI00121758 3 0 Gene Regulation & Transcription
Translationally-controlled tumor protein IPI00129685 3 0 Nandi Structural
Translin IPI00124684 3 0 Gene Regulation & Transcription
trifunctional purine biosynthetic protein adenosine-3
IPI00230612
(+1)
3 0 Metabolism
Tubulin-specific chaperone D IPI00461857 3 0 Structural
ubiquitin carboxyl-terminal hydrolase 19 isoform 2
IPI00955070
(+1)
3 0 Protein Synthesis, Quality Control, & Turnover
ubiquitin specific protease 32 IPI00131330 3 0 Protein Synthesis, Quality Control, & Turnover
Uncharacterized protein C12orf56 homolog
IPI00228534
(+1)
3 0
Teo, Khidekel (2007), Khidekel
(2004),
Unknown
Vesicle transport protein SEC20
IPI00278462
(+1)
3 0 Intracellular Trafficking
Zinc finger RNA-binding protein IPI00131810 3 0 RNA Processing
14-3-3 protein zeta/delta IPI00116498 2 0 Gurcel Signaling
60 kDa SS-A/Ro ribonucleoprotein IPI00116360 2 0 RNA Processing
60S ribosomal protein L10-like
IPI00340103
(+5)
2 0 Protein Synthesis, Quality Control, & Turnover
60S ribosomal protein L11
IPI00331461
(+3)
2 0 Protein Synthesis, Quality Control, & Turnover
60S ribosomal protein L29
IPI00222548
(+1)
2 0 Protein Synthesis, Quality Control, & Turnover
60S ribosomal protein L4 IPI00111412 2 0 Teo, Nandi Protein Synthesis, Quality Control, & Turnover
Active regulator of SIRT1 IPI00226227 2 0 Protein Synthesis, Quality Control, & Turnover
Alanyl-tRNA synthetase, cytoplasmic IPI00321308 2 0 RNA Processing
AP-3 complex subunit beta-1 IPI00130444 2 0 Intracellular Trafficking
AP-3 complex subunit beta-2 IPI00420426 2 0 Intracellular Trafficking
Armadillo repeat-containing X-linked protein 3 IPI00308332 2 0 Unknown
ATP-dependent RNA helicase DDX24 isoform 1
IPI00113576
(+1)
2 0 RNA Processing
ATP-dependent RNA helicase DDX3X IPI00230035 2 0 RNA Processing
411
Identified Proteins (232, 200 new)
Accession
Number
GlcNAlk DMSO Citation Function
ATP-dependent zinc metalloprotease YME1L1 IPI00136555 2 0 Metabolism
Atp8b3 protein IPI00785352 2 0 Metabolism
B-cell differentiation antigen CD72 IPI00314355 2 0 Signaling
Bromodomain adjacent to zinc finger domain, 2B
IPI00622726
(+2)
2 0 Gene Regulation & Transcription
BTB/POZ domain-containing protein KCTD16 IPI00675985 2 0 Signaling
C-C chemokine receptor type 1
IPI00125199
(+1)
2 0 Signaling
Calpain 10, isoform CRA_a IPI00475395 2 0 Protein Synthesis, Quality Control, & Turnover
CD79B antigen IPI00131458 2 0 Signaling
Chromobox protein homolog 5 IPI00123755 2 0 Gene Regulation & Transcription
Coatomer subunit alpha
IPI00229834
(+1)
2 0 Nandi Intracellular Trafficking
Coiled-coil domain-containing protein 82 IPI00109873 2 0 Unknown
Collagen alpha-2(VI) chain
IPI00621027
(+1)
2 0 Structural
Cullin-associated NEDD8-dissociated protein 1 IPI00896727 2 0 Clark Protein Synthesis, Quality Control, & Turnover
Cysteine and glycine-rich protein 2 IPI00470178 2 0 Signaling
cytochrome P450, family 2, subfamily b, polypeptide 13 IPI00877190 2 0 Metabolism
Cytokine receptor common subunit gamma IPI00119612 2 0 Signaling
desmocollin-3 IPI00624693 2 0 Intracellular Trafficking
DNA ligase 1 IPI00473314 2 0 Gene Regulation & Transcription
DnaJ homolog subfamily A member 1 IPI00132208 2 0 Teo Protein Synthesis, Quality Control, & Turnover
Down syndrome cell adhesion molecule homolog IPI00112204 2 0 Signaling
E3 ubiquitin-protein ligase RNF19A IPI00120281 2 0 Protein Synthesis, Quality Control, & Turnover
Ets transcription factor Spi-B IPI00474655 2 0 Gene Regulation & Transcription
Eukaryotic translation initiation factor 3 subunit C IPI00321647 2 0 Protein Synthesis, Quality Control, & Turnover
F-box only protein 7
IPI00169531
(+3)
2 0 Protein Synthesis, Quality Control, & Turnover
412
Identified Proteins (232, 200 new)
Accession
Number
GlcNAlk DMSO Citation Function
General transcription factor IIF subunit 1
IPI00153986
(+1)
2 0 Gene Regulation & Transcription
Glutamate receptor ionotropic, NMDA3A
IPI00112337
(+1)
2 0 Signaling
Glutathione S-transferase Mu 5 IPI00114380 2 0 Dehennaut Metabolism
GMP synthase [glutamine-hydrolyzing] IPI00351252 2 0 Metabolism
Guanine nucleotide-binding protein G(i) subunit alpha-2
IPI00228617
(+1)
2 0 Signaling
Hemochromatosis protein IPI00649703 2 0 Gene Regulation & Transcription
Histone H1.3 IPI00331597 2 0 Teo Gene Regulation & Transcription
Histone H1.4 IPI00223714 2 0 Teo Gene Regulation & Transcription
Hsp90 co-chaperone Cdc37 IPI00117087 2 0 Protein Synthesis, Quality Control, & Turnover
hypothetical protein isoform 2 IPI00752723 2 0 Unknown
Ig kappa chain V-V region MOPC 173 IPI00464382 2 0 Unknown
Inactive dual specificity phosphatase 27 IPI00357029 2 0 Protein Synthesis, Quality Control, & Turnover
Inorganic pyrophosphatase IPI00110684 2 0 Metabolism
Isoform 1 of Alpha-taxilin IPI00342749 2 0 Intracellular Trafficking
Isoform 1 of ATPase family AAA domain-containing protein 3
IPI00126913
(+1)
2 0 Protein Synthesis, Quality Control, & Turnover
Isoform 1 of BAG family molecular chaperone regulator 1 IPI00310293 2 0 Protein Synthesis, Quality Control, & Turnover
Isoform 1 of COP9 signalosome complex subunit 7a
IPI00123465
(+1)
2 0 Signaling
Isoform 1 of Fanconi anemia group D2 protein homolog IPI00353539 2 0 Signaling
Isoform 1 of Guanine nucleotide-binding protein-like 3
IPI00222461
(+1)
2 0 Signaling
Isoform 1 of Heterochromatin protein 1-binding protein 3
IPI00342766
(+3)
2 0 Gene Regulation & Transcription
Isoform 1 of Long-chain fatty acid transport protein 3
IPI00131976
(+1)
2 0 Metabolism
Isoform 1 of Lysine-specific demethylase 2A
IPI00742275
(+1)
2 0 Gene Regulation & Transcription
Isoform 1 of Myosin-XVI
IPI00381066
(+2)
2 0 Structural
Isoform 1 of Neural cell adhesion molecule L1-like protein
IPI00222149
(+1)
2 0 Structural
Isoform 1 of Oxysterol-binding protein-related protein 1
IPI00469318
(+2)
2 0 Intracellular Trafficking
413
Identified Proteins (232, 200 new)
Accession
Number
GlcNAlk DMSO Citation Function
Isoform 1 of Prominin-1
IPI00120066
(+9)
2 0 Structural
Isoform 1 of Protein FAM40A
IPI00223670
(+1)
2 0 Unknown
Isoform 1 of Protocadherin Fat 3 IPI00828569 2 0 Structural
Isoform 1 of SH3 domain-containing YSC84-like protein 1
IPI00114903
(+2)
2 0 Signaling
Isoform 1 of Sprouty-related, EVH1 domain-containing protein 1 IPI00125975 2 0 Signaling
Isoform 1 of T-cell surface glycoprotein CD4 IPI00122806 2 0 Signaling
Isoform 1 of T-complex protein 1 subunit alpha
IPI00459493
(+1)
2 0 Nandi Protein Synthesis, Quality Control, & Turnover
Isoform 2 of Adenylate kinase 2, mitochondrial
IPI00269076
(+1)
2 0 Nandi Signaling
Isoform 2 of Drebrin-like protein
IPI00308222
(+2)
2 0 Structural
Isoform 2 of E3 ubiquitin-protein ligase HERC2
IPI00309574
(+1)
2 0 Protein Synthesis, Quality Control, & Turnover
Isoform 2 of Leucine-rich repeat and coiled-coil domain-containing protein 1
IPI00469945
(+3)
2 0 Signaling
Isoform 2 of Neuropilin and tolloid-like protein 2 IPI00223269 2 0 Signaling
Isoform 2 of Sodium/bile acid cotransporter 7 IPI00828850 2 0 Intracellular Trafficking
Isoform 2 of Tyrosine-protein kinase receptor TYRO3
IPI00126146
(+4)
2 0 Signaling
Isoform Long of Ectonucleoside triphosphate diphosphohydrolase 2 IPI00115089 2 0 Metabolism
Isoform PMX1-A of Paired mesoderm homeobox protein 1
IPI00230653
(+1)
2 0 Gene Regulation & Transcription
Killer cell inhibitory receptor-like protein p91A IPI00453607 2 0 Intracellular Trafficking
killer cell lectin-like receptor subfamily B member 1C isoform 1
IPI00322058
(+1)
2 0 Signaling
Leucine-rich repeat-containing protein 41 IPI00230296 2 0 Protein Synthesis, Quality Control, & Turnover
Leucyl-tRNA synthetase, cytoplasmic IPI00453819 2 0 RNA Processing
Limbic system-associated membrane protein
IPI00222833
(+1)
2 0 Structural
Mannose-P-dolichol utilization defect 1 protein
IPI00331563
(+3)
2 0 Protein Synthesis, Quality Control, & Turnover
MCG127722, isoform CRA_b
IPI00467976
(+2)
2 0 Signaling
Microtubule-associated protein 1S IPI00223621 2 0 Structural
MKIAA1233 protein (Fragment) IPI00354031 2 0 Intracellular Trafficking
414
Identified Proteins (232, 200 new)
Accession
Number
GlcNAlk DMSO Citation Function
N-alpha-acetyltransferase 15, NatA auxiliary subunit IPI00387212 2 0 Protein Synthesis, Quality Control, & Turnover
Nuclear pore complex protein Nup214 IPI00229722 2 0 Teo Intracellular Trafficking
nuclear receptor coactivator 4 IPI00125208 2 0 Signaling
Peripheral clock protein 1
IPI00463392
(+2)
2 0 Unknown
Phosphatidylinositol-5-phosphate 4-kinase type-2 alpha
IPI00115708
(+2)
2 0 Signaling
Podoplanin IPI00230205 2 0 Signaling
Potassium channel subfamily U member 1 IPI00120028 2 0 Signaling
Probable G-protein coupled receptor 115 IPI00134990 2 0 Signaling
Protein
IPI00346330
(+2)
2 0 Unknown
Protein FAM98B IPI00465946 2 0 Teo Unknown
Ras-related protein Rab-1B IPI00133706 2 0 Intracellular Trafficking
Retinitis pigmentosa GTPase regulator (Fragment) IPI00113503 2 0 Signaling
RIKEN cDNA 1700009N14 gene IPI00127109 2 0 Unknown
RIKEN cDNA 1700013E18, isoform CRA_b IPI00944038 2 0 Unknown
RNA polymerase II-associated factor 1 homolog IPI00331654 2 0 RNA Processing
SEC23-interacting protein
IPI00116752
(+1)
2 0 Teo, Nandi Intracellular Trafficking
Sec31a protein
IPI00807797
(+2)
2 0 Clark, Teo Intracellular Trafficking
Semaphorin-4A
IPI00122316
(+2)
2 0 Signaling
similar to Ac2-210 isoform 2 IPI00971257 2 0 Unknown
similar to lipoprotein receptor-related protein IPI00754224 2 0 Unknown
sodium leak channel non-selective protein IPI00742341 2 0 Intracellular Trafficking
Sodium-coupled neutral amino acid transporter 4 IPI00320698 2 0 Intracellular Trafficking
Solute carrier organic anion transporter family member 1A6 IPI00114950 2 0 Intracellular Trafficking
Synaptonemal complex protein 2 IPI00228891 2 0 Signaling
Syndecan-4 IPI00136382 2 0 Signaling
TATA-box-binding protein
IPI00776010
(+1)
2 0 Gene Regulation & Transcription
transcription activator BRG1 isoform 1
IPI00460668
(+2)
2 0 Gene Regulation & Transcription
415
Identified Proteins (232, 200 new)
Accession
Number
GlcNAlk DMSO Citation Function
Transcriptional activator protein Pur-beta IPI00128867 2 0 Clark Gene Regulation & Transcription
Transmembrane protein 200A IPI00405747 2 0 Intracellular Trafficking
Uncharacterized protein
IPI00663331
(+1)
2 0 Unknown
Uncharacterized protein IPI00762823 2 0 Unknown
Uncharacterized protein IPI00378254 2 0 Unknown
Uncharacterized protein IPI00874578 2 0 Unknown
Uncharacterized protein C1orf198 homolog IPI00225267 2 0 Unknown
Uridine 5'-monophosphate synthase IPI00121552 2 0 Metabolism
Vacuolar protein sorting 13D
IPI00649141
(+1)
2 0 Intracellular Trafficking
Vasodilator-stimulated phosphoprotein IPI00624876 2 0 Structural
Vomeronasal 1 receptor, H5 IPI00153504 2 0 Unknown
Vomeronasal receptor V1RH7
IPI00153502
(+1)
2 0 Unknown
Zinc finger CCCH domain-containing protein 11A IPI00421162 2 0 Gene Regulation & Transcription
416
Table 2-2 References
Boyce, M., Carrico, I.S., Ganguli, A.S., Yu, S.-H., Hangauer, M.J., Hubbard, S.C., Kohler, J.J., and Bertozzi, C.R. (2011). Meta-
bolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine sal-
vage pathway. Proc. Natl. Acad. Sci. U.S.a.
Cieniewski-Bernard, C., Bastide, B., Lefebvre, T., Lemoine, J., Mounier, Y., and Michalski, J.-C. (2004). Identification of O-
linked N-acetylglucosamine proteins in rat skeletal muscle using two-dimensional gel electrophoresis and mass spectrometry.
Mol Cell Proteomics 3, 577–585.
Clark, P.M., Dweck, J.F., Mason, D.E., Hart, C.R., Buck, S.B., Peters, E.C., Agnew, B.J., and Hsieh-Wilson, L.C. (2008). Direct
in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. J. Am. Chem. Soc. 130, 11576–11577.
Dehennaut, V., Slomianny, M.-C., Page, A., Vercoutter-Edouart, A.-S., Jessus, C., Michalski, J.-C., Vilain, J.-P., Bodart, J.-F.,
and Lefebvre, T. (2008). Identification of Structural and Functional O-Linked N-Acetylglucosamine-bearing Proteins in
Xenopus laevis Oocyte. Molecular & Cellular Proteomics 7, 2229–2245.
Gurcel, C., Vercoutter-Edouart, A., Fonbonne, C., Mortuaire, M., Salvador, A., Michalski, J., and Lemoine, J. (2008). Identifi-
cation of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Analytical and Bioanalytical Chemistry 390,
2089–2097.
Hanover, J.A., Cohen, C.K., Willingham, M.C., and Park, M.K. (1987). O-linked N-acetylglucosamine is attached to proteins of
the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. J. Biol. Chem. 262, 9887–9894.
Khidekel, N., Ficarro, S.B., Clark, P.M., Bryan, M.C., Swaney, D.L., Rexach, J.E., Sun, Y.E., Coon, J.J., Peters, E.C., and Hsieh-
Wilson, L.C. (2007). Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem
Biol 3, 339–348.
Khidekel, N., Ficarro, S.B., Peters, E.C., and Hsieh-Wilson, L.C. (2004). Exploring the O-GlcNAc proteome: direct identifica-
tion of O-GlcNAc-modified proteins from the brain. Proc Natl Acad Sci USA 101, 13132–13137.
Nandi, A., Sprung, R., Barma, D.K., Zhao, Y., Kim, S.C., Falck, J.R., and Zhao, Y. (2006). Global identification of O-GlcNAc-
modified proteins. Anal Chem 78, 452–458.
Sprung, R., Nandi, A., Chen, Y., Kim, S., Barma, D., Falck, J., and Zhao, Y. (2005). Tagging-via-substrate strategy for probing
O-GlcNAc modified proteins. J. Proteome Res. 4, 950–957.
417
Teo, C.F., Ingale, S., Wolfert, M.A., Elsayed, G.A., Nöt, L.G., Chatham, J.C., Wells, L., and Boons, G.-J. (2010). Glycopeptide-
specific monoclonal antibodies suggest new roles for O-GlcNAc. Nat Chem Biol 6, 338–343.
Vosseller, K., Trinidad, J.C., Chalkley, R.J., Specht, C.G., Thalhammer, A., Lynn, A.J., Snedecor, J.O., Guan, S.,
Medzihradszky, K.F., Maltby, D.A., et al. (2006). O-linked N-acetylglucosamine proteomics of postsynaptic density prepara-
tions using lectin weak affinity chromatography and mass spectrometry. Mol Cell Proteomics 5, 923–934.
Wang, Z., Pandey, A., and Hart, G.W. (2007). Dynamic interplay between O-linked N-acetylglucosaminylation and glycogen
synthase kinase-3-dependent phosphorylation. Mol Cell Proteomics 6, 1365–1379.
Wells, L., Vosseller, K., Cole, R.N., Cronshaw, J.M., Matunis, M.J., and Hart, G.W. (2002). Mapping sites of O-GlcNAc modi-
fication using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804.
418
Table 5-1. Proteins identified using AspAlk enrichment. HCT-15 cells were treated in triplicate with either AspAlk (1
mM, +) or DMSO (-) for 2 hours. At this time the cell lysates were subjected to CuAAC with azido-biotin, followed by enrich-
ment with streptavidin beads and on-bead trypsinolysis. Labeled proteins were selected as those that were represented by at
least 1 unique-peptide in each AspAlk treated sample, a total of at least 3 spectral-counts from the same three samples, at least
a average of 3-times more spectral counts in the AspAlk treated samples compared to DMSO, and a p-value of no greater than
0.05 (t-test). Previously identified aspirin-acetylated proteins are highlighted in blue and the histones confirmed by in-gel
fluorescence are highlighted in green. Proteins previously identified by anti-lysine antibodies in both aspirin-treated and un-
treated cells are highlighted in red.
AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment 1
Ex-
peri-
ment 1
Ex-
peri-
ment 2
Ex-
peri-
ment 2
Ex-
peri-
ment 3
Ex-
peri-
ment 3 Average Average
No.
Ac-
ces-
sion Gene Description - + - + - + - +
Fold
En-
rich-
ment
t-test
(p-
value)
MW
[kDa]
Previous
Identifica-
tion
1 P80188 NGAL Neutrophil gelatinase-associated lipocalin OS=Homo sapiens GN=LCN2 PE=1 SV=2 - [NGAL_HUMAN] 0 11 0 11 0 14 0.00 12.00 N/A 0.00028 22.6
2 E7EQR4E7EQR4 Ezrin OS=Homo sapiens GN=EZR PE=4 SV=2 - [E7EQR4_HUMAN] 0 12 0 8 0 5 0.00 8.33 N/A 0.015 65.5 (2)
3 P49411 EFTU Elongation factor Tu, mitochondrial OS=Homo sapiens GN=TUFM PE=1 SV=2 - [EFTU_HUMAN] 0 9 0 10 0 4 0.00 7.67 N/A 0.014 49.5
4 P06576 ATPB ATP synthase subunit beta, mitochondrial OS=Homo sapiens GN=ATP5B PE=1 SV=3 - [ATPB_HUMAN] 0 5 0 10 0 8 0.00 7.67 N/A 0.0062 56.5
5 P18669 PGAM1Phosphoglycerate mutase 1 OS=Homo sapiens GN=PGAM1 PE=1 SV=2 - [PGAM1_HUMAN] 0 8 0 5 0 9 0.00 7.33 N/A 0.0037 28.8 (2)
6 P40926 MDHM Malate dehydrogenase, mitochondrial OS=Homo sapiens GN=MDH2 PE=1 SV=3 - [MDHM_HUMAN] 0 6 0 8 0 8 0.00 7.33 N/A 0.00039 35.5
7 P15559 NQO1 Isoform 2 of NAD(P)H dehydrogenase [quinone] 1 OS=Homo sapiens GN=NQO1 - [NQO1_HUMAN] 0 8 0 8 0 6 0.00 7.33 N/A 0.00039 27.3
8 P14174 MIF Macrophage migration inhibitory factor OS=Homo sapiens GN=MIF PE=1 SV=4 - [MIF_HUMAN] 0 10 0 6 0 5 0.00 7.00 N/A 0.010 12.5 (2)
9 P21796 VDAC1Voltage-dependent anion-selective channel protein 1 OS=Homo sapiens GN=VDAC1 PE=1 SV=2 - [VDAC1_HUMAN] 0 7 0 8 0 5 0.00 6.67 N/A 0.00 30.8
10 P25205 MCM3 DNA replication licensing factor MCM3 OS=Homo sapiens GN=MCM3 PE=1 SV=3 - [MCM3_HUMAN] 0 9 0 8 0 3 0.00 6.67 N/A 0.023 90.9
11 Q01813 K6PP 6-phosphofructokinase type C OS=Homo sapiens GN=PFKP PE=1 SV=2 - [K6PP_HUMAN] 0 9 0 6 0 4 0.00 6.33 N/A 0.012 85.5
12 I3L397 I3L397 Eukaryotic translation initiation factor 5A-1 (Fragment) OS=Homo sapiens GN=EIF5A PE=4 SV=1 - [I3L397_HUMAN] 0 6 0 6 0 5 0.00 5.67 N/A 0.000070 16.1
13 P31948 STIP1 Stress-induced-phosphoprotein 1 OS=Homo sapiens GN=STIP1 PE=1 SV=1 - [STIP1_HUMAN] 0 4 0 6 0 6 0.00 5.33 N/A 0.0013 62.6
14 P04792 HSPB1 Heat shock protein beta-1 OS=Homo sapiens GN=HSPB1 PE=1 SV=2 - [HSPB1_HUMAN] 0 5 0 4 0 6 0.00 5.00 N/A 0.0010 22.8
15 P31946 1433B Isoform Short of 14 protein beta/alpha OS=Homo sapiens GN=YWHAB - [1433B_HUMAN] 0 6 0 3 0 5 0.00 4.67 N/A 0.0061 27.8
16 B4DFL2 B4DFL2Isocitrate dehydrogenase [NADP] OS=Homo sapiens GN=IDH2 PE=2 SV=1 - [B4DFL2_HUMAN] 0 5 0 7 0 2 0.00 4.67 N/A 0.033 45.2
17 E7EU96 E7EU96Casein kinase II subunit alpha OS=Homo sapiens GN=CSNK2A1 PE=4 SV=1 - [E7EU96_HUMAN] 0 7 0 5 0 2 0.00 4.67 N/A 0.033 45.3
18 P24752 THIL Acetyl-CoA acetyltransferase, mitochondrial OS=Homo sapiens GN=ACAT1 PE=1 SV=1 - [THIL_HUMAN] 0 4 0 5 0 5 0.00 4.67 N/A 0.00015 45.2
19 P32322 P5CR1 Pyrroline-5-carboxylate reductase 1, mitochondrial OS=Homo sapiens GN=PYCR1 PE=1 SV=2 - [P5CR1_HUMAN] 0 3 0 5 0 5 0.00 4.33 N/A 0.0029 33.3
20 Q9H2U2 IPYR2 Isoform 3 of Inorganic pyrophosphatase 2, mitochondrial OS=Homo sapiens GN=PPA2 - [IPYR2_HUMAN] 0 4 0 7 0 2 0.00 4.33 N/A 0.041 34.6
21 P43487 RANG Ran-specific GTPase-activating protein OS=Homo sapiens GN=RANBP1 PE=1 SV=1 - [RANG_HUMAN] 0 5 0 4 0 3 0.00 4.00 N/A 0.0023 23.3
22 F5GYZ3 NONO Non-POU domain-containing octamer-binding protein OS=Homo sapiens GN=NONO PE=4 SV=1 - [F5GYZ3_HUMAN] 0 4 0 5 0 3 0.00 4.00 N/A 0.0023 43.8
23 P63244 GBLP Guanine nucleotide-binding protein subunit beta -like 1 OS=Homo sapiens GN=GNB2L1 PE=1 SV=3 - [GBLP_HUMAN] 0 5 0 3 0 4 0.00 4.00 N/A 0.0023 35.1
24 P61981 1433G 14 protein gamma OS=Homo sapiens GN=YWHAG PE=1 SV=2 - [1433G_HUMAN] 0 4 0 3 0 5 0.00 4.00 N/A 0.0023 28.3
419
AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment 1
Ex-
peri-
ment 1
Ex-
peri-
ment 2
Ex-
peri-
ment 2
Ex-
peri-
ment 3
Ex-
peri-
ment 3 Average Average
No.
Ac-
ces-
sion Gene Description - + - + - + - +
Fold
En-
rich-
ment
t-test
(p-
value)
MW
[kDa]
Previous
Identifica-
tion
25 Q7L1Q6 BZW1 Isoform 2 of Basic leucine zipper and W2 domain-containing protein 1 OS=Homo sapiens GN=BZW1 - [BZW1_HUMAN] 0 3 0 4 0 4 0.00 3.67 N/A 0.00039 40.5
26 Q7KZF4SND1 Staphylococcal nuclease domain-containing protein 1 OS=Homo sapiens GN=SND1 PE=1 SV=1 - [SND1_HUMAN] 0 3 0 5 0 2 0.00 3.33 N/A 0.019 101.9
27 Q9NZL9 MAT2B Isoform 4 of Methionine adenosyltransferase 2 subunit beta OS=Homo sapiens GN=MAT2B - [MAT2B_HUMAN] 0 4 0 4 0 2 0.00 3.33 N/A 0.0075 34.6
28 Q5VU59Q5VU59 Tropomyosin 3 OS=Homo sapiens GN=TPM3 PE=2 SV=1 - [Q5VU59_HUMAN] 0 3 0 3 0 3 0.00 3.00 N/A 0.0 27.2
29 P49458 SRP09 Signal recognition particle 9 kDa protein OS=Homo sapiens GN=SRP9 PE=1 SV=2 - [SRP09_HUMAN] 0 3 0 3 0 3 0.00 3.00 N/A 0.0 10.1 (2)
30 P50454 SERPH Serpin H1 OS=Homo sapiens GN=SERPINH1 PE=1 SV=2 - [SERPH_HUMAN] 0 2 0 5 0 2 0.00 3.00 N/A 0.040 46.4
31 Q13907 IDI1 Isopentenyl-diphosphate Delta-isomerase 1 OS=Homo sapiens GN=IDI1 PE=1 SV=2 - [IDI1_HUMAN] 0 4 0 4 0 1 0.00 3.00 N/A 0.040 26.3
32 P62258 1433E Isoform SV of 14 protein epsilon OS=Homo sapiens GN=YWHAE - [1433E_HUMAN] 0 3 0 3 0 3 0.00 3.00 N/A 0.0 26.5
33 B4E2W0B4E2W0 3-ketoacyl-CoA thiolase OS=Homo sapiens GN=HADHB PE=2 SV=1 - [B4E2W0_HUMAN] 0 3 0 3 0 3 0.00 3.00 N/A 0.0 48.8
34 P36957 ODO2 Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial OS=Homo sapiens GN=DLST PE=1 SV=4 - [ODO2_HUMAN] 0 2 0 4 0 2 0.00 2.67 N/A 0.016 48.7
35 E9PG15 E9PG15 14 protein theta (Fragment) OS=Homo sapiens GN=YWHAQ PE=4 SV=1 - [E9PG15_HUMAN] 0 3 0 3 0 2 0.00 2.67 N/A 0.0013 17.0
36 P37802 TAGL2 Transgelin OS=Homo sapiens GN=TAGLN2 PE=1 SV=3 - [TAGL2_HUMAN] 0 3 0 2 0 2 0.00 2.33 N/A 0.0022 22.4
37 H7BZJ3 H7BZJ3Thioredoxin (Fragment) OS=Homo sapiens GN=PDIA3 PE=3 SV=1 - [H7BZJ3_HUMAN] 0 2 0 2 0 3 0.00 2.33 N/A 0.0022 13.5
38 G3V3U4G3V3U4 Proteasome subunit alpha type OS=Homo sapiens GN=PSMA6 PE=3 SV=1 - [G3V3U4_HUMAN] 0 2 0 3 0 2 0.00 2.33 N/A 0.0022 11.6
39 E9PBF6 E9PBF6Lamin-B1 OS=Homo sapiens GN=LMNB1 PE=3 SV=1 - [E9PBF6_HUMAN] 0 3 0 2 0 2 0.00 2.33 N/A 0.0022 44.6
40 Q08257 QOR Isoform 2 of Quinone oxidoreductase OS=Homo sapiens GN=CRYZ - [QOR_HUMAN] 0 3 0 2 0 2 0.00 2.33 N/A 0.0022 20.4
41 Q5SRN7Q5SRN7 HLA class I histocompatibility antigen, A-68 alpha chain OS=Homo sapiens GN=HLA-A PE=3 SV=2 - [Q5SRN7_HUMAN] 0 3 0 3 0 1 0.00 2.33 N/A 0.025 34.2
42 P26583 HMGB2 High mobility group protein B2 OS=Homo sapiens GN=HMGB2 PE=1 SV=2 - [HMGB2_HUMAN] 0 3 0 1 0 3 0.00 2.33 N/A 0.025 24.0
43 D6R9P3 D6R9P3 Heterogeneous nuclear ribonucleoprotein A/B OS=Homo sapiens GN=HNRNPAB PE=4 SV=1 - [D6R9P3_HUMAN] 0 3 0 3 0 1 0.00 2.33 N/A 0.025 30.3
44 B4E132 B4E132Uncharacterized protein OS=Homo sapiens GN=DDX3Y PE=2 SV=1 - [B4E132_HUMAN] 0 2 0 2 0 2 0.00 2.00 N/A 0.0 44.8
45 P30086 PEBP1 Phosphatidylethanolamine-binding protein 1 OS=Homo sapiens GN=PEBP1 PE=1 SV=3 - [PEBP1_HUMAN] 0 3 0 2 0 1 0.00 2.00 N/A 0.026 21.0
46 Q9NTK5 OLA1 Obg-like ATPase 1 OS=Homo sapiens GN=OLA1 PE=1 SV=2 - [OLA1_HUMAN] 0 3 0 1 0 2 0.00 2.00 N/A 0.026 44.7
47 E9PIE4 E9PIE4 Mitochondrial carrier homolog 2 OS=Homo sapiens GN=MTCH2 PE=3 SV=1 - [E9PIE4_HUMAN] 0 3 0 2 0 1 0.00 2.00 N/A 0.026 17.6
48 P33176 KINH Kinesin-1 heavy chain OS=Homo sapiens GN=KIF5B PE=1 SV=1 - [KINH_HUMAN] 0 2 0 2 0 2 0.00 2.00 N/A 0.0 109.6
49 E9PMW7 E9PMW7 Elongation factor 1-delta OS=Homo sapiens GN=EEF1D PE=4 SV=1 - [E9PMW7_HUMAN] 0 2 0 2 0 2 0.00 2.00 N/A 0.0 14.2
50 B7Z6B8 B7Z6B82,4-dienoyl-CoA reductase, mitochondrial OS=Homo sapiens GN=DECR1 PE=2 SV=1 - [B7Z6B8_HUMAN] 0 2 0 2 0 2 0.00 2.00 N/A 0.0 35.0
51 Q9Y277VDAC3Voltage-dependent anion-selective channel protein 3 OS=Homo sapiens GN=VDAC3 PE=1 SV=1 - [VDAC3_HUMAN] 0 2 0 2 0 1 0.00 1.67 N/A 0.0075 30.6
52 Q13404 UB2V1 Ubiquitin-conjugating enzyme E2 variant 1 OS=Homo sapiens GN=UBE2V1 PE=1 SV=2 - [UB2V1_HUMAN] 0 2 0 2 0 1 0.00 1.67 N/A 0.0075 16.5
53 P00491 PNPH Purine nucleoside phosphorylase OS=Homo sapiens GN=PNP PE=1 SV=2 - [PNPH_HUMAN] 0 2 0 2 0 1 0.00 1.67 N/A 0.0075 32.1
54 K7EN27K7EN27 Protein DJ-1 (Fragment) OS=Homo sapiens GN=PARK7 PE=4 SV=1 - [K7EN27_HUMAN] 0 2 0 1 0 2 0.00 1.67 N/A 0.0075 17.0
55 P17931 LEG3 Galectin OS=Homo sapiens GN=LGALS3 PE=1 SV=5 - [LEG3_HUMAN] 0 1 0 2 0 2 0.00 1.67 N/A 0.0075 26.1
56 Q01469 FABP5 Fatty acid-binding protein, epidermal OS=Homo sapiens GN=FABP5 PE=1 SV=3 - [FABP5_HUMAN] 0 2 0 2 0 1 0.00 1.67 N/A 0.0075 15.2
57 P67936 TPM4 Tropomyosin alpha chain OS=Homo sapiens GN=TPM4 PE=1 SV=3 - [TPM4_HUMAN] 0 2 0 1 0 1 0.00 1.33 N/A 0.016 28.5
420
AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment 1
Ex-
peri-
ment 1
Ex-
peri-
ment 2
Ex-
peri-
ment 2
Ex-
peri-
ment 3
Ex-
peri-
ment 3 Average Average
No.
Ac-
ces-
sion Gene Description - + - + - + - +
Fold
En-
rich-
ment
t-test
(p-
value)
MW
[kDa]
Previous
Identifica-
tion
58 P25787 PSA2 Proteasome subunit alpha type OS=Homo sapiens GN=PSMA2 PE=1 SV=2 - [PSA2_HUMAN] 0 2 0 1 0 1 0.00 1.33 N/A 0.016 25.9
59 P30533 AMRP Alpha -macroglobulin receptor-associated protein OS=Homo sapiens GN=LRPAP1 PE=1 SV=1 - [AMRP_HUMAN] 0 2 0 1 0 1 0.00 1.33 N/A 0.016 41.4
60 D6RAS3D6RAS3 tRNA (cytosine(34)-C(5))-methyltransferase OS=Homo sapiens GN=NSUN2 PE=4 SV=1 - [D6RAS3_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 23.4
61 Q14247 SRC8 Src substrate cortactin OS=Homo sapiens GN=CTTN PE=1 SV=2 - [SRC8_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 61.5
62 H0YEL5 H0YEL5Peptidyl-prolyl cis-trans isomerase H (Fragment) OS=Homo sapiens GN=PPIH PE=3 SV=1 - [H0YEL5_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 10.3
63 Q00688 FKBP3 Peptidyl-prolyl cis-trans isomerase FKBP3 OS=Homo sapiens GN=FKBP3 PE=1 SV=1 - [FKBP3_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 25.2
64 C9JVB6 C9JVB6Mitochondrial ribonuclease P protein 1 (Fragment) OS=Homo sapiens GN=TRMT10C PE=4 SV=1 - [C9JVB6_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 36.8
65 P05455 SSB Lupus La protein OS=Homo sapiens GN=SSB PE=4 SV=1 - [LA_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 41.8
66 E9PCI9 FPPS Farnesyl pyrophosphate synthase OS=Homo sapiens GN=FDPS PE=3 SV=1 - [E9PCI9_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 40.5
67 P04080 CYTB Cystatin-B OS=Homo sapiens GN=CSTB PE=1 SV=2 - [CYTB_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 11.1
68 E9PEU4 COPD Coatomer subunit delta OS=Homo sapiens GN=ARCN1 PE=4 SV=1 - [E9PEU4_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 47.2
69 Q9NX58 LYAR Cell growth-regulating nucleolar protein OS=Homo sapiens GN=LYAR PE=1 SV=2 - [LYAR_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 43.6
70 E9PP31 E9PP31Caprin-1 OS=Homo sapiens GN=CAPRIN1 PE=4 SV=1 - [E9PP31_HUMAN] 0 1 0 1 0 1 0.00 1.00 N/A 0.0 19.4
71 P38646 GRP75 Stress-70 protein, mitochondrial OS=Homo sapiens GN=HSPA9 PE=1 SV=2 - [GRP75_HUMAN] 1 16 0 11 0 6 0.33 11.00 33.00 0.021 73.6
72 G5E9B2CTT8 Chaperonin containing TCP1, subunit 8 (Theta), isoform CRA_a OS=Homo sapiens GN=CCT8 PE=3 SV=1 - [G5E9B2_HUMAN] 0 12 1 10 0 9 0.33 10.33 31.00 0.00045 59.4
73 G5EA52G5EA52 Protein disulfide isomerase family A, member 3, isoform CRA_b OS=Homo sapiens GN=PDIA3 PE=3 SV=1 - [G5EA52_HUMAN] 0 9 1 11 0 10 0.33 10.00 30.00 0.00013 54.9
74 P10809 CH60 60 kDa heat shock protein, mitochondrial OS=Homo sapiens GN=HSPD1 PE=1 SV=2 - [CH60_HUMAN] 0 18 2 21 0 17 0.67 18.67 28.00 0.000 61.0 (2)
75 P02545 LMNA Isoform C of Prelamin-A/C OS=Homo sapiens GN=LMNA - [LMNA_HUMAN] 0 11 0 9 1 8 0.33 9.33 28.00 0.00067 65.1
76 E7ERW2E7ERW2 Aspartate aminotransferase OS=Homo sapiens GN=GOT2 PE=3 SV=1 - [E7ERW2_HUMAN] 0 7 1 8 0 6 0.33 7.00 21.00 0.00056 43.0
77 P17987 TCPA T-complex protein 1 subunit alpha OS=Homo sapiens GN=TCP1 PE=1 SV=1 - [TCPA_HUMAN] 0 9 1 8 0 3 0.33 6.67 20.00 0.028 60.3
78 P25705 ATPA ATP synthase subunit alpha, mitochondrial OS=Homo sapiens GN=ATP5A1 PE=1 SV=1 - [ATPA_HUMAN] 0 9 2 11 0 10 0.67 10.00 15.00 0.00045 59.7
79 P22314 UBA1 Ubiquitin-like modifier-activating enzyme 1 OS=Homo sapiens GN=UBA1 PE=1 SV=3 - [UBA1_HUMAN] 1 7 0 6 0 2 0.33 5.00 15.00 0.041 117.8
80 F5GY37 F5GY37 Prohibitin OS=Homo sapiens GN=PHB2 PE=4 SV=1 - [F5GY37_HUMAN] 0 3 1 6 0 4 0.33 4.33 13.00 0.013 29.7
81 P51659 DHB4 Peroxisomal multifunctional enzyme type 2 OS=Homo sapiens GN=HSD17B4 PE=1 SV=3 - [DHB4_HUMAN] 0 3 0 5 1 5 0.33 4.33 13.00 0.0058 79.6
82 P07737 PROF1 Profilin-1 OS=Homo sapiens GN=PFN1 PE=1 SV=2 - [PROF1_HUMAN] 0 5 1 4 0 3 0.33 4.00 12.00 0.0053 15.0 (2)
83 P11021 GRP78 78 kDa glucose-regulated protein OS=Homo sapiens GN=HSPA5 PE=1 SV=2 - [GRP78_HUMAN] 0 15 3 10 0 8 1.00 11.00 11.00 0.012 72.3
84 Q07021 C1QBPComplement component 1 Q subcomponent-binding protein, mitochondrial OS=Homo sapiens GN=C1QBP PE=1 SV=1 - [C1QBP_HUMAN] 0 3 1 5 0 3 0.33 3.67 11.00 0.011 31.3
85 B7Z254 B7Z254Protein disulfide-isomerase A6 OS=Homo sapiens GN=PDIA6 PE=2 SV=1 - [B7Z254_HUMAN] 0 7 2 7 0 5 0.67 6.33 9.50 0.0039 47.8
86 P62937 PPIA Peptidyl-prolyl cis-trans isomerase A OS=Homo sapiens GN=PPIA PE=1 SV=2 - [PPIA_HUMAN] 0 7 2 5 0 7 0.67 6.33 9.50 0.0039 18.0
87 P23284 PPIB Peptidyl-prolyl cis-trans isomerase B OS=Homo sapiens GN=PPIB PE=1 SV=2 - [PPIB_HUMAN] 0 12 5 16 0 13 1.67 13.67 8.20 0.0043 23.7
88 P14866 HNRPL Heterogeneous nuclear ribonucleoprotein L OS=Homo sapiens GN=HNRNPL PE=1 SV=2 - [HNRPL_HUMAN] 1 5 1 7 0 4 0.67 5.33 8.00 0.008 64.1
89 Q9UQ80 PA2G4 Proliferation-associated protein 2G4 OS=Homo sapiens GN=PA2G4 PE=1 SV=3 - [PA2G4_HUMAN] 0 3 1 3 0 2 0.33 2.67 8.00 0.008 43.8
90 P06493 CDK1 Cyclin-dependent kinase 1 OS=Homo sapiens GN=CDK1 PE=1 SV=3 - [CDK1_HUMAN] 0 4 1 2 0 2 0.33 2.67 8.00 0.04 34.1
421
AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk AspAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment 1
Ex-
peri-
ment 1
Ex-
peri-
ment 2
Ex-
peri-
ment 2
Ex-
peri-
ment 3
Ex-
peri-
ment 3 Average Average
No.
Ac-
ces-
sion Gene Description - + - + - + - +
Fold
En-
rich-
ment
t-test
(p-
value)
MW
[kDa]
Previous
Identifica-
tion
91 P52272 HNRPMIsoform 2 of Heterogeneous nuclear ribonucleoprotein M OS=Homo sapiens GN=HNRNPM - [HNRPM_HUMAN] 0 7 3 7 0 9 1.00 7.67 7.67 0.0052 73.6
92 P42704 LPPRC Leucine-rich PPR motif-containing protein, mitochondrial OS=Homo sapiens GN=LRPPRC PE=1 SV=3 - [LPPRC_HUMAN] 0 10 3 8 0 4 1.00 7.33 7.33 0.035 157.8
93 B8ZZ51 B8ZZ51Malate dehydrogenase, cytoplasmic OS=Homo sapiens GN=MDH1 PE=4 SV=1 - [B8ZZ51_HUMAN] 1 4 1 5 0 5 0.67 4.67 7.00 0.0011 18.7
94 P40939 ECHA Trifunctional enzyme subunit alpha, mitochondrial OS=Homo sapiens GN=HADHA PE=1 SV=2 - [ECHA_HUMAN] 1 8 3 11 0 7 1.33 8.67 6.50 0.0079 82.9
95 P11142 HSP7C Heat shock cognate 71 kDa protein OS=Homo sapiens GN=HSPA8 PE=1 SV=1 - [HSP7C_HUMAN] 3 20 7 22 0 20 3.33 20.67 6.20 0.0013 70.9
96 Q5T6W5 Q5T6W5 Heterogeneous nuclear ribonucleoprotein K OS=Homo sapiens GN=HNRNPK PE=2 SV=1 - [Q5T6W5_HUMAN] 0 11 3 7 1 6 1.33 8.00 6.00 0.019 47.5
97 F5H897 F5H897Heat shock protein 75 kDa, mitochondrial OS=Homo sapiens GN=TRAP1 PE=3 SV=1 - [F5H897_HUMAN] 1 4 1 5 0 3 0.67 4.00 6.00 0.0075 74.2
98 F8VZY9 F8VZY9Keratin, type I cytoskeletal 18 OS=Homo sapiens GN=KRT18 PE=3 SV=1 - [F8VZY9_HUMAN] 1 9 1 9 2 5 1.33 7.67 5.75 0.0100 43.7
99 P05787 K2C8 Keratin, type II cytoskeletal 8 OS=Homo sapiens GN=KRT8 PE=1 SV=7 - [K2C8_HUMAN] 3 26 2 24 7 15 4.00 21.67 5.42 0.0089 53.7
100 Q07065 CKAP4 Cytoskeleton-associated protein 4 OS=Homo sapiens GN=CKAP4 PE=1 SV=2 - [CKAP4_HUMAN] 0 7 4 9 0 5 1.33 7.00 5.25 0.033 66.0
101 E9PH38 E9PH38Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform OS=Homo sapiens GN=PPP2R1A PE=4 SV=1 - [E9PH38_HUMAN] 0 2 1 1 0 2 0.33 1.67 5.00 0.047 56.8
102 P51148 RAB5C Ras-related protein Rab-5C OS=Homo sapiens GN=RAB5C PE=1 SV=2 - [RAB5C_HUMAN] 1 1 0 2 0 2 0.33 1.67 5.00 0.047 23.5
103 P00558 PGK1 Phosphoglycerate kinase 1 OS=Homo sapiens GN=PGK1 PE=1 SV=3 - [PGK1_HUMAN] 0 2 1 1 0 2 0.33 1.67 5.00 0.047 44.6
104 Q86UP2 KTN1 Isoform 2 of Kinectin OS=Homo sapiens GN=KTN1 - [KTN1_HUMAN] 0 2 1 1 0 2 0.33 1.67 5.00 0.047 149.5
105 P07814 SYEP Bifunctional glutamate/proline--tRNA ligase OS=Homo sapiens GN=EPRS PE=1 SV=5 - [SYEP_HUMAN] 0 2 1 2 0 1 0.33 1.67 5.00 0.047 170.5
106 P60174-1 TPIS Isoform 2 of Triosephosphate isomerase OS=Homo sapiens GN=TPI1 - [TPIS_HUMAN] 0 9 4 7 1 9 1.67 8.33 5.00 0.0083 26.7
107 P09211 GSTP1 Glutathione S-transferase P OS=Homo sapiens GN=GSTP1 PE=1 SV=2 - [GSTP1_HUMAN] 1 8 4 6 0 6 1.67 6.67 4.00 0.022 23.3
108 P06899 H2B1J Histone H2B type 1-J OS=Homo sapiens GN=HIST1H2BJ PE=1 SV=3 - [H2B1J_HUMAN] 1 5 2 5 1 6 1.33 5.33 4.00 0.0011 13.9
109 K7EMV3K7EMV3 Histone H3.3 OS=Homo sapiens GN=H3F3B PE=4 SV=1 - [K7EMV3_HUMAN] 1 3 0 5 2 4 1.00 4.00 4.00 0.021 10.3
110 P13667 PDIA4 Protein disulfide-isomerase A4 OS=Homo sapiens GN=PDIA4 PE=1 SV=2 - [PDIA4_HUMAN] 1 12 5 9 1 6 2.33 9.00 3.86 0.038 72.9
111 P04075 ALDOAFructose-bisphosphate aldolase A OS=Homo sapiens GN=ALDOA PE=1 SV=2 - [ALDOA_HUMAN] 3 15 7 11 0 11 3.33 12.33 3.70 0.021 39.4
112 Q8NC51 PAIRB Isoform 4 of Plasminogen activator inhibitor 1 RNA-binding protein OS=Homo sapiens GN=SERBP1 - [PAIRB_HUMAN] 1 4 2 3 0 3 1.00 3.33 3.33 0.025 42.4
113 Q8NE71 ABCF1 Isoform 2 of ATP-binding cassette sub-family F member 1 OS=Homo sapiens GN=ABCF1 - [ABCF1_HUMAN] 1 4 2 3 0 3 1.00 3.33 3.33 0.025 91.6
114 P14618 KPYM Pyruvate kinase isozymes M1/M2 OS=Homo sapiens GN=PKM PE=1 SV=4 - [KPYM_HUMAN] 4 22 13 24 2 15 6.33 20.33 3.21 0.032 57.9 (2)
115 P60842 IF4A1 Eukaryotic initiation factor 4A-I OS=Homo sapiens GN=EIF4A1 PE=1 SV=1 - [IF4A1_HUMAN] 1 7 3 7 2 5 2.00 6.33 3.17 0.0080 46.1
116 P06733 ENOA Alpha-enolase OS=Homo sapiens GN=ENO1 PE=1 SV=2 - [ENOA_HUMAN] 2 20 12 14 2 14 5.33 16.00 3.00 0.05 47.1 (2)
117 P06748 NPM Isoform 2 of Nucleophosmin OS=Homo sapiens GN=NPM1 - [NPM_HUMAN] 3 15 8 14 2 10 4.33 13.00 3.00 0.023 29.4
118 G3V576 G3V576 Heterogeneous nuclear ribonucleoproteins C1/C2 OS=Homo sapiens GN=HNRNPC PE=4 SV=1 - [G3V576_HUMAN] 1 7 2 4 2 4 1.67 5.00 3.00 0.034 25.2
119 P22234 PUR6 Multifunctional protein ADE2 OS=Homo sapiens GN=PAICS PE=1 SV=3 - [PUR6_HUMAN] 1 4 2 4 1 4 1.33 4.00 3.00 0.0013 47.0
120 A8MXH2 A8MXH2 Nucleosome assembly protein 1-like 4 (Fragment) OS=Homo sapiens GN=NAP1L4 PE=3 SV=2 - [A8MXH2_HUMAN] 1 2 1 2 0 2 0.67 2.00 3.00 0.016 18.1
422
Table 5-1 References
1 Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975). Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975). Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975). Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975). Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975). Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975). Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975). Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975). Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975). Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975). Roth, G. J.; Stanford N.; Majerus P .W.; Acetylation of prostaglandin synthase by aspirin. Proc Natl Acad Sci USA 72, 3073-3076 (1975).
2 Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011). Marimuthu, S.; Chivukula, R.S.V.; Alfonso, L.F.; Maridani, M.; Hagen, F.K.; Bhat, G.J.; Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets. Int J Oncol 39, 1273-1283 (2011).
3 Hawkins, D., Pinckard, R. N. & Farr, R. S. Acetylation of human serum albumin by acetylsalicylic acid. Science 160, 780–781 (1968). Hawkins, D., Pinckard, R. N. & Farr, R. S. Acetylation of human serum albumin by acetylsalicylic acid. Science 160, 780–781 (1968). Hawkins, D., Pinckard, R. N. & Farr, R. S. Acetylation of human serum albumin by acetylsalicylic acid. Science 160, 780–781 (1968). Hawkins, D., Pinckard, R. N. & Farr, R. S. Acetylation of human serum albumin by acetylsalicylic acid. Science 160, 780–781 (1968). Hawkins, D., Pinckard, R. N. & Farr, R. S. Acetylation of human serum albumin by acetylsalicylic acid. Science 160, 780–781 (1968). Hawkins, D., Pinckard, R. N. & Farr, R. S. Acetylation of human serum albumin by acetylsalicylic acid. Science 160, 780–781 (1968). Hawkins, D., Pinckard, R. N. & Farr, R. S. Acetylation of human serum albumin by acetylsalicylic acid. Science 160, 780–781 (1968). Hawkins, D., Pinckard, R. N. & Farr, R. S. Acetylation of human serum albumin by acetylsalicylic acid. Science 160, 780–781 (1968). Hawkins, D., Pinckard, R. N. & Farr, R. S. Acetylation of human serum albumin by acetylsalicylic acid. Science 160, 780–781 (1968). Hawkins, D., Pinckard, R. N. & Farr, R. S. Acetylation of human serum albumin by acetylsalicylic acid. Science 160, 780–781 (1968).
4 Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968). Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968). Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968). Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968). Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968). Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968). Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968). Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968). Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968). Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968). Pinckard, R. N., Hawkins, D. & Farr, R. S. In vitro acetylation of plasma proteins, enzymes and DNA by aspirin. Nature 219, 68–69 (1968).
423
Table 6-1. Proteins identified using 6AzGlcNAc enrichment. NIH 3T3 were treated in triplicate with either
Ac36AzGlcNAc (200 µM, +) or Ac4GlcNAc (200 µM, -) for 16 hours. At this time the cell lysates were subjected to CuAAC with
alkyne-biotin, followed by enrichment with streptavidin beads and on-bead trypsinolysis. Labeled proteins were selected as
those that were represented by at least 1 unique-peptide in each Ac36AzGlcNAc treated sample, a total of at least 3 spectral-
counts from the same three samples, and at least a total of 3 times more spectral counts in the Ac36AzGlcNAc treated samples
compared to Ac4GlcNAc. Blue indicates proteins previously identified in O-GlcNAc proteomic studies, purple indicates pro-
teins identified in both O-GlcNAc and mucin O-linked proteomic studies and red indicates proteins identified in only O-linked
mucin proteomic studies. Novel proteins that were identified in this study are indicated in white.
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
P27546-2 MAP4 Isoform 2 of Microtubule-associated protein 4 0 31 0 37 0 35 0 103 N/A 4.107E-05 117 ✓ ✓ ✓ (9)(19)(23)(26)
P58871 TB182 182 kDa tankyrase-1-binding protein 0 23 0 24 0 25 0 72 N/A 2.002E-06 182 ✓ ✓ ✓ (1)(20)(23)(26)
Q3THK7 GUAA GMP synthase [glutamine-hydrolyzing] 0 19 0 19 0 21 0 59 N/A 7.862E-06 77 ✓ ✓ (26)
Q9QUR6 PPCE Prolyl endopeptidase 0 15 0 22 0 17 0 54 N/A 9.839E-04 81 ✓ ✓ (26) (17)
P40124 CAP1 Adenylyl cyclase-associated protein 1 0 18 0 15 0 20 0 53 N/A 2.626E-04 52 ✓ ✓ ✓ (9)(23)(26)
Q3UZ39 LRRF1 Leucine-rich repeat flightless-interacting protein 1 0 15 0 20 0 15 0 50 N/A 5.620E-04 79 ✓ ✓ (23)(26)
Q61584-5 FXR1 Isoform D of Fragile X mental retardation syndrome-related protein 1 0 17 0 17 0 13 0 47 N/A 3.001E-04 70 ✓ ✓ (9)
P30416 FKBP4 Peptidyl-prolyl cis-trans isomerase FKBP4 0 16 0 16 0 13 0 45 N/A 1.151E-04 52 ✓ ✓ (5)(9)(23)(26)
Q99PG2 OGFR Opioid growth factor receptor 0 15 0 12 0 17 0 44 N/A 5.419E-04 71 ✓ ✓ (9)(23)
Q00519 XDH Xanthine dehydrogenase/oxidase 0 17 0 16 0 11 0 44 N/A 1.387E-03 147 ✓ ✓ (26)
B1AU75 B1AU75 Nuclear autoantigenic sperm protein 0 17 0 14 0 13 0 44 N/A 2.588E-04 84 ✓ ✓ (26)
A2A6U3 A2A6U3 Septin 9 0 18 0 16 0 10 0 44 N/A 3.650E-03 64 ✓ ✓ (26)
Q9DCL9 PUR6 Multifunctional protein ADE2 0 16 0 17 0 10 0 43 N/A 2.797E-03 47 ✓ ✓ (18)(19)(23)(26)
Q8VCQ8 Q8VCQ8 Caldesmon 1 0 15 0 12 0 15 0 42 N/A 1.510E-04 60 ✓ ✓ ✓ (26)
Q9ERG0 LIMA1 LIM domain and actin-binding protein 1 0 16 0 13 0 12 0 41 N/A 3.411E-04 84 ✓ ✓ (9)(23)(26)
Q8BTI8-3 SRRM2 Isoform 3 of Serine/arginine repetitive matrix protein 2 0 15 0 13 0 13 0 41 N/A 3.344E-05 285 ✓ ✓ (9)(23)
Q3UMF0-4 COBL1 Isoform 4 of Cordon-bleu protein-like 1 0 14 0 12 0 11 0 37 N/A 1.517E-04 130 ✓ ✓ ✓
O35286 DHX15 Putative pre-mRNA-splicing factor ATP-dependent RNA helicase 0 11 0 12 0 13 0 36 N/A 3.166E-05 91 ✓ (9)(14)(23)(26)
Q99K48 NONO Non-POU domain-containing octamer-binding protein 0 11 0 13 0 11 0 35 N/A 6.260E-05 55 ✓ ✓ ✓
(9)(19)(23)(24)(
26)
Q60865 CAPR1 Caprin-1 0 12 0 15 0 8 0 35 N/A 4.524E-03 78 ✓ ✓ ✓ (19)(23)(26)
P51125-3 ICAL Isoform 3 of Calpastatin 0 10 0 14 0 10 0 34 N/A 1.051E-03 80 ✓ ✓ (26)
424
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Q3UMF0-3 COBL1 Isoform 3 of Cordon-bleu protein-like 1 0 13 0 11 0 10 0 34 N/A 2.114E-04 129 ✓ ✓ ✓
Q7TQH0-2 ATX2L Isoform 2 of Ataxin-2-like protein 0 7 0 15 0 11 0 33 N/A 8.885E-03 113 ✓ ✓ ✓ (19)(23)
Q8BK67 RCC2 Protein RCC2 GN=Rcc2 0 7 0 11 0 14 0 32 N/A 6.251E-03 56 ✓ ✓ (9)(23)
Q9Z1F9 SAE2 SUMO-activating enzyme subunit 2 0 11 0 11 0 10 0 32 N/A 5.685E-06 71 ✓ ✓ ✓ (26)
Q8C7R4 UBA6 Ubiquitin-like modifier-activating enzyme 6 0 11 0 13 0 7 0 31 N/A 4.237E-03 118 ✓ ✓ (9)(23)
Q62418-3 DBNL Isoform 3 of Drebrin-like protein 0 8 0 8 0 14 0 30 N/A 7.490E-03 48 ✓ ✓ ✓ (9)(23)(26)
Q8K298 ANLN Actin-binding protein anillin 0 10 0 9 0 10 0 29 N/A 8.416E-06 123 ✓ ✓ (9)(23)(26)
Q80YR5 SAFB2 Scaffold attachment factor B2 0 8 0 8 0 13 0 29 N/A 4.395E-03 112 ✓ ✓ (9)(23)
Q8R5H1-5 UBP15 Isoform 5 of Ubiquitin carboxyl-terminal hydrolase 15 0 11 0 10 0 8 0 29 N/A 3.936E-04 109 ✓ (23)
Q60710 SAMH1 SAM domain and HD domain-containing protein 1 0 6 0 8 0 15 0 29 N/A 2.395E-02 73 ✓ ✓ ✓ (23)
Q9JLV1 BAG3 BAG family molecular chaperone regulator 3 0 9 0 9 0 10 0 28 N/A 9.679E-06 62 ✓ ✓ ✓ (9)(23)(26) (17)
Q91W50 CSDE1 Cold shock domain-containing protein E1 0 9 0 10 0 9 0 28 N/A 9.679E-06 89 ✓ ✓ (9)(19)(23)
Q61191 HCFC1 Host cell factor 1 0 13 0 11 0 4 0 28 N/A 2.676E-02 210 ✓ ✓ ✓
(1)(2)(9)(13)(19)
(20)(21)(22)(23)
(24)(26)
Q3TW96 UAP1L UDP-N-acetylhexosamine pyrophosphorylase-like protein 1 0 11 0 11 0 6 0 28 N/A 4.992E-03 57 ✓ ✓ ✓
O09106 HDAC1 Histone deacetylase 1 0 11 0 10 0 6 0 27 N/A 4.150E-03 55 ✓ ✓ (9)(19)(23)
Q9QXS6-3 DREB Isoform E2 of Drebrin 0 9 0 8 0 10 0 27 N/A 9.888E-05 72 ✓ ✓ ✓ (23)
Q3U4W8 Q3U4W8 Ubiquitin carboxyl-terminal hydrolase 0 8 0 9 0 10 0 27 N/A 9.888E-05 93 ✓ ✓
G5E8E1 G5E8E1 Leucine rich repeat (In FLII) interacting protein 1, isoform CRA_e 0 8 0 10 0 9 0 27 N/A 9.888E-05 49 ✓ ✓ ✓
Q7TQI3 OTUB1 Ubiquitin thioesterase OTUB1 0 9 0 10 0 6 0 25 N/A 2.272E-03 31 ✓ ✓ (5)(9)(23)(26)
O70318 E41L2 Band 4.1-like protein 2 0 12 0 9 0 4 0 25 N/A 2.335E-02 110 ✓ ✓ ✓ (20)(23)
Q9CT10 RANB3 Ran-binding protein 3 0 9 0 9 0 7 0 25 N/A 2.356E-04 53 ✓ ✓
Q05CL8 LARP7 La-related protein 7 0 8 0 9 0 7 0 24 N/A 1.573E-04 65 ✓ ✓ (9)(23)
Q8R050-2 ERF3A Isoform 2 of Eukaryotic peptide chain release factor GTP-binding subunit ERF3A 0 9 0 8 0 7 0 24 N/A 1.573E-04 69 ✓ ✓ (23)(26)
P31230 AIMP1 Aminoacyl tRNA synthase complex-interacting multifunctional protein 1 0 8 0 8 0 8 0 24 N/A 0.000E+00 34 ✓ ✓ ✓ (1)
Q9CPV4-3 GLOD4 Isoform 3 of Glyoxalase domain-containing protein 4 0 7 0 10 0 6 0 23 N/A 3.098E-03 31 ✓ ✓ (9)(23)(26)
Q9CZD3 SYG Glycine--tRNA ligase 0 6 0 6 0 11 0 23 N/A 1.003E-02 82 ✓ ✓ ✓ (14)(23)(26)
P51859 HDGF Hepatoma-derived growth factor 0 7 0 8 0 7 0 22 N/A 2.526E-05 26 ✓ ✓ ✓ (9)(23)(26)
G3X9V0 G3X9V0 MCG22048, isoform CRA_a 0 6 0 8 0 8 0 22 N/A 3.882E-04 26 ✓ ✓
O70310 NMT1 Glycylpeptide N-tetradecanoyltransferase 1 0 6 0 5 0 10 0 21 N/A 1.016E-02 57 ✓ ✓ (9)(23) (17)
Q6PAM1 TXLNA Alpha-taxilin 0 6 0 8 0 7 0 21 N/A 2.655E-04 62 ✓ (9)(23)(26)
425
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
Q8WTY4 CPIN1 Anamorsin 0 10 0 4 0 7 0 21 N/A 1.559E-02 33 ✓ (26)
P46664 PURA2 Adenylosuccinate synthetase isozyme 2 0 6 0 9 0 6 0 21 N/A 2.192E-03 50 ✓ (23)
O54988-2 SLK Isoform 2 of STE20-like serine/threonine-protein kinase 0 10 0 7 0 4 0 21 N/A 1.559E-02 138 ✓ (14)(26)
Q9DBG5 PLIN3 Perilipin-3 0 8 0 6 0 7 0 21 N/A 2.655E-04 47 ✓ ✓ ✓ (1)(9)(20)(23)
Q64012-2 RALY Isoform 1 of RNA-binding protein Raly 0 7 0 7 0 7 0 21 N/A 0.000E+00 31 ✓ ✓
J3QNB1 J3QNB1 La-related protein 1 0 6 0 7 0 8 0 21 N/A 2.655E-04 121 ✓ ✓
Q8JZK9 HMCS1 Hydroxymethylglutaryl-CoA synthase, cytoplasmic 0 7 0 7 0 6 0 20 N/A 3.688E-05 58 ✓ ✓ (5)
P70372 ELAV1 ELAV-like protein 1 0 6 0 8 0 6 0 20 N/A 5.620E-04 36 ✓ ✓ (19)(26)
Q8R1X6 SPG20 Spartin 0 9 0 8 0 3 0 20 N/A 2.292E-02 73 ✓ ✓ (1)(20)
Q9Z0E6 GBP2 Interferon-induced guanylate-binding protein 2 0 8 0 8 0 4 0 20 N/A 7.490E-03 67 ✓ ✓
Q9WTK5 NFKB2 Nuclear factor NF-kappa-B p100 subunit 0 6 0 7 0 6 0 19 N/A 4.520E-05 97 ✓ ✓ ✓ (9)(23)(26)
A2AMW0 A2AMW0 Capping protein (Actin filament) muscle Z-line, beta 0 7 0 8 0 4 0 19 N/A 6.214E-03 29 ✓ ✓ (5)(26)
P45377 ALD2 Aldose reductase-related protein 2 0 8 0 6 0 5 0 19 N/A 1.991E-03 36 ✓ ✓ ✓ (26)
Q60967 PAPS1 Bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase 1 0 7 0 6 0 6 0 19 N/A 4.520E-05 71 ✓ ✓
D3YXK2 SAFB1 Scaffold attachment factor B1 0 6 0 6 0 7 0 19 N/A 4.520E-05 105 ✓ ✓ ✓
Q8C052 MAP1S Microtubule-associated protein 1S 0 6 0 6 0 6 0 18 N/A 0.000E+00103 ✓ ✓ (9)(23)(26)
Q9Z1D1 EIF3G Eukaryotic translation initiation factor 3 subunit G 0 6 0 7 0 5 0 18 N/A 4.841E-04 36 ✓ ✓ ✓ (9)(19)(23)(26)
Q8C156 CND2 Condensin complex subunit 2 0 6 0 6 0 6 0 18 N/A 0.000E+00 82 ✓ ✓ (23)
P35235 PTN11 Tyrosine-protein phosphatase non-receptor type 11 0 6 0 5 0 7 0 18 N/A 4.841E-04 68 ✓ ✓ (14)(23)
Q8BVY0 Q8BVY0 Protein Rsl1d1 0 6 0 6 0 6 0 18 N/A 0.000E+00 50 ✓ ✓
Q6PHZ2-2 KCC2D Isoform 2 of Calcium/calmodulin-dependent protein kinase type II subunit delta 0 7 0 6 0 5 0 18 N/A 4.841E-04 54 ✓ ✓
E9Q066 E9Q066 La-related protein 4 0 7 0 5 0 6 0 18 N/A 4.841E-04 80 ✓ ✓
Q8BMK4 CKAP4 Cytoskeleton-associated protein 4 0 6 0 6 0 5 0 17 N/A 7.021E-05 64 ✓ (16)
Q08093 CNN2 Calponin-2 0 7 0 6 0 4 0 17 N/A 3.016E-03 33 ✓ ✓ ✓ (9)(23)(26)
Q91Z38 TTC1 Tetratricopeptide repeat protein 1 0 6 0 6 0 5 0 17 N/A 7.021E-05 33 ✓ ✓ (9)(23)
O70551 SRPK1 SRSF protein kinase 1 0 3 0 6 0 8 0 17 N/A 1.754E-02 73 ✓ ✓ (9)(23)
Q9DAW9 CNN3 Calponin-3 GN=Cnn3 PE=2 SV=1 - [CNN3_MOUSE] 0 5 0 4 0 8 0 17 N/A 9.206E-03 36 ✓ (9)
P24288 BCAT1 Branched-chain-amino-acid aminotransferase, cytosolic 0 5 0 6 0 5 0 16 N/A 8.922E-05 43 ✓ (9)(23)(26)
Q6ZPJ3 UBE2O Ubiquitin-conjugating enzyme E2 O 0 5 0 5 0 6 0 16 N/A 8.922E-05 141 ✓ (9)(23)
Q9Z110-2 P5CS Isoform Short of Delta-1-pyrroline-5-carboxylate synthase 0 7 0 5 0 4 0 16 N/A 3.772E-03 87 ✓ ✓ (23)(26)
P32921-2 SYWC Isoform 2 of Tryptophan--tRNA ligase, cytoplasmic 0 6 0 3 0 7 0 16 N/A 1.135E-02 54 ✓ ✓ (14)(23)
426
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
Q8K354 CYTB Carbonyl reductase [NADPH] 3 0 4 0 6 0 6 0 16 N/A 1.324E-03 31 ✓ ✓
Q6NZD2 Q6NZD2 Sorting nexin 1 0 7 0 5 0 4 0 16 N/A 3.772E-03 59 ✓ ✓ ✓
G3UZI2 G3UZI2 Heterogeneous nuclear ribonucleoprotein Q 0 5 0 6 0 5 0 16 N/A 8.922E-05 59 ✓
P23198 CBX3 Chromobox protein homolog 3 0 5 0 5 0 5 0 15 N/A 0.000E+00 21 ✓ ✓ (9)(23)(26)
P97452 BOP1 Ribosome biogenesis protein BOP1 0 6 0 5 0 4 0 15 N/A 9.781E-04 83 ✓ (9)(23)
Q9JLM8 DCLK1 Serine/threonine-protein kinase DCLK1 0 4 0 6 0 5 0 15 N/A 9.781E-04 84 ✓ ✓ (5)(20)
Q9D819 IPYR Inorganic pyrophosphatase 0 6 0 4 0 5 0 15 N/A 9.781E-04 33 ✓ (23)(26)
Q64737 PUR2 Trifunctional purine biosynthetic protein adenosine-3 0 6 0 5 0 4 0 15 N/A 9.781E-04 107 ✓ (23)(26)
O09172 GSH0 Glutamate--cysteine ligase regulatory subunit 0 5 0 6 0 4 0 15 N/A 9.781E-04 31 ✓ ✓ (23)(26)
P61759 PFD3 Prefoldin subunit 3 0 4 0 6 0 5 0 15 N/A 9.781E-04 22 ✓
E9PZM7 E9PZM7 Protein Scaf11 0 7 0 3 0 5 0 15 N/A 1.235E-02 162 ✓
P42567 EPS15 Epidermal growth factor receptor substrate 15 0 3 0 6 0 5 0 14 N/A 6.122E-03 98 ✓ (9)(23)
B2RRE7 OTUD4 OTU domain-containing protein 4 0 4 0 6 0 4 0 14 N/A 2.192E-03 123 ✓ ✓ (9)(23)
Q8R2M2 TDIF2 Deoxynucleotidyltransferase terminal-interacting protein 2 0 5 0 5 0 4 0 14 N/A 1.510E-04 84 ✓ ✓ (23)
Q9WU78 PDC6I Programmed cell death 6-interacting protein 0 5 0 5 0 4 0 14 N/A 1.510E-04 96 ✓ ✓ ✓
Q62426 CYTB Cystatin-B 0 4 0 6 0 4 0 14 N/A 2.192E-03 11 ✓ ✓
P98078 DAB2 Disabled homolog 2 0 4 0 7 0 3 0 14 N/A 1.780E-02 82 ✓ ✓ ✓
F6T2Z7 F6T2Z7 Protein Cald1 (Fragment) 0 5 0 3 0 6 0 14 N/A 6.122E-03 41 ✓ ✓ ✓
Q8K310 MATR3 Matrin-3 0 3 0 5 0 5 0 13 N/A 2.890E-03 95 ✓ ✓ (9)(23)(26)
Q8CI11 GNL3 Guanine nucleotide-binding protein-like 3 0 4 0 4 0 5 0 13 N/A 2.020E-04 61 ✓ (9)(23)(26)
P70288 HDAC2 Histone deacetylase 2 0 5 0 4 0 4 0 13 N/A 2.020E-04 55 ✓ ✓ (9)(23)
P37913 DNLI1 DNA ligase 1 0 6 0 3 0 4 0 13 N/A 7.966E-03 102 ✓ (26)
P49586 PCY1A Choline-phosphate cytidylyltransferase A 0 4 0 6 0 3 0 13 N/A 7.966E-03 42 ✓ ✓ (23)
Q6P5B5 Q6P5B5 Fragile X mental retardation syndrome-related protein 2 0 3 0 5 0 5 0 13 N/A 2.890E-03 74 ✓ ✓
Q61081 CDC37 Hsp90 co-chaperone Cdc37 0 3 0 5 0 4 0 12 N/A 2.278E-03 45 ✓ ✓ (9)(23)(26)
Q9QXD8 LIMD1 LIM domain-containing protein 1 0 4 0 5 0 3 0 12 N/A 2.278E-03 71 ✓ (9)(23)
Q8BK64 AHSA1 Activator of 90 kDa heat shock protein ATPase homolog 1 0 5 0 4 0 3 0 12 N/A 2.278E-03 38 ✓ (9)(23)
Q5UE59 Q5UE59 Kinesin light chain 1 0 3 0 3 0 6 0 12 N/A 1.613E-02 62 ✓ ✓
O89110 CASP8 Caspase-8 0 5 0 4 0 3 0 12 N/A 2.278E-03 55 ✓ ✓
O08529 CAN2 Calpain-2 catalytic subunit 0 4 0 5 0 3 0 12 N/A 2.278E-03 80 ✓ ✓
B7ZP47 B7ZP47 Wapal protein 0 3 0 3 0 6 0 12 N/A 1.613E-02 133 ✓ ✓
427
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
Q9JKF1 IQGA1 Ras GTPase-activating-like protein IQGAP1 0 4 0 4 0 3 0 11 N/A 3.882E-04 189 ✓ (14)(23) (17)
Q6PGH2 HN1L Hematological and neurological expressed 1-like protein 0 3 0 4 0 4 0 11 N/A 3.882E-04 20 ✓ ✓ (9)(23)(26) (16)
Q810D6 GRWD1 Glutamate-rich WD repeat-containing protein 1 0 4 0 4 0 3 0 11 N/A 3.882E-04 49 ✓ (9)(23)
P42227-2 STAT3 Isoform Stat3B of Signal transducer and activator of transcription 3 0 4 0 4 0 3 0 11 N/A 3.882E-04 83 ✓ ✓ (9)
P19096 FAS Fatty acid synthase 0 2 0 5 0 4 0 11 N/A 1.417E-02 272 ✓ ✓ (5)(8)(23)
Q7TSJ2-3 MAP6 Isoform 3 of Microtubule-associated protein 6 0 3 0 5 0 3 0 11 N/A 5.328E-03 33 ✓ ✓ ✓ (5)
Q9CXW3 CYBP Calcyclin-binding protein 0 3 0 3 0 5 0 11 N/A 5.328E-03 27 ✓ ✓ (26)
P52479 UBP10 Ubiquitin carboxyl-terminal hydrolase 10 0 5 0 3 0 3 0 11 N/A 5.328E-03 87 ✓ (23)(26)
O08915 AIP AH receptor-interacting protein 0 3 0 5 0 3 0 11 N/A 5.328E-03 38 ✓ (20)
Q9DBC7 KAP0 cAMP-dependent protein kinase type I-alpha regulatory subunit 0 4 0 4 0 3 0 11 N/A 3.882E-04 43 ✓ ✓
Q9D4G5 Q9D4G5 Protein Pop1 0 4 0 4 0 3 0 11 N/A 3.882E-04 114 ✓
Q8C650-2 SEP10 Isoform 2 of Septin-10 0 3 0 4 0 4 0 11 N/A 3.882E-04 50 ✓
Q5SSZ5-2 TENS3 Isoform 2 of Tensin-3 0 4 0 4 0 3 0 11 N/A 3.882E-04 59 ✓
O55131 SEPT7 Septin-7 0 3 0 5 0 3 0 11 N/A 5.328E-03 51 ✓
G3X8Y3 G3X8Y3 N-alpha-acetyltransferase 15, NatA auxiliary subunit 0 4 0 4 0 3 0 11 N/A 3.882E-04 101 ✓
E9Q2X6 E9Q2X6 Structural maintenance of chromosomes protein 0 2 0 4 0 5 0 11 N/A 1.417E-02 144 ✓
E9PX53 E9PX53 Serine/threonine-protein phosphatase 4 regulatory subunit 1 0 5 0 3 0 3 0 11 N/A 5.328E-03 104 ✓
Q8C1A5 THOP1 Thimet oligopeptidase 0 4 0 4 0 2 0 10 N/A 7.490E-03 78 ✓ (9)(23) (17)
P62774 MTPN Myotrophin 0 2 0 3 0 5 0 10 N/A 1.944E-02 13 ✓ ✓ (9)(23)
Q9QYA2 TOM40 Mitochondrial import receptor subunit TOM40 homolog 0 5 0 3 0 2 0 10 N/A 1.944E-02 38 ✓ (23)
Q9QWF0 CAF1A Chromatin assembly factor 1 subunit A 0 4 0 3 0 3 0 10 N/A 5.620E-04 102 ✓ (23)
Q9CXF4 TBC15 TBC1 domain family member 15 0 2 0 4 0 4 0 10 N/A 7.490E-03 77 ✓ (23)
Q3UY34 CL043 Uncharacterized protein C12orf43 homolog 0 4 0 3 0 3 0 10 N/A 5.620E-04 28 ✓ ✓ (23)
P63037 DNJA1 DnaJ homolog subfamily A member 1 0 5 0 2 0 3 0 10 N/A 1.944E-02 45 ✓ ✓ (19)(26)
Q9JLM9 GRB14 Growth factor receptor-bound protein 14 0 4 0 3 0 3 0 10 N/A 5.620E-04 61 ✓
B1AXN9 B1AXN9 Ribosomal protein S6 kinase alpha-3 0 4 0 3 0 3 0 10 N/A 5.620E-04 81 ✓
A2AMY5 A2AMY5 Ubiquitin-associated protein 2 0 4 0 3 0 3 0 10 N/A 5.620E-04 118 ✓ ✓ ✓
Q9DBR1-2 XRN2 Isoform 2 of 5'-3' exoribonuclease 2 0 3 0 3 0 3 0 9 N/A 0.000E+00108 ✓ ✓ (9)(23)
Q9DBR0 AKAP8 A-kinase anchor protein 8 0 2 0 3 0 4 0 9 N/A 6.533E-03 76 ✓ ✓ (9)(23)
Q91XI1 DUS3L tRNA-dihydrouridine(47) synthase [NAD(P)(+)]-like 0 3 0 3 0 3 0 9 N/A 0.000E+00 71 ✓ (9)(23)
Q9R0P5 DEST Destrin 0 4 0 3 0 2 0 9 N/A 6.533E-03 19 ✓ ✓ ✓ (26)
428
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
P68037 UB2L3 Ubiquitin-conjugating enzyme E2 L3 0 3 0 3 0 3 0 9 N/A 0.000E+00 18 ✓ ✓ (26)
Q9CWE0 FA54B Protein FAM54B 0 4 0 3 0 2 0 9 N/A 6.533E-03 32 ✓ ✓ (23)
Q9CR86 CHSP1 Calcium-regulated heat stable protein 1 0 2 0 5 0 2 0 9 N/A 3.994E-02 16 ✓ ✓ (23)
Q0VGB7 PP4R2 Serine/threonine-protein phosphatase 4 regulatory subunit 2 0 2 0 2 0 5 0 9 N/A 3.994E-02 46 ✓ (23)
Q99K51 PLST Plastin-3 0 2 0 3 0 4 0 9 N/A 6.533E-03 71 ✓ ✓
Q8K3A9 MEPCE 7SK snRNA methylphosphate capping enzyme 0 2 0 5 0 2 0 9 N/A 3.994E-02 72 ✓
Q8BIJ7 RUFY1 RUN and FYVE domain-containing protein 1 0 4 0 1 0 4 0 9 N/A 3.994E-02 80 ✓
Q6ZQK5 ACAP2 Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 2 0 4 0 4 0 1 0 9 N/A 3.994E-02 87 ✓ ✓
Q5SSZ5 TENS3 Tensin-3 0 3 0 4 0 2 0 9 N/A 6.533E-03 156 ✓
E9QP59 E9QP59 Inner nuclear membrane protein Man1 0 3 0 3 0 3 0 9 N/A 0.000E+00100 ✓ ✓
E9Q7G0 E9Q7G0 Protein Numa1 0 3 0 4 0 2 0 9 N/A 6.533E-03 236 ✓ ✓
B7ZCP4 B7ZCP4 Copine I 0 3 0 4 0 2 0 9 N/A 6.533E-03 53 ✓ ✓
A2AVJ7 A2AVJ7 Ribosome binding protein 1 0 2 0 4 0 3 0 9 N/A 6.533E-03 158 ✓ ✓
E9Q3T0 E9Q3T0 Uncharacterized protein 0 3 0 3 0 3 0 9 N/A 0.000E+00 11
Q9D6Z1 NOP56 Nucleolar protein 56 0 3 0 3 0 2 0 8 N/A 1.324E-03 64 ✓ (9)(23)(26) (16)
Q9EP82 WDR4 tRNA (guanine-N(7)-)-methyltransferase subunit WDR4 0 2 0 3 0 3 0 8 N/A 1.324E-03 46 ✓ (9)(23)
Q9CR00 PSMD9 26S proteasome non-ATPase regulatory subunit 9 0 3 0 3 0 2 0 8 N/A 1.324E-03 25 ✓ ✓ (9)(23)
Q8BY71 HAT1 Histone acetyltransferase type B catalytic subunit 0 3 0 3 0 2 0 8 N/A 1.324E-03 49 ✓ (9)(23)
P43247 MSH2 DNA mismatch repair protein Msh2 0 2 0 3 0 3 0 8 N/A 1.324E-03 104 ✓ ✓ (9)(23)
A2BE28-2 LAS1L Isoform 2 of Ribosomal biogenesis protein LAS1L 0 2 0 4 0 2 0 8 N/A 1.613E-02 88 ✓ (9)(23)
Q8VCF0 MAVS Mitochondrial antiviral-signaling protein 0 3 0 3 0 2 0 8 N/A 1.324E-03 53 ✓ ✓ ✓ (5)(9)(20)(23)
Q6NZF1 ZC11A Zinc finger CCCH domain-containing protein 11A 0 2 0 4 0 2 0 8 N/A 1.613E-02 86 ✓ (26)
Q9CYA6 ZCHC8 Zinc finger CCHC domain-containing protein 8 0 1 0 3 0 4 0 8 N/A 3.902E-02 78 ✓ ✓ (23)
Q8BQ30 PPR18 Phostensin 0 2 0 3 0 3 0 8 N/A 1.324E-03 66 ✓ (23)
Q6NSQ7 LTV1 Protein LTV1 homolog 0 2 0 3 0 3 0 8 N/A 1.324E-03 54 ✓ ✓ (23)
Q9EP97 SENP3 Sentrin-specific protease 3 0 3 0 3 0 2 0 8 N/A 1.324E-03 64 ✓
Q921Q7 RIN1 Ras and Rab interactor 1 0 1 0 3 0 4 0 8 N/A 3.902E-02 83 ✓
Q8C8U0 LIPB1 Liprin-beta-1 0 2 0 4 0 2 0 8 N/A 1.613E-02 109 ✓
Q3V4D5 Q3V4D5 N-acetyltransferase ARD1 homolog 0 3 0 3 0 2 0 8 N/A 1.324E-03 25 ✓
H3BKN0 H3BKN0 tRNA (cytosine(34)-C(5))-methyltransferase 0 4 0 2 0 2 0 8 N/A 1.613E-02 81 ✓
P35278 RAB5C Ras-related protein Rab-5C 0 3 0 1 0 3 0 7 N/A 2.490E-02 23 ✓ ✓ (9)(23)(26)
429
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
Q6P5E6 GGA2 ADP-ribosylation factor-binding protein GGA2 0 2 0 2 0 3 0 7 N/A 2.192E-03 66 ✓ (9)(23)
Q9JKP5 MBNL1 Muscleblind-like protein 1 GN=Mbnl1 PE=1 SV=1 - [MBNL1_MOUSE 0 2 0 3 0 2 0 7 N/A 2.192E-03 37 ✓ ✓ ✓ (9)(23)
O88622-2 PARG Isoform 2 of Poly(ADP-ribose) glycohydrolase 0 3 0 3 0 1 0 7 N/A 2.490E-02 104 ✓ ✓ (9)(23)
O54692 ZW10 Centromere/kinetochore protein zw10 homolog 0 2 0 3 0 2 0 7 N/A 2.192E-03 88 ✓ (9)(23)
Q5PSV9 MDC1 Mediator of DNA damage checkpoint protein 1 0 2 0 3 0 2 0 7 N/A 2.192E-03 185 ✓ ✓ (9)(14)(23)
Q9WVG6-2 CARM1 Isoform 2 of Histone-arginine methyltransferase CARM1 0 2 0 3 0 2 0 7 N/A 2.192E-03 63 ✓ ✓ ✓ (5)(9)(19)(23)
Q9WVQ5 MTNB Probable methylthioribulose-1-phosphate dehydratase 0 3 0 3 0 1 0 7 N/A 2.490E-02 27 ✓ (23)
Q6P5D8 SMHD1 Structural maintenance of chromosomes flexible hinge domain-containing protein 1 0 2 0 3 0 2 0 7 N/A 2.192E-03 226 ✓ ✓ (23)
Q3THS6 METK2 S-adenosylmethionine synthase isoform type-2 0 2 0 3 0 2 0 7 N/A 2.192E-03 44 ✓ ✓ (20)
Q921K2 Q921K2 Poly (ADP-ribose) polymerase family, member 1 0 3 0 2 0 2 0 7 N/A 2.192E-03 113 ✓ ✓
Q8VE88-2 F1142 Isoform 2 of Protein FAM114A2 0 2 0 3 0 2 0 7 N/A 2.192E-03 53 ✓ ✓
Q8BP48 AMPM1 Methionine aminopeptidase 1 0 2 0 3 0 2 0 7 N/A 2.192E-03 43 ✓ ✓
Q3TT92 Q3TT92 Dihydropyrimidinase-related protein 3 0 1 0 3 0 3 0 7 N/A 2.490E-02 62 ✓ ✓
P35123 UBP4 Ubiquitin carboxyl-terminal hydrolase 4 0 1 0 3 0 3 0 7 N/A 2.490E-02 108 ✓ ✓
F7AC41 F7AC41 Protein Pus7 (Fragment) 0 3 0 3 0 1 0 7 N/A 2.490E-02 73 ✓
A2A5R8 A2A5R8 Double-stranded RNA-binding protein Staufen homolog 1 0 3 0 3 0 1 0 7 N/A 2.490E-02 54 ✓
Q62348 TSN Translin 0 2 0 2 0 2 0 6 N/A 0.000E+00 26 ✓ ✓ ✓ (9)(23)(26)
Q9Z2X8 KEAP1 Kelch-like ECH-associated protein 1 0 3 0 1 0 2 0 6 N/A 2.572E-02 70 ✓ (9)(23)
Q9Z1Z0-2 USO1 Isoform 2 of General vesicular transport factor p115 0 2 0 3 0 1 0 6 N/A 2.572E-02 100 ✓ ✓ (9)(23)
Q9JJY4 DDX20 Probable ATP-dependent RNA helicase DDX20 0 2 0 2 0 2 0 6 N/A 0.000E+00 92 ✓ (9)(23)
Q8K1R7 NEK9 Serine/threonine-protein kinase Nek9 0 2 0 3 0 1 0 6 N/A 2.572E-02 107 ✓ ✓ (9)(23)
Q8BIW1 PRUNE Protein prune homolog 0 2 0 2 0 2 0 6 N/A 0.000E+00 50 ✓ (9)(23)
P61290 PSME3 Proteasome activator complex subunit 3 0 3 0 1 0 2 0 6 N/A 2.572E-02 30 ✓ (9)(23)
P42669 PURA Transcriptional activator protein Pur-alpha 0 3 0 2 0 1 0 6 N/A 2.572E-02 35 ✓ ✓ (9)(20)(23)
Q76MZ3 2AAA Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform 0 2 0 2 0 2 0 6 N/A 0.000E+00 65 ✓ ✓ (4)(14)
Q64337 SQSTM Sequestosome-1 0 3 0 2 0 1 0 6 N/A 2.572E-02 48 ✓ ✓ (23)
Q8BFS6-2 CPPED Isoform 2 of Calcineurin-like phosphoesterase domain-containing protein 1 0 2 0 2 0 2 0 6 N/A 0.000E+00 33 ✓ (23)
Q80XI4 PI42B Phosphatidylinositol 5-phosphate 4-kinase type-2 beta 0 1 0 3 0 2 0 6 N/A 2.572E-02 47 ✓ (23)
Q80WT5-2 AFTIN Isoform 2 of Aftiphilin 0 2 0 2 0 2 0 6 N/A 0.000E+00 98 ✓ (23)
Q8BQM4 HEAT3 HEAT repeat-containing protein 3 0 2 0 2 0 2 0 6 N/A 0.000E+00 74 ✓ (23)
P34022 RANG Ran-specific GTPase-activating protein 0 1 0 2 0 3 0 6 N/A 2.572E-02 24 ✓ (19)(23)(26)
430
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
Q9CZP7-2 CD37L Isoform 2 of Hsp90 co-chaperone Cdc37-like 1 0 3 0 2 0 1 0 6 N/A 2.572E-02 35 ✓
Q8R409 HEXI1 Protein HEXIM1 0 2 0 3 0 1 0 6 N/A 2.572E-02 40 ✓
Q8K0C9 GMDS GDP-mannose 4,6 dehydratase 0 2 0 2 0 2 0 6 N/A 0.000E+00 42 ✓ ✓
Q60848-2 HELLS Isoform 2 of Lymphocyte-specific helicase 0 3 0 2 0 1 0 6 N/A 2.572E-02 94 ✓
P33174 KIF4 Chromosome-associated kinesin KIF4 0 3 0 1 0 2 0 6 N/A 2.572E-02 139 ✓
G3UW40 G3UW40 MCG4620, isoform CRA_b 0 2 0 2 0 2 0 6 N/A 0.000E+00 93 ✓ ✓
E9Q7L0 E9Q7L0 Protein Ogdhl 0 2 0 2 0 2 0 6 N/A 0.000E+00117 ✓
D3YX62 D3YX62 Heme oxygenase 2 (Fragment) 0 1 0 3 0 2 0 6 N/A 2.572E-02 26 ✓
B7ZCL8 B7ZCL8 55 kDa erythrocyte membrane protein 0 3 0 1 0 2 0 6 N/A 2.572E-02 50 ✓
Q8BKC5 IPO5 Importin-5 0 1 0 2 0 2 0 5 N/A 7.490E-03 124 ✓ ✓ (9)(23) (17)
Q9JIH2 NUP50 Nuclear pore complex protein Nup50 0 2 0 2 0 1 0 5 N/A 7.490E-03 50 ✓ ✓ (9)(23)
Q8C4J7 TBL3 Transducin beta-like protein 3 0 2 0 1 0 2 0 5 N/A 7.490E-03 88 ✓ (9)(23)
Q8BFY6 PEF1 Peflin 0 2 0 2 0 1 0 5 N/A 7.490E-03 29 ✓ (9)(23)
Q7TNV0 DEK Protein DEK 0 2 0 1 0 2 0 5 N/A 7.490E-03 43 ✓ ✓ (9)(23)
Q91VE6-2 MK67I Isoform 2 of MKI67 FHA domain-interacting nucleolar phosphoprotein 0 1 0 2 0 2 0 5 N/A 7.490E-03 31 ✓ (23)
O89032-3 SPD2A Isoform 3 of SH3 and PX domain-containing protein 2A 0 2 0 2 0 1 0 5 N/A 7.490E-03 119 ✓ ✓
O55137 ACOT1 Acyl-coenzyme A thioesterase 1 0 2 0 1 0 2 0 5 N/A 7.490E-03 46 ✓ ✓
G3X8Q0 G3X8Q0 Trans-acting transcription factor 1 0 2 0 1 0 2 0 5 N/A 7.490E-03 80 ✓ ✓ ✓
E9Q310 E9Q310 Glucocorticoid receptor (Fragment) 0 2 0 2 0 1 0 5 N/A 7.490E-03 52 ✓
D3Z2M0 D3Z2M0 Cytoplasmic tRNA 2-thiolation protein 2 (Fragment) 0 2 0 2 0 1 0 5 N/A 7.490E-03 27 ✓
F8VQ29 F8VQ29 Protein Iqgap3 0 1 0 2 0 2 0 5 N/A 7.490E-03 185
Q61687 ATRX Transcriptional regulator ATRX 0 1 0 1 0 2 0 4 N/A 1.613E-02 278 ✓ (9)(23)(26)
Q9WTX5 SKP1 S-phase kinase-associated protein 1 0 2 0 1 0 1 0 4 N/A 1.613E-02 19 ✓ ✓ (9)(23)
Q9D5T0 ATAD1 ATPase family AAA domain-containing protein 1 0 1 0 2 0 1 0 4 N/A 1.613E-02 41 ✓ ✓ (9)(23)
Q8VCH8 UBXN4 UBX domain-containing protein 4 0 2 0 1 0 1 0 4 N/A 1.613E-02 56 ✓ (9)(23)
Q8CG48 SMC2 Structural maintenance of chromosomes protein 2 0 1 0 2 0 1 0 4 N/A 1.613E-02 134 ✓ (9)(23)
P15307 REL Proto-oncogene c-Rel 0 1 0 2 0 1 0 4 N/A 1.613E-02 65 ✓ ✓ ✓ (9)(23)
Q9D8S9 BOLA1 BolA-like protein 1 0 1 0 1 0 2 0 4 N/A 1.613E-02 14 (9)(23)
A2AAW9 A2AAW9 Eukaryotic translation initiation factor 2 subunit 3, X-linked 0 1 0 1 0 2 0 4 N/A 1.613E-02 37 ✓ ✓ (26)
Q9JJV2-3 PROF2 Isoform 3 of Profilin-2 0 1 0 1 0 2 0 4 N/A 1.613E-02 9.8 ✓ (23)
Q8BI72 CARF CDKN2A-interacting protein 0 2 0 1 0 1 0 4 N/A 1.613E-02 60 ✓ ✓ (23)
431
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
Q03963 E2AK2 Interferon-induced, double-stranded RNA-activated protein kinase 0 2 0 1 0 1 0 4 N/A 1.613E-02 58 ✓ ✓ (23)
P00493 HPRT Hypoxanthine-guanine phosphoribosyltransferase 0 1 0 1 0 2 0 4 N/A 1.613E-02 25 ✓ ✓ (19)
Q62419 SH3G1 Endophilin-A2 0 1 0 1 0 2 0 4 N/A 1.613E-02 42 ✓ (1)(20)
Q9JLJ5 ELOV1 Elongation of very long chain fatty acids protein 1 0 2 0 1 0 1 0 4 N/A 1.613E-02 33 ✓ ✓ ✓
J3KMM5 J3KMM5 Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 0 2 0 1 0 1 0 4 N/A 1.613E-02 110 ✓
E9Q9C5 E9Q9C5 V-type proton ATPase 16 kDa proteolipid subunit (Fragment) 0 2 0 1 0 1 0 4 N/A 1.613E-02 15 ✓ ✓
Q9R0X4 ACOT9 Acyl-coenzyme A thioesterase 9, mitochondrial 0 2 0 1 0 1 0 4 N/A 1.613E-02 51 ✓ ✓
Q9CR51 VATG1 V-type proton ATPase subunit G 1 0 1 0 1 0 2 0 4 N/A 1.613E-02 14 ✓ ✓
Q91WG2-2 RABE2 Isoform 3 of Rab GTPase-binding effector protein 2 0 2 0 1 0 1 0 4 N/A 1.613E-02 54 ✓
Q60760-3 GRB10 Isoform 3 of Growth factor receptor-bound protein 10 0 2 0 1 0 1 0 4 N/A 1.613E-02 61 ✓
P35550 FBRL rRNA 2'-O-methyltransferase fibrillarin 0 1 0 2 0 1 0 4 N/A 1.613E-02 34 ✓
O70325-2 GPX41 Isoform Cytoplasmic of Phospholipid hydroperoxide glutathione peroxidase, mitochondrial 0 2 0 1 0 1 0 4 N/A 1.613E-02 20 ✓
O70305-2 ATX2 Isoform 2 of Ataxin-2 0 1 0 1 0 2 0 4 N/A 1.613E-02 129 ✓ ✓ ✓
E9Q242 E9Q242 Adenylosuccinate lyase 0 2 0 1 0 1 0 4 N/A 1.613E-02 53 ✓ ✓
Q64514-2 TPP2 Isoform Short of Tripeptidyl-peptidase 2 0 1 0 1 0 1 0 3 N/A 0.000E+00138 ✓ ✓ (9)(23)
Q3UHX0 NOL8 Nucleolar protein 8 0 1 0 1 0 1 0 3 N/A 0.000E+00129 ✓ ✓ (9)(23)
Q3U5F4 YRDC YrdC domain-containing protein, mitochondrial 0 1 0 1 0 1 0 3 N/A 0.000E+00 29 ✓ (9)(23)
P47856-2 GFPT1 Isoform 2 of Glutamine--fructose-6-phosphate aminotransferase [isomerizing] 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 77 ✓ (9)(23)
Q69Z38 PEAK1 Pseudopodium-enriched atypical kinase 1 0 1 0 1 0 1 0 3 N/A 0.000E+00191 ✓ ✓ (9)(20)(23)
Q99LS3 SERB Phosphoserine phosphatase 0 1 0 1 0 1 0 3 N/A 0.000E+00 25 ✓ ✓ (26)
Q80YR4-2 ZN598 Isoform 2 of Zinc finger protein 598 0 1 0 1 0 1 0 3 N/A 0.000E+00 96 ✓ ✓ (23)
P59708 PM14 Pre-mRNA branch site protein p14 0 1 0 1 0 1 0 3 N/A 0.000E+00 15 ✓ (23)
G3X972 G3X972 Protein Sec24c 0 1 0 1 0 1 0 3 N/A 0.000E+00119 ✓ ✓ ✓ (2)
G3X928 G3X928 SEC23-interacting protein 0 1 0 1 0 1 0 3 N/A 0.000E+00111 ✓ ✓ ✓ (2)
Q3TJZ6 FA98A Protein FAM98A 0 1 0 1 0 1 0 3 N/A 0.000E+00 55 (19)
Q9DBR7 MYPT1 Protein phosphatase 1 regulatory subunit 12A 0 1 0 1 0 1 0 3 N/A 0.000E+00115 ✓ ✓ ✓
(1)(5)(19)(20)(2
6)
Q9CRD0-3 OCAD1 Isoform 3 of OCIA domain-containing protein 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 21 ✓
Q99NB8 UBQL4 Ubiquilin-4 0 1 0 1 0 1 0 3 N/A 0.000E+00 64 ✓ ✓ ✓
Q91YS8 KCC1A Calcium/calmodulin-dependent protein kinase type 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 42 ✓ ✓
Q8K124 PKHO2 Pleckstrin homology domain-containing family O member 2 0 1 0 1 0 1 0 3 N/A 0.000E+00 54 ✓
432
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
Q8C0V0 TLK1 Serine/threonine-protein kinase tousled-like 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 87 ✓
Q8BIW9 CTF18 Chromosome transmission fidelity protein 18 homolog 0 1 0 1 0 1 0 3 N/A 0.000E+00108 ✓
Q62074 KPCI Protein kinase C iota type 0 1 0 1 0 1 0 3 N/A 0.000E+00 68 ✓
Q3V3Y9 Q3V3Y9 Kinesin-like protein KIF1C 0 1 0 1 0 1 0 3 N/A 0.000E+00 47 ✓
Q3TZX8-3 NOL9 Isoform 3 of Polynucleotide 5'-hydroxyl-kinase NOL9 0 1 0 1 0 1 0 3 N/A 0.000E+00 71 ✓ ✓
Q3TFP0 Q3TFP0 FUS interacting protein (Serine-arginine rich) 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 22 ✓
P70445 4EBP2 Eukaryotic translation initiation factor 4E-binding protein 2 0 1 0 1 0 1 0 3 N/A 0.000E+00 13 ✓ ✓
P63166 SUMO1 Small ubiquitin-related modifier 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 12 ✓
P31938 MP2K1 Dual specificity mitogen-activated protein kinase kinase 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 43 ✓
O09110-2 MP2K3 Isoform 1 of Dual specificity mitogen-activated protein kinase kinase 3 0 1 0 1 0 1 0 3 N/A 0.000E+00 36 ✓
J3QP68 J3QP68 Uncharacterized protein 0 1 0 1 0 1 0 3 N/A 0.000E+00 41 ✓
J3JS94 J3JS94 L antigen family member 3 0 1 0 1 0 1 0 3 N/A 0.000E+00 12 ✓ ✓
H3BKK2 H3BKK2 Protein D2Ertd750e (Fragment) 0 1 0 1 0 1 0 3 N/A 0.000E+00 21 ✓
G3UZ44 G3UZ44 Paired mesoderm homeobox protein 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 23 ✓
E9Q986 E9Q986 Catenin delta-1 0 1 0 1 0 1 0 3 N/A 0.000E+00 92 ✓
E9Q7M2 E9Q7M2 Protein Tsc22d2 0 1 0 1 0 1 0 3 N/A 0.000E+00 78 ✓ ✓
E9Q5L7 E9Q5L7 PHD finger protein 10 0 1 0 1 0 1 0 3 N/A 0.000E+00 27 ✓ ✓
D3Z4W3 D3Z4W3 Proline-rich AKT1 substrate 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 9.7 ✓
D3YVJ7 D3YVJ7 Protein Akr1b3 (Fragment) 0 1 0 1 0 1 0 3 N/A 0.000E+00 20 ✓
D3YUC9 D3YUC9 Methionine-R-sulfoxide reductase B3, mitochondrial 0 1 0 1 0 1 0 3 N/A 0.000E+00 12 ✓ ✓
A2AG83 A2AG83 26S proteasome non-ATPase regulatory subunit 10 0 1 0 1 0 1 0 3 N/A 0.000E+00 16 ✓ ✓
A2A4Z1 A2A4Z1 Ubiquitin-conjugating enzyme E2 C 0 1 0 1 0 1 0 3 N/A 0.000E+00 19 ✓
O54931-2 AKAP2 Isoform 2 of A-kinase anchor protein 2 0 23 1 26 0 26 1 75 75.00 1.977E-05 97 ✓ ✓ ✓ (9)(23)(26)
P07742 RIR1 Ribonucleoside-diphosphate reductase large subunit 0 17 1 21 0 20 1 58 58.00 1.083E-04 90 ✓ ✓ (14)(23)(26)
Q61033 LAP2A Lamina-associated polypeptide 2, isoforms alpha/zeta 1 20 0 19 0 12 1 51 51.00 2.785E-03 75 ✓ ✓ ✓ (23)(26)
P80314 TCPB T-complex protein 1 subunit beta 1 16 0 16 0 18 1 50 50.00 2.566E-05 57 ✓ ✓ (5)(26)
Q6DFW4 NOP58 Nucleolar protein 58 1 17 0 14 0 17 1 48 48.00 1.193E-04 60 ✓ ✓ ✓ (9)(23)(26)
P60335 PCBP1 Poly(rC)-binding protein 1 0 14 1 16 0 17 1 47 47.00 8.364E-05 38 ✓ ✓ ✓ (9)(14)(23)(26)
Q60854 SPB6 Serpin B6 0 13 1 20 0 13 1 46 46.00 3.126E-03 43 ✓ ✓ (26)
Q80X90 FLNB Filamin-B 0 14 1 11 0 15 1 40 40.00 4.786E-04 278 ✓ ✓ (9)(23)(26)
Q61990 PCBP2 Poly(rC)-binding protein 2 0 9 1 14 0 13 1 36 36.00 1.724E-03 38 ✓ ✓ ✓ (5)(14)(26) (17)
433
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
Q8BFW7 LPP Lipoma-preferred partner homolog 0 13 1 13 0 10 1 36 36.00 3.790E-04 66 ✓ ✓ ✓ (9)(20)(23)(26) (16)
Q62167 DDX3X ATP-dependent RNA helicase DDX3X 0 13 1 13 0 10 1 36 36.00 3.790E-04 73 ✓ ✓ (9)(23)(26)
Q9D0E1-2 HNRPM Isoform 2 of Heterogeneous nuclear ribonucleoprotein M 1 25 1 25 0 20 2 70 35.00 1.828E-04 74 ✓ ✓ (23)
Q60598 SRC8 Src substrate cortactin 0 10 1 10 0 13 1 33 33.00 5.368E-04 61 ✓ ✓ (20)(26)
Q61074 PPM1G Protein phosphatase 1G 0 11 1 7 0 10 1 28 28.00 1.956E-03 59 ✓ (9)(23)
O08553 DPYL2 Dihydropyrimidinase-related protein 2 0 9 1 6 0 12 1 27 27.00 7.966E-03 62 ✓ (1)(20)
A2AFJ1 A2AFJ1 Histone-binding protein RBBP7 0 10 1 7 0 9 1 26 26.00 9.045E-04 47 ✓ ✓ (26)
P70698 PYRG1 CTP synthase 1 1 9 0 9 0 8 1 26 26.00 6.015E-05 67 ✓ ✓ (23)(26)
E9PVC5 E9PVC5 Eukaryotic translation initiation factor 4 gamma 1 0 6 1 10 0 10 1 26 26.00 3.736E-03 175 ✓ ✓ ✓ (12)
Q8CGC7 SYEP Bifunctional glutamate/proline--tRNA ligase 1 16 1 13 0 14 2 43 21.50 1.317E-04 170 ✓ (14)(19)(23)(26)
Q61699-2 HS105 Isoform HSP105-beta of Heat shock protein 105 kDa 0 20 3 21 0 20 3 61 20.33 5.199E-05 92 ✓ ✓ (5)(23)
Q91VI7 RINI Ribonuclease inhibitor 0 8 1 6 0 6 1 20 20.00 1.052E-03 50 ✓ ✓ (23)
Q9CQX2 CYB5B Cytochrome b5 type B 1 5 0 9 0 5 1 19 19.00 1.201E-02 16 ✓ ✓ (26)
D3Z5M2 D3Z5M2 Protein Gm10110 0 14 2 12 0 9 2 35 17.50 2.337E-03 68 ✓ ✓
Q8CI51 PDLI5 PDZ and LIM domain protein 5 0 11 2 10 0 13 2 34 17.00 6.454E-04 63 ✓ ✓ ✓ (1)(23)(26)
Q99K70 RRAGC Ras-related GTP-binding protein C 0 7 1 5 0 5 1 17 17.00 2.019E-03 44 ✓ ✓ (9)(23)
P16045 LEG1 Galectin-1 1 35 2 35 3 29 6 99 16.50 1.184E-04 15 ✓ ✓ (23) (17)
Q8BGJ5 Q8BGJ5 MCG13402, isoform CRA_a 0 14 1 13 2 22 3 49 16.33 6.185E-03 57 ✓ ✓ (26)
Q3U0V1 FUBP2 Far upstream element-binding protein 2 1 26 2 29 2 25 5 80 16.00 3.656E-05 77 ✓ ✓ (14)(23)(26)
P06151 LDHA L-lactate dehydrogenase A chain 1 17 1 13 1 15 3 45 15.00 2.655E-04 37 ✓ ✓ ✓
(6)(9)(23)(24)(2
6) (17)
Q8R3C0 MCMBP Mini-chromosome maintenance complex-binding protein 0 5 1 7 0 3 1 15 15.00 1.780E-02 73 ✓ (9)(23)
Q61029-3 LAP2B Isoform Epsilon of Lamina-associated polypeptide 2, isoforms beta/delta/epsilon/gamma 1 10 1 11 0 7 2 28 14.00 2.253E-03 46 ✓ ✓ ✓ (26)
Q9JIF0-3 ANM1 Isoform 3 of Protein arginine N-methyltransferase 1 0 15 2 14 1 12 3 41 13.67 2.749E-04 40 ✓ ✓ (26) (17)
Q6IRU2 TPM4 Tropomyosin alpha-4 chain 1 13 1 15 1 12 3 40 13.33 1.517E-04 29 ✓ ✓ (9)(23)(26)
P25206 MCM3 DNA replication licensing factor MCM3 0 6 1 4 0 3 1 13 13.00 1.324E-02 92 ✓ ✓ (9)(23)
Q91V92 ACLY ATP-citrate synthase 1 2 0 5 0 5 1 12 12.00 2.539E-02 120 ✓ ✓ (9)(14)(23)(26) (17)
Q501J6 DDX17 Probable ATP-dependent RNA helicase DDX17 0 3 0 3 1 5 1 11 11.00 1.106E-02 72 ✓ ✓ ✓ (5)(9)(23)(26)
Q9WUM4 COR1C Coronin-1C 0 6 2 5 0 9 2 20 10.00 1.201E-02 53 ✓ ✓ (14)(26)
Q8VDM4 PSMD2 26S proteasome non-ATPase regulatory subunit 2 0 11 3 9 0 9 3 29 9.67 1.961E-03 100 ✓ ✓ (9)(19)(23)
Q8BGD9 IF4B Eukaryotic translation initiation factor 4B 2 15 2 17 1 13 5 45 9.00 3.755E-04 69 ✓ ✓ (26)
434
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
P63242 IF5A1 Eukaryotic translation initiation factor 5A-1 1 8 1 6 0 4 2 18 9.00 1.135E-02 17 ✓ (14)(23)(26) (17)
Q9WVA4 TAGL2 Transgelin-2 0 6 2 7 0 5 2 18 9.00 3.772E-03 22 ✓ ✓ (23)(26)
P30681 HMGB2 High mobility group protein B2 1 3 0 4 0 2 1 9 9.00 1.613E-02 24 ✓ (9)(23)(26)
P14733 LMNB1 Lamin-B1 0 4 1 3 0 2 1 9 9.00 1.613E-02 67 ✓ ✓ ✓ (9)(23)
Q11011 PSA Puromycin-sensitive aminopeptidase 0 4 1 3 0 2 1 9 9.00 1.613E-02 103 ✓ ✓ (5)(23)
Q921F2 TADBP TAR DNA-binding protein 43 0 3 1 3 0 3 1 9 9.00 1.324E-03 45 ✓ ✓ (14)(26)
A1BN54 A1BN54 Alpha actinin 1a 0 21 3 23 4 17 7 61 8.71 1.083E-03 103 ✓ ✓ (26)
Q791V5 MTCH2 Mitochondrial carrier homolog 2 1 4 1 6 0 6 2 16 8.00 3.320E-03 34 ✓ ✓ (9)(23)
Q9CPP0 NPM3 Nucleoplasmin-3 0 2 1 3 0 3 1 8 8.00 7.763E-03 19 ✓ (9)(19)(23)(26)
H7BWX9 H7BWX9 Small ubiquitin-related modifier 2 0 3 1 3 0 2 1 8 8.00 7.763E-03 6.0 ✓
Q62523 ZYX Zyxin 0 4 2 5 0 4 2 13 6.50 7.933E-03 61 ✓ ✓ ✓ (1)(9)(23)(26)
P61979-3 HNRPK Isoform 3 of Heterogeneous nuclear ribonucleoprotein K 5 21 3 16 1 17 9 54 6.00 1.434E-03 49 ✓ ✓
(5)(8)(14)(23)(2
4)(26)
G3UX26 G3UX26 Voltage-dependent anion-selective channel protein 2 (Fragment) 5 23 3 20 4 26 12 69 5.75 4.815E-04 30 ✓ ✓
Q8BGQ7 SYAC Alanine--tRNA ligase, cytoplasmic 1 5 1 8 1 4 3 17 5.67 1.780E-02 107 ✓ ✓ (23)(26) (17)
P17751 TPIS Triosephosphate isomerase 2 12 3 9 1 11 6 32 5.33 1.193E-03 32 ✓ ✓ (5)(19)(23)(26) (17)
Q9CQ65 MTAP S-methyl-5'-thioadenosine phosphorylase 2 11 3 14 3 16 8 41 5.13 1.798E-03 31 ✓ ✓ (9)(23)(26) (17)
Q8VHX6-2 FLNC Isoform 2 of Filamin-C 0 2 1 1 0 2 1 5 5.00 4.742E-02 287 ✓ (9)(23)
Q9CZY3 UB2V1 Ubiquitin-conjugating enzyme E2 variant 1 0 2 0 2 1 1 1 5 5.00 4.742E-02 16 ✓ (23)
P07356 ANXA2 Annexin A2 3 23 7 25 5 24 15 72 4.80 1.240E-04 39 ✓ ✓ (8)(9)(23)(26) (17)
P68254-2 1433T Isoform 2 of 14-3-3 protein theta 1 9 1 10 3 5 5 24 4.80 1.910E-02 28 ✓ ✓ ✓ (5)(8)(23)(26) (17)
Q61024 ASNS Asparagine synthetase [glutamine-hydrolyzing] 0 8 3 11 4 14 7 33 4.71 1.472E-02 64 ✓ ✓ (9)(23)(26)
Q02053 UBA1 Ubiquitin-like modifier-activating enzyme 1 0 7 4 9 2 12 6 28 4.67 1.680E-02 118 ✓ (5)(9)(14)(23)
P18760 COF1 Cofilin-1 3 9 2 10 1 9 6 28 4.67 3.882E-04 19 ✓ ✓ ✓ (14)(19)(23)(26)
P46935 NEDD4 E3 ubiquitin-protein ligase NEDD4 8 33 10 44 8 43 26 120 4.62 9.338E-04 103 ✓ ✓ (1)(20)(26)
Q60749 KHDR1 KH domain-containing, RNA-binding, signal transduction-associated protein 1 1 3 1 4 1 6 3 13 4.33 1.944E-02 48 ✓ ✓ (14)(26)
J3QPE8 J3QPE8 MCG16555 4 9 1 10 2 10 7 29 4.14 1.473E-03 31 ✓ ✓ ✓
A2AL12 A2AL12 Heterogeneous nuclear ribonucleoprotein A3 4 13 3 13 3 14 10 40 4.00 2.920E-05 35 ✓ ✓ (5)(26)
Q7TPV4 MBB1A Myb-binding protein 1A 4 12 5 8 0 14 9 34 3.78 2.335E-02 152 ✓ (26)
Q9Z2X1 HNRPF Heterogeneous nuclear ribonucleoprotein F 5 21 7 25 6 21 18 67 3.72 3.567E-04 46 ✓ (23)(26) (17)
P58252 EF2 Elongation factor 2 7 20 4 23 7 20 18 63 3.50 4.472E-04 95 ✓ ✓
(5)(8)(14)(23)(2
6) (17)
435
No.
6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc 6AzGlcNAc
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intracel-
lular
Exclu-
sively
Extracellu-
lar, Lyso-
somal,
Lumenal Both
GlcN
Az
Gal-
NAz
Previous O-
GlcNAc
Proteomics
Identification
Previous
Mucin
Pro-
teomics
Identifica-
tion
359
360
361
362
363
364
365
366
E9PVM7 E9PVM7 Glutathione S-transferase Mu 5 (Fragment) 1 3 1 2 0 2 2 7 3.50 2.411E-02 26 ✓
B7FAU9 B7FAU9 Filamin, alpha 2 19 8 16 5 17 15 52 3.47 3.159E-03 280 ✓ ✓ (26)
P16858 G3P Glyceraldehyde-3-phosphate dehydrogenase 20 64 21 67 20 67 61 198 3.25 1.697E-06 36 ✓ ✓
(1)(2)(4)(6)(8)(2
3)(24)(26)
E9Q7H5 E9Q7H5 Uncharacterized protein 5 11 3 11 3 13 11 35 3.18 1.058E-03 33 ✓ (5)
P57780 ACTN4 Alpha-actinin-4 0 8 2 10 6 7 8 25 3.13 4.531E-02 105 ✓ (9)(23) (17)
P10107 ANXA1 Annexin A1 5 16 5 19 6 14 16 49 3.06 1.798E-03 39 ✓ ✓ (26) (17)
E9Q070 E9Q070 Uncharacterized protein 1 3 1 4 1 2 3 9 3.00 2.572E-02 34 ✓
Q8C1B7-3 SEP11 Isoform 3 of Septin-11 0 2 1 2 1 2 2 6 3.00 1.613E-02 49 ✓
436
Table 6.2. Proteins identified using GlcNAz enrichment. NIH 3T3 were treated in triplicate with either Ac4GlcNAz
(200 µM, +) or Ac4GlcNAc (200 µM, -) for 16 hours. At this time the cell lysates were subjected to CuAAC with alkyne-biotin,
followed by enrichment with streptavidin beads and on-bead trypsinolysis. Labeled proteins were selected as those that were
represented by at least 1 unique-peptide in each Ac4GlcNAz treated sample, a total of at least 3 spectral-counts from the same
three samples, and at least a total of 3 times more spectral counts in the Ac4GlcNAz treated samples compared to Ac4GlcNAc.
Blue indicates proteins previously identified in O-GlcNAc proteomic studies, purple indicates proteins identified in both O-
GlcNAc and O-linked mucin proteomic studies and red indicates proteins identified in only O-linked mucin proteomic studies.
Novel proteins that were identified in this study are indicated in white.
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
P27546-2 MAP4 Isoform 2 of Microtubule-associated protein 4 0 35 0 31 0 35 0 101 N/A 1.461E-05 117 ✓ ✓ ✓ (19)(23)(26)
Q80X50-5 UBP2L Isoform 5 of Ubiquitin-associated protein 2-like 0 28 0 26 0 30 0 84 N/A 1.716E-05 117 ✓ ✓
(5)(9)(19)(23
)(26)
Q80X50-2 UBP2L Isoform 2 of Ubiquitin-associated protein 2-like 0 27 0 25 0 29 0 81 N/A 1.983E-05 107 ✓ ✓
(5)(9)(19)(23
)(26)
P30416 FKBP4 Peptidyl-prolyl cis-trans isomerase FKBP4 0 21 0 21 0 18 0 60 N/A 3.688E-05 52 ✓ ✓ (5)(23)(26)
Q61191 HCFC1 Host cell factor 1 0 17 0 18 0 25 0 60 N/A 1.358E-03 210 ✓ ✓ ✓
(1)(2)(13)(19
)(20)(21)(22)
(23)(24)(26)
Q3THK7 GUAA GMP synthase [glutamine-hydrolyzing] 0 20 0 16 0 19 0 55 N/A 1.077E-04 77 ✓ ✓ (26)
Q8VCQ8 Q8VCQ8 Caldesmon 1 0 15 0 17 0 17 0 49 N/A 1.647E-05 60 ✓ ✓ ✓ (26)
P40124 CAP1 Adenylyl cyclase-associated protein 1 0 17 0 15 0 15 0 47 N/A 1.944E-05 52 ✓ ✓ ✓ (23)(26)
Q9QUR6 PPCE Prolyl endopeptidase 0 14 0 12 0 20 0 46 N/A 3.098E-03 81 ✓ ✓ (26) (17)
Q3UZ39 LRRF1 Leucine-rich repeat flightless-interacting protein 1 0 14 0 14 0 18 0 46 N/A 3.264E-04 79 ✓ ✓ (23)(26)
Q3TLH4-5 PRC2C Isoform 5 of Protein PRRC2C 0 18 0 17 0 11 0 46 N/A 2.175E-03 302 ✓ ✓ (19)(23)(26)
Q61584-5 FXR1 Isoform D of Fragile X mental retardation syndrome-related protein 1 0 14 0 13 0 18 0 45 N/A 6.030E-04 70 ✓ ✓
Q9ERG0 LIMA1 LIM domain and actin-binding protein 1 0 15 0 15 0 14 0 44 N/A 1.595E-06 84 ✓ ✓ (9)(23)(26)
Q63850 NUP62 Nuclear pore glycoprotein p62 0 13 0 16 0 14 0 43 N/A 8.387E-05 53 ✓ ✓
(2)(19)(23)(2
6) (17)
G3X928 G3X928 SEC23-interacting protein 0 13 0 15 0 12 0 40 N/A 1.116E-04 111 ✓ ✓ ✓ (2)(9)
O88532 ZFR Zinc finger RNA-binding protein 0 13 0 11 0 15 0 39 N/A 3.546E-04 117 ✓ ✓
(1)(5)(12)(13
)(19)(20)(26)
437
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Q99PG2 OGFR Opioid growth factor receptor 0 11 0 18 0 10 0 39 N/A 6.672E-03 71 ✓ ✓
Q8K4Z5 SF3A1 Splicing factor 3A subunit 1 0 13 0 12 0 13 0 38 N/A 2.864E-06 89 ✓ ✓ (23)(26) (17)
Q9DCL9 PUR6 Multifunctional protein ADE2 0 14 0 10 0 14 0 38 N/A 6.852E-04 47 ✓ ✓
(18)(19)(23)(
26)
Q9Z110-2 P5CS Isoform Short of Delta-1-pyrroline-5-carboxylate synthase 0 8 0 12 0 13 0 33 N/A 1.971E-03 87 ✓ ✓ (9)(23)(26)
A2AMY5 A2AMY5 Ubiquitin-associated protein 2 0 12 0 13 0 8 0 33 N/A 1.971E-03 118 ✓ ✓ ✓
B1AU75 B1AU75 Nuclear autoantigenic sperm protein 0 7 0 15 0 10 0 32 N/A 1.025E-02 84 ✓ ✓ (26)
Q3TW96 UAP1L UDP-N-acetylhexosamine pyrophosphorylase-like protein 1 0 11 0 10 0 10 0 31 N/A 6.452E-06 57 ✓ ✓ ✓ (9)
Q9Z1F9 SAE2 SUMO-activating enzyme subunit 2 0 8 0 11 0 12 0 31 N/A 1.006E-03 71 ✓ ✓ ✓ (26)
Q9QZM0 UBQL2 Ubiquilin-2 0 9 0 10 0 12 0 31 N/A 3.035E-04 67 ✓ ✓
Q9CZD3 SYG Glycine--tRNA ligase 0 10 0 9 0 11 0 30 N/A 6.521E-05 82 ✓ ✓ ✓
(9)(14)(23)(2
6)
Q6XLQ8 Q6XLQ8 Calumenin 0 9 0 8 0 13 0 30 N/A 2.814E-03 37 ✓ ✓ (9)
O70318 E41L2 Band 4.1-like protein 2 0 9 0 13 0 8 0 30 N/A 2.814E-03 110 ✓ ✓ ✓ (20)(23)
P58871 TB182 182 kDa tankyrase-1-binding protein 0 8 0 10 0 12 0 30 N/A 9.781E-04 182 ✓ ✓ ✓
(1)(20)(23)(2
6)
O35887 CALU Calumenin 0 10 0 8 0 11 0 29 N/A 3.936E-04 37 ✓ ✓ (23) (16)(17)
Q9DBR7 MYPT1 Protein phosphatase 1 regulatory subunit 12A 0 9 0 13 0 7 0 29 N/A 5.397E-03 115 ✓ ✓ ✓
(1)(5)(9)(19)(
20)(26)
Q5SUT0 Q5SUT0 Ewing sarcoma breakpoint region 1 0 8 0 11 0 9 0 28 N/A 4.511E-04 65 ✓ ✓ (9)(26)
Q3UMF0-4 COBL1 Isoform 4 of Cordon-bleu protein-like 1 0 8 0 10 0 10 0 28 N/A 1.510E-04 130 ✓ ✓ ✓
A2A6U3 A2A6U3 Septin 9 0 8 0 8 0 11 0 27 N/A 8.438E-04 64 ✓ ✓ (26)
Q80ZX0 Q80ZX0 Protein Sec24b 0 10 0 9 0 8 0 27 N/A 9.888E-05 136 ✓ ✓ (2)
G3X972 G3X972 Protein Sec24c 0 7 0 11 0 9 0 27 N/A 1.462E-03 119 ✓ ✓ ✓ (2)
Q8CGF7 TCRG1 Transcription elongation regulator 1 0 9 0 9 0 9 0 27 N/A 0.000E+00 124 ✓ ✓ (19)(23)
Q9CT10 RANB3 Ran-binding protein 3 0 9 0 9 0 9 0 27 N/A 0.000E+00 53 ✓ ✓
Q3UMF0-3 COBL1 Isoform 3 of Cordon-bleu protein-like 1 0 8 0 9 0 10 0 27 N/A 9.888E-05 129 ✓ ✓ ✓
E9Q4Q2 E9Q4Q2 Splicing factor 1 0 10 0 7 0 10 0 27 N/A 8.438E-04 60 ✓ ✓
Q00519 XDH Xanthine dehydrogenase/oxidase 0 12 0 4 0 10 0 26 N/A 2.265E-02 147 ✓ ✓ (9)(26)
Q60865 CAPR1 Caprin-1 0 8 0 8 0 10 0 26 N/A 2.020E-04 78 ✓ ✓ ✓ (19)(23)(26)
E9Q3G8 E9Q3G8 Protein Nup153 0 8 0 8 0 9 0 25 N/A 1.520E-05 152 ✓ ✓ (2)(12)(20)
438
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
Q7TQH0-2 ATX2L Isoform 2 of Ataxin-2-like protein 0 8 0 9 0 8 0 25 N/A 1.520E-05 113 ✓ ✓ ✓ (19)(23)
Q80U93 NU214 Nuclear pore complex protein Nup214 0 7 0 10 0 8 0 25 N/A 6.996E-04 213 ✓ ✓
(1)(2)(19)(20
)(23)(26)
Q7M6Y3-6 PICA Isoform 6 of Phosphatidylinositol-binding clathrin assembly protein 0 4 0 8 0 12 0 24 N/A 2.572E-02 71 ✓ ✓ (17)
Q6PFD9 Q6PFD9 Nucleoporin 98 0 8 0 11 0 5 0 24 N/A 9.890E-03 125 ✓ ✓ (9)(20)
Q6PB44-2 PTN23 Isoform 2 of Tyrosine-protein phosphatase non-receptor type 23 0 10 0 7 0 7 0 24 N/A 1.324E-03 185 ✓ ✓ (9)
Q99K48 NONO Non-POU domain-containing octamer-binding protein 0 5 0 9 0 10 0 24 N/A 6.352E-03 55 ✓ ✓ ✓
(19)(23)(24)(
26)
P10852 4F2 4F2 cell-surface antigen heavy chain 0 9 0 5 0 9 0 23 N/A 4.535E-03 58 ✓ ✓ (10)(17)
Q9QXS6-3 DREB Isoform E2 of Drebrin 0 9 0 5 0 9 0 23 N/A 4.535E-03 72 ✓ ✓ ✓ (9)(23)
O09106 HDAC1 Histone deacetylase 1 0 8 0 5 0 10 0 23 N/A 6.185E-03 55 ✓ ✓ (9)(19)(23)
P98078 DAB2 Disabled homolog 2 0 6 0 10 0 7 0 23 N/A 3.098E-03 82 ✓ ✓ ✓
Q9WVG6-2 CARM1 Isoform 2 of Histone-arginine methyltransferase CARM1 0 7 0 6 0 9 0 22 N/A 1.143E-03 63 ✓ ✓ ✓ (5)(19)(23)
Q8K310 MATR3 Matrin-3 0 7 0 8 0 7 0 22 N/A 2.526E-05 95 ✓ ✓ (23)(26)
Q62418-3 DBNL Isoform 3 of Drebrin-like protein 0 6 0 6 0 10 0 22 N/A 5.328E-03 48 ✓ ✓ ✓ (23)(26)
Q8BK67 RCC2 Protein RCC2 0 9 0 5 0 8 0 22 N/A 3.650E-03 56 ✓ ✓ (23)
Q9JLM8 DCLK1 Serine/threonine-protein kinase DCLK1 0 7 0 7 0 7 0 21 N/A 0.000E+00 84 ✓ ✓ (5)(20)
Q8CH18 CCAR1 Cell division cycle and apoptosis regulator protein 1 0 8 0 8 0 5 0 21 N/A 2.192E-03 132 ✓ ✓ (19)(23)
Q8R317-2 UBQL1 Isoform 2 of Ubiquilin-1 0 6 0 5 0 10 0 21 N/A 1.016E-02 59 ✓ ✓
Q8R317 UBQL1 Ubiquilin-1 0 6 0 5 0 10 0 21 N/A 1.016E-02 62 ✓ ✓
F6T2Z7 F6T2Z7 Protein Cald1 (Fragment) 0 6 0 6 0 9 0 21 N/A 2.192E-03 41 ✓ ✓ ✓
P54728 RD23B UV excision repair protein RAD23 homolog B 0 8 0 6 0 6 0 20 N/A 5.620E-04 44 ✓ ✓ (5)(23) (17)
P45377 ALD2 Aldose reductase-related protein 2 0 6 0 4 0 10 0 20 N/A 1.944E-02 36 ✓ ✓ ✓ (9)(26)
Q8R050-2 ERF3A Isoform 2 of Eukaryotic peptide chain release factor GTP-binding subunit ERF3A 0 7 0 6 0 7 0 20 N/A 3.688E-05 69 ✓ ✓ (9)(23)(26)
P70372 ELAV1 ELAV-like protein 1 0 8 0 6 0 6 0 20 N/A 5.620E-04 36 ✓ ✓ (9)(19)(26)
P51125-3 ICAL Isoform 3 of Calpastatin 0 5 0 8 0 7 0 20 N/A 1.641E-03 80 ✓ ✓ (26)
P51859 HDGF Hepatoma-derived growth factor 0 7 0 7 0 6 0 20 N/A 3.688E-05 26 ✓ ✓ ✓ (23)(26)
Q9DBG5 PLIN3 Perilipin-3 0 5 0 6 0 9 0 20 N/A 5.167E-03 47 ✓ ✓ ✓
(1)(9)(20)(23
)
Q921F4 HNRLL Heterogeneous nuclear ribonucleoprotein L-like 0 8 0 8 0 4 0 20 N/A 7.490E-03 64 ✓
439
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
Q7TQI3 OTUB1 Ubiquitin thioesterase OTUB1 0 5 0 6 0 8 0 19 N/A 1.991E-03 31 ✓ ✓
(5)(9)(23)(26
)
Q05CL8 LARP7 La-related protein 7 0 5 0 6 0 8 0 19 N/A 1.991E-03 65 ✓ ✓ (23)
G5E8E1 G5E8E1 Leucine rich repeat (In FLII) interacting protein 1, isoform CRA_e 0 5 0 6 0 8 0 19 N/A 1.991E-03 49 ✓ ✓ ✓
G3UWA6 G3UWA6 4F2 cell-surface antigen heavy chain 0 7 0 4 0 7 0 18 N/A 3.883E-03 62 ✓ ✓ (10)
Q8BYK6 YTHD3 YTH domain family protein 3 0 6 0 6 0 6 0 18 N/A 0.000E+00 64 ✓ ✓ (1)(19)(20)
Q8C052 MAP1S Microtubule-associated protein 1S 0 6 0 6 0 5 0 17 N/A 7.021E-05 103 ✓ ✓ (9)(23)(26)
A2AMW0 A2AMW0 Capping protein (Actin filament) muscle Z-line, beta ] 0 6 0 7 0 4 0 17 N/A 3.016E-03 29 ✓ ✓ (5)(26)
Q64012-2 RALY Isoform 1 of RNA-binding protein Raly 0 7 0 3 0 7 0 17 N/A 1.316E-02 31 ✓ ✓
Q60967 PAPS1 Bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase 1 0 7 0 4 0 6 0 17 N/A 3.016E-03 71 ✓ ✓
O08529 CAN2 Calpain-2 catalytic subunit 0 8 0 3 0 6 0 17 N/A 1.754E-02 80 ✓ ✓
Q9Z1D1 EIF3G Eukaryotic translation initiation factor 3 subunit G 0 6 0 7 0 3 0 16 N/A 1.135E-02 36 ✓ ✓ ✓
(9)(19)(23)(2
6)
Q60737 CSK21 Casein kinase II subunit alpha 0 7 0 5 0 4 0 16 N/A 3.772E-03 45 ✓ ✓ (5)
P31230 AIMP1 Aminoacyl tRNA synthase complex-interacting multifunctional protein 1 0 6 0 5 0 5 0 16 N/A 8.922E-05 34 ✓ ✓ ✓ (1)
Q8K3Z9 PO121 Nuclear envelope pore membrane protein POM 121 0 5 0 5 0 6 0 16 N/A 8.922E-05 121 ✓ ✓ (1)
P19096 FAS Fatty acid synthase 0 4 0 6 0 5 0 15 N/A 9.781E-04 272 ✓ ✓ (5)(8)(9)(23)
P23198 CBX3 Chromobox protein homolog 3 0 5 0 4 0 6 0 15 N/A 9.781E-04 21 ✓ ✓ (23)(26)
Q64337 SQSTM Sequestosome-1 0 4 0 5 0 6 0 15 N/A 9.781E-04 48 ✓ ✓ (23)
Q8C7R4 UBA6 Ubiquitin-like modifier-activating enzyme 6 0 3 0 4 0 8 0 15 N/A 3.069E-02 118 ✓ ✓ (23)
Q3THS6 METK2 S-adenosylmethionine synthase isoform type-2 0 4 0 4 0 7 0 15 N/A 7.490E-03 44 ✓ ✓ (20)
Q6PHZ2-2 KCC2D Isoform 2 of Calcium/calmodulin-dependent protein kinase type II subunit delta 0 6 0 4 0 5 0 15 N/A 9.781E-04 54 ✓ ✓
P47930 FOSL2 Fos-related antigen 2 0 5 0 6 0 4 0 15 N/A 9.781E-04 35 ✓ ✓
J3QNB1 J3QNB1 La-related protein 1 0 7 0 3 0 5 0 15 N/A 1.235E-02 121 ✓ ✓
Q9JLV1 BAG3 BAG family molecular chaperone regulator 3 0 4 0 5 0 5 0 14 N/A 1.510E-04 62 ✓ ✓ ✓ (23)(26) (17)
Q9R0P5 DEST Destrin 0 4 0 4 0 6 0 14 N/A 2.192E-03 19 ✓ ✓ ✓ (9)(26)
P59326 YTHD1 YTH domain family protein 1 0 4 0 5 0 5 0 14 N/A 1.510E-04 61 ✓ ✓ (9)(19)
Q3TN34 Q3TN34 JRAB 0 4 0 7 0 3 0 14 N/A 1.780E-02 108 ✓ ✓ (26)
Q8BI72 CARF CDKN2A-interacting protein 0 4 0 4 0 6 0 14 N/A 2.192E-03 60 ✓ ✓ (23)
Q8K354 CBR3 Carbonyl reductase [NADPH] 3 0 4 0 4 0 6 0 14 N/A 2.192E-03 31 ✓ ✓
Q9WV55 VAPA Vesicle-associated membrane protein-associated protein A 0 4 0 3 0 6 0 13 N/A 7.966E-03 28 ✓ (9)(26)
440
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
Q99JF8 PSIP1 PC4 and SFRS1-interacting protein 0 4 0 5 0 4 0 13 N/A 2.020E-04 60 ✓ (9)(23)
Q8BVY0 Q8BVY0 Protein Rsl1d1 0 3 0 3 0 7 0 13 N/A 3.138E-02 50 ✓ ✓ (9)
B7ZCP4 B7ZCP4 Copine I 0 5 0 4 0 4 0 13 N/A 2.020E-04 53 ✓ ✓ (9)
P68037 UB2L3 Ubiquitin-conjugating enzyme E2 L3 0 5 0 3 0 5 0 13 N/A 2.890E-03 18 ✓ ✓ (26)
Q61081 CDC37 Hsp90 co-chaperone Cdc37 0 6 0 3 0 4 0 13 N/A 7.966E-03 45 ✓ ✓ (23)(26)
Q80YR5 SAFB2 Scaffold attachment factor B2 0 5 0 3 0 5 0 13 N/A 2.890E-03 112 ✓ ✓ (23)
Q60710 SAMH1 SAM domain and HD domain-containing protein 1 0 4 0 4 0 5 0 13 N/A 2.020E-04 73 ✓ ✓ ✓ (23)
Q91W50 CSDE1 Cold shock domain-containing protein E1 0 4 0 4 0 5 0 13 N/A 2.020E-04 89 ✓ ✓ (19)(23)
Q8R1X6 SPG20 Spartin 0 6 0 3 0 4 0 13 N/A 7.966E-03 73 ✓ ✓ (1)(9)(20)
Q921K2 Q921K2 Poly (ADP-ribose) polymerase family, member 1 0 4 0 4 0 5 0 13 N/A 2.020E-04 113 ✓ ✓
Q6NZD2 Q6NZD2 Sorting nexin 1 0 5 0 4 0 4 0 13 N/A 2.020E-04 59 ✓ ✓ ✓
O89110 CASP8 Caspase-8 0 3 0 5 0 5 0 13 N/A 2.890E-03 55 ✓ ✓
D3YXK2 SAFB1 Scaffold attachment factor B1 0 6 0 3 0 4 0 13 N/A 7.966E-03 105 ✓ ✓ ✓
O70310 NMT1 Glycylpeptide N-tetradecanoyltransferase 1 0 6 0 4 0 2 0 12 N/A 2.572E-02 57 ✓ ✓ (23) (17)
P49586 PCY1A Choline-phosphate cytidylyltransferase A 0 3 0 5 0 4 0 12 N/A 2.278E-03 42 ✓ ✓ (23)
Q6NXL1 Q6NXL1 Protein Sec24d 0 2 0 5 0 5 0 12 N/A 1.613E-02 113 ✓ ✓ (2)
P01899 HA11 H-2 class I histocompatibility antigen, D-B alpha chain 0 4 0 4 0 4 0 12 N/A 0.000E+00 41 ✓ ✓
P01897 HA1L H-2 class I histocompatibility antigen, L-D alpha chain 0 4 0 4 0 4 0 12 N/A 0.000E+00 41 ✓ ✓
Q99K51 PLST Plastin-3 0 2 0 5 0 5 0 12 N/A 1.613E-02 71 ✓ ✓
Q62426 CYTB Cystatin-B 0 4 0 5 0 3 0 12 N/A 2.278E-03 11 ✓ ✓
Q9CXW3 CYBP Calcyclin-binding protein 0 4 0 4 0 3 0 11 N/A 3.882E-04 27 ✓ ✓ (9)(26)
Q9WTK5 NFKB2 Nuclear factor NF-kappa-B p100 subunit 0 3 0 4 0 4 0 11 N/A 3.882E-04 97 ✓ ✓ ✓ (9)(23)(26)
O70305-2 ATX2 Isoform 2 of Ataxin-2 0 3 0 3 0 5 0 11 N/A 5.328E-03 129 ✓ ✓ ✓ (9)
O55137 ACOT1 Acyl-coenzyme A thioesterase 1 0 4 0 3 0 4 0 11 N/A 3.882E-04 46 ✓ ✓ (9)
A2AVJ7 A2AVJ7 Ribosome binding protein 1 0 2 0 4 0 5 0 11 N/A 1.417E-02 158 ✓ ✓ (9)
P83741-4 WNK1 Isoform 4 of Serine/threonine-protein kinase WNK1 0 2 0 5 0 4 0 11 N/A 1.417E-02 225 ✓ ✓
(5)(13)(19)(2
3)
Q76MZ3 2AAA Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform 0 4 0 3 0 4 0 11 N/A 3.882E-04 65 ✓ ✓ (4)(9)(14)
Q08093 CNN2 Calponin-2 0 4 0 3 0 4 0 11 N/A 3.882E-04 33 ✓ ✓ ✓ (23)(26)
P70288 HDAC2 Histone deacetylase 2 0 5 0 3 0 3 0 11 N/A 5.328E-03 55 ✓ ✓ (23)
O70551 SRPK1 SRSF protein kinase 1 0 4 0 4 0 3 0 11 N/A 3.882E-04 73 ✓ ✓ (23)
441
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
P32921-2 SYWC Isoform 2 of Tryptophan--tRNA ligase, cytoplasmic 0 4 0 2 0 5 0 11 N/A 1.417E-02 54 ✓ ✓ (14)(23)
Q9Z1A1 Q9Z1A1 Protein Tfg 0 4 0 4 0 3 0 11 N/A 3.882E-04 43 ✓ ✓
G3X9V0 G3X9V0 MCG22048, isoform CRA_a 0 3 0 3 0 5 0 11 N/A 5.328E-03 26 ✓ ✓
F6TQN9 F6TQN9 Disabled homolog 2 (Fragment) 0 3 0 5 0 3 0 11 N/A 5.328E-03 68 ✓ ✓
E9Q7W0 E9Q7W0 Recombining-binding protein suppressor of hairless 0 4 0 3 0 4 0 11 N/A 3.882E-04 54 ✓ ✓
E9Q7G0 E9Q7G0 Protein Numa1 0 3 0 5 0 3 0 11 N/A 5.328E-03 236 ✓ ✓
Q8C156 CND2 Condensin complex subunit 2 0 3 0 3 0 4 0 10 N/A 5.620E-04 82 ✓ ✓ (9)(23)
Q99NB8 UBQL4 Ubiquilin-4 0 2 0 3 0 5 0 10 N/A 1.944E-02 64 ✓ ✓ ✓ (9)
G3X8Q0 G3X8Q0 Trans-acting transcription factor 1 0 4 0 3 0 3 0 10 N/A 5.620E-04 80 ✓ ✓ ✓ (9)
Q9D5T0 ATAD1 ATPase family AAA domain-containing protein 1 0 3 0 4 0 3 0 10 N/A 5.620E-04 41 ✓ ✓ (23)
Q91Z38 TTC1 Tetratricopeptide repeat protein 1 0 2 0 3 0 5 0 10 N/A 1.944E-02 33 ✓ ✓ (23)
Q8BTI8-3 SRRM2 Isoform 3 of Serine/arginine repetitive matrix protein 2 0 2 0 5 0 3 0 10 N/A 1.944E-02 285 ✓ ✓ (23)
Q8BJU0-2 SGTA Isoform 2 of Small glutamine-rich tetratricopeptide repeat-containing protein alpha 0 3 0 3 0 4 0 10 N/A 5.620E-04 34 ✓ (23)
Q8CFQ9 Q8CFQ9 Fusion, derived from t(12;16) malignant liposarcoma (Human) 0 3 0 3 0 4 0 10 N/A 5.620E-04 53 ✓ ✓
P54729 NUB1 NEDD8 ultimate buster 1 0 4 0 4 0 2 0 10 N/A 7.490E-03 70 ✓
O70494-2 SP3 Isoform 2 of Transcription factor Sp3 0 3 0 3 0 4 0 10 N/A 5.620E-04 78 ✓ ✓
E9Q066 E9Q066 La-related protein 4 0 3 0 3 0 4 0 10 N/A 5.620E-04 80 ✓ ✓
P61222 ABCE1 ATP-binding cassette sub-family E member 1 0 2 0 3 0 4 0 9 N/A 6.533E-03 67 ✓ (9)(23)(26)
Q5UE59 Q5UE59 Kinesin light chain 1 0 2 0 3 0 4 0 9 N/A 6.533E-03 62 ✓ ✓ (9)
Q7TSJ2-3 MAP6 Isoform 3 of Microtubule-associated protein 6 0 3 0 3 0 3 0 9 N/A 0.000E+00 33 ✓ ✓ ✓ (5)
Q924B0 Q924B0 Inositol (Myo)-1(Or 4)-monophosphatase 1 0 3 0 2 0 4 0 9 N/A 6.533E-03 30 ✓ (26)
Q6PDL0 DC1L2 Cytoplasmic dynein 1 light intermediate chain 2 0 5 0 2 0 2 0 9 N/A 3.994E-02 54 ✓ (23)
P04095 PR2C2 Prolactin-2C2 0 5 0 2 0 2 0 9 N/A 3.994E-02 25 ✓ ✓
Q9WU78 PDC6I Programmed cell death 6-interacting protein 0 2 0 3 0 4 0 9 N/A 6.533E-03 96 ✓ ✓ ✓
Q9R0X4 ACOT9 Acyl-coenzyme A thioesterase 9, mitochondrial 0 3 0 3 0 3 0 9 N/A 0.000E+00 51 ✓ ✓
Q9DBC7 KAP0 cAMP-dependent protein kinase type I-alpha regulatory subunit 0 2 0 3 0 4 0 9 N/A 6.533E-03 43 ✓ ✓
Q91YT7 Q91YT7 Protein Ythdf2 0 3 0 3 0 3 0 9 N/A 0.000E+00 62 ✓ ✓
Q8VE88-2 F1142 Isoform 2 of Protein FAM114A2 0 3 0 3 0 3 0 9 N/A 0.000E+00 53 ✓ ✓
D3YUW8 D3YUW8 Pogo transposable element with ZNF domain 0 4 0 3 0 2 0 9 N/A 6.533E-03 145 ✓ ✓
Q3UPH1 PRRC1 Protein PRRC1 0 3 0 2 0 3 0 8 N/A 1.324E-03 46 ✓ ✓ (9)(19)(26) (17)
442
N
o.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
Q8BH97 RCN3 Reticulocalbin-3 0 2 0 2 0 4 0 8 N/A 1.613E-02 38 ✓ ✓ (17)
E9QP49 E9QP49 EH domain-binding protein 1-like protein 1 0 3 0 2 0 3 0 8 N/A 1.324E-03 185 ✓ (9)
Q8VCF0 MAVS Mitochondrial antiviral-signaling protein 0 2 0 3 0 3 0 8 N/A 1.324E-03 53 ✓ ✓ ✓ (5)(20)(23)
O09172 GSH0 Glutamate--cysteine ligase regulatory subunit 0 2 0 2 0 4 0 8 N/A 1.613E-02 31 ✓ ✓ (23)(26)
Q9Z1Z0-2 USO1 Isoform 2 of General vesicular transport factor p115 0 1 0 3 0 4 0 8 N/A 3.902E-02 100 ✓ ✓ (23)
Q8K327 CHAP1 Chromosome alignment-maintaining phosphoprotein 1 0 2 0 4 0 2 0 8 N/A 1.613E-02 88 ✓ (23)
Q3UY34 CL043 Uncharacterized protein C12orf43 homolog 0 3 0 3 0 2 0 8 N/A 1.324E-03 28 ✓ ✓ (23)
Q3UPF5-2 ZCCHV Isoform 2 of Zinc finger CCCH-type antiviral protein 1 0 2 0 4 0 2 0 8 N/A 1.613E-02 88 ✓ (23)
P43247 MSH2 DNA mismatch repair protein Msh2 0 3 0 2 0 3 0 8 N/A 1.324E-03 104 ✓ ✓ (23)
P63037 DNJA1 DnaJ homolog subfamily A member 1 0 4 0 2 0 2 0 8 N/A 1.613E-02 45 ✓ ✓ (19)(26)
Q99LA2 Q99LA2 Protein Zfp207 0 2 0 2 0 4 0 8 N/A 1.613E-02 50 ✓ ✓
Q6P5B5 Q6P5B5 Fragile X mental retardation syndrome-related protein 2 0 2 0 3 0 3 0 8 N/A 1.324E-03 74 ✓ ✓
Q6NSQ7 LTV1 Protein LTV1 homolog 0 3 0 1 0 3 0 7 N/A 2.490E-02 54 ✓ ✓ (9)(23)
P15307 REL Proto-oncogene c-Rel 0 3 0 3 0 1 0 7 N/A 2.490E-02 65 ✓ ✓ ✓ (9)(23)
P05627 JUN Transcription factor AP-1 0 3 0 1 0 3 0 7 N/A 2.490E-02 36 ✓ ✓ (9)(23)
Q9WV92-3 E41L3 Isoform 3 of Band 4.1-like protein 3 0 3 0 2 0 2 0 7 N/A 2.192E-03 102 ✓ ✓ (9)
Q6P4T3 Q6P4T3 Eyes absent 3 homolog (Drosophila) 0 2 0 2 0 3 0 7 N/A 2.192E-03 58 ✓ ✓ (9)
Q8JZK9 HMCS1 Hydroxymethylglutaryl-CoA synthase, cytoplasmic 0 2 0 2 0 3 0 7 N/A 2.192E-03 58 ✓ ✓ (5)
O55091 IMPCT Protein IMPACT 0 2 0 2 0 3 0 7 N/A 2.192E-03 36 ✓ (26)
Q9CPV4-3 GLOD4 Isoform 3 of Glyoxalase domain-containing protein 4 0 2 0 2 0 3 0 7 N/A 2.192E-03 31 ✓ ✓ (23)(26)
Q9JIH2 NUP50 Nuclear pore complex protein Nup50 0 3 0 1 0 3 0 7 N/A 2.490E-02 50 ✓ ✓ (23)
Q6P5D8 SMHD1 Structural maintenance of chromosomes flexible hinge domain-containing protein 1 0 2 0 2 0 3 0 7 N/A 2.192E-03 226 ✓ ✓ (23)
Q3TYX3 SMYD5 SET and MYND domain-containing protein 5 0 3 0 1 0 3 0 7 N/A 2.490E-02 47 ✓ (23)
Q69Z38 PEAK1 Pseudopodium-enriched atypical kinase 1 0 3 0 2 0 2 0 7 N/A 2.192E-03 191 ✓ ✓ (20)(23)
A2ATI9 A2ATI9 Golgi reassembly stacking protein 2 0 1 0 3 0 3 0 7 N/A 2.490E-02 45 ✓ ✓ (13)
Q99P91 GPNMB Transmembrane glycoprotein NMB 0 2 0 2 0 3 0 7 N/A 2.192E-03 64 ✓ ✓
Q5SUH7 Q5SUH7 Clathrin interactor 1 0 1 0 3 0 3 0 7 N/A 2.490E-02 68 ✓ ✓
E9QP59 E9QP59 Inner nuclear membrane protein Man1 0 2 0 3 0 2 0 7 N/A 2.192E-03 100 ✓ ✓
E9Q5E0 E9Q5E0 Myocyte-specific enhancer factor 2D 0 3 0 2 0 2 0 7 N/A 2.192E-03 54 ✓ ✓
P08207 S10AA Protein S100-A10 0 3 0 2 0 2 0 7 N/A 2.192E-03 11 ✓
443
N
o.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
Q62433 NDRG1 Protein NDRG1 0 2 0 2 0 2 0 6 N/A 0.000E+00 43 (23) (16)(17)
A2AAW9 A2AAW9 Eukaryotic translation initiation factor 2 subunit 3, X-linked 0 2 0 1 0 3 0 6 N/A 2.572E-02 37 ✓ ✓ (9)(26)
Q9CYA6 ZCHC8 Zinc finger CCHC domain-containing protein 8 0 1 0 3 0 2 0 6 N/A 2.572E-02 78 ✓ ✓ (9)(23)
Q9CR86 CHSP1 Calcium-regulated heat stable protein 1 0 2 0 2 0 2 0 6 N/A 0.000E+00 16 ✓ ✓ (9)(23)
Q03963 E2AK2 Interferon-induced, double-stranded RNA-activated protein kinase 0 2 0 3 0 1 0 6 N/A 2.572E-02 58 ✓ ✓ (9)(23)
Q8VDM6 HNRL1 Heterogeneous nuclear ribonucleoprotein U-like protein 1 0 2 0 2 0 2 0 6 N/A 0.000E+00 96 ✓ ✓ (9)
Q8K0C9 GMDS GDP-mannose 4,6 dehydratase 0 1 0 2 0 3 0 6 N/A 2.572E-02 42 ✓ ✓ (9)
Q3TL72 Q3TL72 NEDD8-activating enzyme E1 catalytic subunit 0 3 0 2 0 1 0 6 N/A 2.572E-02 50 ✓ (9)
B7ZCL8 B7ZCL8 55 kDa erythrocyte membrane protein 0 1 0 2 0 3 0 6 N/A 2.572E-02 50 ✓ (9)
Q8K298 ANLN Actin-binding protein anillin 0 2 0 1 0 3 0 6 N/A 2.572E-02 123 ✓ ✓ (23)(26)
Q9CWE0 FA54B Protein FAM54B 0 2 0 2 0 2 0 6 N/A 0.000E+00 32 ✓ ✓ (23)
P62774 MTPN Myotrophin 0 2 0 2 0 2 0 6 N/A 0.000E+00 13 ✓ ✓ (23)
O88622-2 PARG Isoform 2 of Poly(ADP-ribose) glycohydrolase 0 2 0 2 0 2 0 6 N/A 0.000E+00 104 ✓ ✓ (23)
P42669 PURA Transcriptional activator protein Pur-alpha 0 1 0 2 0 3 0 6 N/A 2.572E-02 35 ✓ ✓ (20)(23)
Q6NZN0-5 RBM26 Isoform 5 of RNA-binding protein 26 0 2 0 2 0 2 0 6 N/A 0.000E+00 111 ✓ ✓ (19)(23)
Q5SFM8-2 RBM27 Isoform 2 of RNA-binding protein 27 0 1 0 2 0 3 0 6 N/A 2.572E-02 113 ✓ ✓ (19)(23)
Q5PSV9 MDC1 Mediator of DNA damage checkpoint protein 1 0 3 0 1 0 2 0 6 N/A 2.572E-02 185 ✓ ✓ (14)(23)
P35235 PTN11 Tyrosine-protein phosphatase non-receptor type 11 0 2 0 1 0 3 0 6 N/A 2.572E-02 68 ✓ ✓ (14)(23)
P0C7T6 ATX1L Ataxin-1-like 0 2 0 2 0 2 0 6 N/A 0.000E+00 73 ✓ ✓ (1)(9)
Q8C2Q3 RBM14 RNA-binding protein 14 0 2 0 3 0 1 0 6 N/A 2.572E-02 69 ✓ ✓
(1)(20)(23)(2
4)
Q8BH80 Q8BH80 Vesicle-associated membrane protein, associated protein B and C 0 2 0 1 0 3 0 6 N/A 2.572E-02 27 ✓
Q9JJU8 SH3L1 SH3 domain-binding glutamic acid-rich-like protein 0 2 0 2 0 2 0 6 N/A 0.000E+00 13 ✓
P35123 UBP4 Ubiquitin carboxyl-terminal hydrolase 4 0 1 0 2 0 3 0 6 N/A 2.572E-02 108 ✓ ✓
E9Q7C1 E9Q7C1 Mediator of RNA polymerase II transcription subunit 15 0 2 0 2 0 2 0 6 N/A 0.000E+00 83 ✓ ✓
E9Q5L7 E9Q5L7 PHD finger protein 10 0 2 0 2 0 2 0 6 N/A 0.000E+00 27 ✓ ✓
B1ATZ0 B1ATZ0 HGF-regulated tyrosine kinase substrate 0 2 0 2 0 2 0 6 N/A 0.000E+00 86 ✓ ✓
P17047 LAMP2 Lysosome-associated membrane glycoprotein 2 0 1 0 2 0 2 0 5 N/A 7.490E-03 46 ✓ ✓ (9)(23) (3)(16)(17)
Q8VIJ6 SFPQ Splicing factor, proline- and glutamine-rich 0 2 0 2 0 1 0 5 N/A 7.490E-03 75 ✓ ✓
(5)(9)(19)(20
)(23)(24) (17)
Q8BPB5 FBLN3 EGF-containing fibulin-like extracellular matrix protein 1 0 2 0 1 0 2 0 5 N/A 7.490E-03 55 ✓ ✓ (16)
444
N
o.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
P15379-2 CD44 Isoform 13 of CD44 antigen 0 1 0 2 0 2 0 5 N/A 7.490E-03 40 ✓ ✓ (23) (10)(16)
Q9WTX5 SKP1 S-phase kinase-associated protein 1 0 2 0 1 0 2 0 5 N/A 7.490E-03 19 ✓ ✓ (9)(23)
Q9JKP5 MBNL1 Muscleblind-like protein 1 0 2 0 2 0 1 0 5 N/A 7.490E-03 37 ✓ ✓ ✓ (9)(23)
Q9CU62 SMC1A Structural maintenance of chromosomes protein 1A 0 1 0 2 0 2 0 5 N/A 7.490E-03 143 ✓ (9)(23)
Q8BT60 CPNE3 Copine-3 0 2 0 2 0 1 0 5 N/A 7.490E-03 60 ✓ (9)(23)
P11983-2 TCPA Isoform 2 of T-complex protein 1 subunit alpha 0 2 0 2 0 1 0 5 N/A 7.490E-03 55 ✓
(9)(14)(23)(2
6)
Q9Z1B5 MD2L1 Mitotic spindle assembly checkpoint protein MAD2A 0 2 0 1 0 2 0 5 N/A 7.490E-03 24 ✓ ✓ (9)
H3BJU7 H3BJU7 Rho guanine nucleotide exchange factor 2 0 2 0 1 0 2 0 5 N/A 7.490E-03 109 ✓ (9)
Q61595-2 KTN1 Isoform 2 of Kinectin 0 2 0 2 0 1 0 5 N/A 7.490E-03 138 ✓ (23)
Q9CR00 PSMD9 26S proteasome non-ATPase regulatory subunit 9 0 2 0 2 0 1 0 5 N/A 7.490E-03 25 ✓ ✓ (23)
Q8R2M2 TDIF2 Deoxynucleotidyltransferase terminal-interacting protein 2 0 1 0 2 0 2 0 5 N/A 7.490E-03 84 ✓ ✓ (23)
Q80YR4-2 ZN598 Isoform 2 of Zinc finger protein 598 0 2 0 2 0 1 0 5 N/A 7.490E-03 96 ✓ ✓ (23)
Q7TNV0 DEK Protein DEK 0 2 0 1 0 2 0 5 N/A 7.490E-03 43 ✓ ✓ (23)
B1AR09 B1AR09 Myeloid/lymphoid or mixed lineage-leukemia translocation to 6 homolog (Drosophila) 0 1 0 2 0 2 0 5 N/A 7.490E-03 111 ✓ ✓ (20)
Q8VI36-2 PAXI Isoform Alpha of Paxillin 0 1 0 2 0 2 0 5 N/A 7.490E-03 61 ✓ ✓ (14)(23)
P80317 TCPZ T-complex protein 1 subunit zeta 0 1 0 2 0 2 0 5 N/A 7.490E-03 58 ✓ (14)
Q8BP48 AMPM1 Methionine aminopeptidase 1 0 2 0 2 0 1 0 5 N/A 7.490E-03 43 ✓ ✓
Q8BH93 MISSL MAPK-interacting and spindle-stabilizing protein-like 0 2 0 1 0 2 0 5 N/A 7.490E-03 24 ✓ ✓
Q62219-6 TGFI1 Isoform 6 of Transforming growth factor beta-1-induced transcript 1 protein 0 2 0 2 0 1 0 5 N/A 7.490E-03 6.6 ✓
F6TWX0 F6TWX0 Nuclear transcription factor Y subunit alpha (Fragment) 0 2 0 2 0 1 0 5 N/A 7.490E-03 23 ✓ ✓
E9Q242 E9Q242 Adenylosuccinate lyase 0 2 0 2 0 1 0 5 N/A 7.490E-03 53 ✓ ✓
B7ZP47 B7ZP47 Wapal protein 0 2 0 2 0 1 0 5 N/A 7.490E-03 133 ✓ ✓
P11438 LAMP1 Lysosome-associated membrane glycoprotein 1 0 2 0 1 0 1 0 4 N/A 1.613E-02 44 ✓ ✓ (23) (3)(15)(16)
Q925B0-2 PAWR Isoform 2 of PRKC apoptosis WT1 regulator protein 0 2 0 1 0 1 0 4 N/A 1.613E-02 31 ✓ (9)(23)
Q8CDN6 TXNL1 Thioredoxin-like protein 1 0 1 0 2 0 1 0 4 N/A 1.613E-02 32 ✓ (9)(23)
Q3B7Z2-2 OSBP1 Isoform 2 of Oxysterol-binding protein 1 0 1 0 1 0 2 0 4 N/A 1.613E-02 62 ✓ (9)(23)
Q80UU9 PGRC2 Membrane-associated progesterone receptor component 2 0 2 0 1 0 1 0 4 N/A 1.613E-02 23 ✓ (9)
Q9Z0E6 GBP2 Interferon-induced guanylate-binding protein 2 0 2 0 1 0 1 0 4 N/A 1.613E-02 67 ✓ ✓ (9)
Q9R1J0 NSDHL Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating 0 1 0 1 0 2 0 4 N/A 1.613E-02 41 ✓ ✓ (9)
Q8BIH0-2 SP130 Isoform 2 of Histone deacetylase complex subunit SAP130 0 1 0 2 0 1 0 4 N/A 1.613E-02 93 ✓ ✓ (9)
445
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
Q8K1M6-4 DNM1L Isoform 4 of Dynamin-1-like protein 0 1 0 2 0 1 0 4 N/A 1.613E-02 69 ✓ (5)(9)
P13439 UMPS Uridine 5'-monophosphate synthase 0 1 0 2 0 1 0 4 N/A 1.613E-02 52 ✓ (26)
P35278 RAB5C Ras-related protein Rab-5C 0 1 0 1 0 2 0 4 N/A 1.613E-02 23 ✓ ✓ (23)(26)
Q64514-2 TPP2 Isoform Short of Tripeptidyl-peptidase 2 0 2 0 1 0 1 0 4 N/A 1.613E-02 138 ✓ ✓ (23)
Q60953-2 PML Isoform 2 of Protein PML 0 1 0 2 0 1 0 4 N/A 1.613E-02 93 ✓ (23)
P51432 PLCB3 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3 0 1 0 2 0 1 0 4 N/A 1.613E-02 139 ✓ (23)
O88291 ZN326 DBIRD complex subunit ZNF326 0 2 0 1 0 1 0 4 N/A 1.613E-02 65 ✓ (23)
Q8BQM4 HEAT3 HEAT repeat-containing protein 3 0 1 0 1 0 2 0 4 N/A 1.613E-02 74 ✓ (23)
Q9JKV1 ADRM1 Proteasomal ubiquitin receptor ADRM1 0 2 0 1 0 1 0 4 N/A 1.613E-02 42 ✓ ✓ (20)(23)
P00493 HPRT Hypoxanthine-guanine phosphoribosyltransferase 0 1 0 2 0 1 0 4 N/A 1.613E-02 25 ✓ ✓ (19)
Q922Y1 UBXN1 UBX domain-containing protein 1 0 1 0 1 0 2 0 4 N/A 1.613E-02 34 ✓ (1)(23)
E9Q9C5 E9Q9C5 V-type proton ATPase 16 kDa proteolipid subunit (Fragment) 0 1 0 1 0 2 0 4 N/A 1.613E-02 15 ✓ ✓
Q8K1M3 Q8K1M3 Protein kinase, cAMP dependent regulatory, type II alpha 0 1 0 1 0 2 0 4 N/A 1.613E-02 46 ✓
Q80U35 ARHGH Rho guanine nucleotide exchange factor 17 0 1 0 1 0 2 0 4 N/A 1.613E-02 222 ✓
Q6ZQK5 ACAP2 Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 2 0 1 0 1 0 2 0 4 N/A 1.613E-02 87 ✓ ✓
Q6Q2Z6 ACOT5 Acyl-coenzyme A thioesterase 5 0 1 0 1 0 2 0 4 N/A 1.613E-02 47 ✓
G3UW40 G3UW40 MCG4620, isoform CRA_b 0 2 0 1 0 1 0 4 N/A 1.613E-02 93 ✓ ✓
P02469 LAMB1 Laminin subunit beta-1 0 1 0 1 0 1 0 3 N/A 0.000E+00 197 ✓ (9) (16)(17)
Q6PGH2 HN1L Hematological and neurological expressed 1-like protein 0 1 0 1 0 1 0 3 N/A 0.000E+00 20 ✓ ✓ (9)(23)(26) (16)
Q62348 TSN Translin 0 1 0 1 0 1 0 3 N/A 0.000E+00 26 ✓ ✓ ✓ (9)(23)(26)
Q9Z2M7 PMM2 Phosphomannomutase 2 0 1 0 1 0 1 0 3 N/A 0.000E+00 28 ✓ (9)(23)
Q8K1R7 NEK9 Serine/threonine-protein kinase Nek9 0 1 0 1 0 1 0 3 N/A 0.000E+00 107 ✓ ✓ (9)(23)
Q9JIF7 COPB Coatomer subunit beta 0 1 0 1 0 1 0 3 N/A 0.000E+00 107 ✓ ✓ (9)(14)(23)
Q91YS8 KCC1A Calcium/calmodulin-dependent protein kinase type 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 42 ✓ ✓ (9)
Q61164 CTCF Transcriptional repressor CTCF 0 1 0 1 0 1 0 3 N/A 0.000E+00 84 ✓ (9)
Q3TT92 Q3TT92 Dihydropyrimidinase-related protein 3 0 1 0 1 0 1 0 3 N/A 0.000E+00 62 ✓ ✓ (9)
Q99LS3 SERB Phosphoserine phosphatase 0 1 0 1 0 1 0 3 N/A 0.000E+00 25 ✓ ✓ (26)
Q9QYC0-2 ADDA Isoform 2 of Alpha-adducin 0 1 0 1 0 1 0 3 N/A 0.000E+00 70 ✓ (23)
Q9DBR1-2 XRN2 Isoform 2 of 5'-3' exoribonuclease 2 0 1 0 1 0 1 0 3 N/A 0.000E+00 108 ✓ ✓ (23)
Q8CIN4 PAK2 Serine/threonine-protein kinase PAK 2 0 1 0 1 0 1 0 3 N/A 0.000E+00 58 ✓ (23)
446
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
Q6P5G6 UBXN7 UBX domain-containing protein 7 0 1 0 1 0 1 0 3 N/A 0.000E+00 52 ✓ ✓ (23)
Q3UHX0 NOL8 Nucleolar protein 8 0 1 0 1 0 1 0 3 N/A 0.000E+00 129 ✓ ✓ (23)
P46061 RAGP1 Ran GTPase-activating protein 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 64 ✓ (23)
O08997 ATOX1 Copper transport protein ATOX1 0 1 0 1 0 1 0 3 N/A 0.000E+00 7.3 ✓ (23)
D3YUC9 D3YUC9 Methionine-R-sulfoxide reductase B3, mitochondrial 0 1 0 1 0 1 0 3 N/A 0.000E+00 12 ✓ ✓
Q9JLJ5 ELOV1 Elongation of very long chain fatty acids protein 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 33 ✓ ✓ ✓
P46978 STT3A Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A 0 1 0 1 0 1 0 3 N/A 0.000E+00 81 ✓
P14719-2 ILRL1 Isoform B of Interleukin-1 receptor-like 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 39 ✓ ✓
Q9CR51 VATG1 V-type proton ATPase subunit G 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 14 ✓ ✓
Q60929-2 MEF2A Isoform 2 of Myocyte-specific enhancer factor 2A 0 1 0 1 0 1 0 3 N/A 0.000E+00 53 ✓ ✓
Q5SWD9-2 TSR1 Isoform 2 of Pre-rRNA-processing protein TSR1 homolog 0 1 0 1 0 1 0 3 N/A 0.000E+00 78 ✓
Q3TZX8-3 NOL9 Isoform 3 of Polynucleotide 5'-hydroxyl-kinase NOL9 0 1 0 1 0 1 0 3 N/A 0.000E+00 71 ✓ ✓
J3JS94 J3JS94 L antigen family member 3 0 1 0 1 0 1 0 3 N/A 0.000E+00 12 ✓ ✓
A2AG83 A2AG83 26S proteasome non-ATPase regulatory subunit 10 0 1 0 1 0 1 0 3 N/A 0.000E+00 16 ✓ ✓
F8WGW3 F8WGW3 S1 RNA-binding domain-containing protein 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 110
O54931-2 AKAP2 Isoform 2 of A-kinase anchor protein 2 0 15 1 17 0 17 1 49 49.00 2.785E-05 97 ✓ ✓ ✓ (9)(23)(26)
Q6DFW4 NOP58 Nucleolar protein 58 1 13 0 15 0 14 1 42 42.00 3.344E-05 60 ✓ ✓ ✓ (9)(23)(26)
Q8BFW7 LPP Lipoma-preferred partner homolog 0 11 1 15 0 14 1 40 40.00 4.786E-04 66 ✓ ✓ ✓
(9)(20)(23)(2
6) (16)
P80314 TCPB T-complex protein 1 subunit beta 1 10 0 9 0 19 1 38 38.00 1.819E-02 57 ✓ ✓ (5)(26)
E9PVC5 E9PVC5 Eukaryotic translation initiation factor 4 gamma 1 0 9 1 11 0 12 1 32 32.00 3.937E-04 175 ✓ ✓ ✓ (9)(12)
Q61033 LAP2A Lamina-associated polypeptide 2, isoforms alpha/zeta 1 11 0 8 0 13 1 32 32.00 2.274E-03 75 ✓ ✓ ✓ (23)(26)
P60335 PCBP1 Poly(rC)-binding protein 1 0 10 1 10 0 11 1 31 31.00 2.920E-05 38 ✓ ✓ ✓
(9)(14)(23)(2
6)
P25206 MCM3 DNA replication licensing factor MCM3 0 9 1 11 0 10 1 30 30.00 1.315E-04 92 ✓ ✓ (9)(23)
Q9D0E1-2 HNRPM Isoform 2 of Heterogeneous nuclear ribonucleoprotein M 1 14 1 17 0 26 2 57 28.50 7.165E-03 74 ✓ ✓ (9)(23)
P70698 PYRG1 CTP synthase 1 1 8 0 6 0 10 1 24 24.00 3.098E-03 67 ✓ ✓ (23)(26)
Q62167 DDX3X ATP-dependent RNA helicase DDX3X 0 6 1 5 0 11 1 22 22.00 2.061E-02 73 ✓ ✓ (23)(26)
Q60598 SRC8 Src substrate cortactin 0 8 1 6 0 8 1 22 22.00 7.163E-04 61 ✓ ✓ (20)(26)
Q8VHR5 P66B Transcriptional repressor p66-beta 0 5 1 8 0 8 1 21 21.00 3.198E-03 65 ✓ ✓
(1)(12)(20)(2
3)
447
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
Q80X90 FLNB Filamin-B 0 4 1 8 0 8 1 20 20.00 9.969E-03 278 ✓ ✓ (23)(26)
Q91VI7 RINI Ribonuclease inhibitor 0 6 1 5 0 8 1 19 19.00 3.126E-03 50 ✓ ✓ (23)
Q99K70 RRAGC Ras-related GTP-binding protein C 0 6 1 4 0 8 1 18 18.00 9.206E-03 44 ✓ ✓ (23)
Q9CQX2 CYB5B Cytochrome b5 type B 1 7 0 5 0 5 1 17 17.00 2.019E-03 16 ✓ ✓ (26)
Q61699-2 HS105 Isoform HSP105-beta of Heat shock protein 105 kDa 0 19 3 10 0 21 3 50 16.67 1.132E-02 92 ✓ ✓ (5)(9)(23)
Q8CI51 PDLI5 PDZ and LIM domain protein 5 0 5 2 13 0 15 2 33 16.50 2.980E-02 63 ✓ ✓ ✓
(1)(9)(23)(26
)
Q60854 SPB6 Serpin B6 0 5 1 5 0 6 1 16 16.00 4.472E-04 43 ✓ ✓ (26)
Q61029-3 LAP2B Isoform Epsilon of Lamina-associated polypeptide 2, isoforms beta/delta/epsilon/gamma 1 9 1 10 0 11 2 30 15.00 1.510E-04 46 ✓ ✓ ✓ (26)
P06151 LDHA L-lactate dehydrogenase A chain 1 12 1 15 1 15 3 42 14.00 2.020E-04 37 ✓ ✓ ✓
(6)(23)(24)(2
6) (17)
Q61990 PCBP2 Poly(rC)-binding protein 2 0 4 1 5 0 5 1 14 14.00 7.779E-04 38 ✓ ✓ ✓ (5)(14)(26) (17)
Q501J6 DDX17 Probable ATP-dependent RNA helicase DDX17 0 4 0 4 1 5 1 13 13.00 1.058E-03 72 ✓ ✓ ✓
(5)(9)(23)(26
)
A2AFJ1 A2AFJ1 Histone-binding protein RBBP7 0 3 1 3 0 6 1 12 12.00 2.539E-02 47 ✓ ✓ (26)
P16045 LEG1 Galectin-1 1 32 2 25 3 13 6 70 11.67 1.870E-02 15 ✓ ✓ (23) (17)
Q3U0V1 FUBP2 Far upstream element-binding protein 2 1 18 2 19 2 17 5 54 10.80 1.647E-05 77 ✓ ✓ (14)(23)(26)
Q9JIF0-3 ANM1 Isoform 3 of Protein arginine N-methyltransferase 1 0 10 2 9 1 13 3 32 10.67 1.921E-03 40 ✓ ✓ (26) (17)
Q9WUM4 COR1C Coronin-1C 0 6 2 8 0 6 2 20 10.00 3.126E-03 53 ✓ ✓ (14)(26)
D3Z5M2 D3Z5M2 Protein Gm10110 0 6 2 5 0 8 2 19 9.50 6.859E-03 68 ✓
Q91V92 ACLY ATP-citrate synthase 1 2 0 3 0 4 1 9 9.00 1.613E-02 120 ✓ ✓
(9)(14)(23)(2
6) (17)
Q11011 PSA Puromycin-sensitive aminopeptidase 0 3 1 3 0 3 1 9 9.00 1.324E-03 103 ✓ ✓ (5)(23)
Q8BGQ7 SYAC Alanine--tRNA ligase, cytoplasmic 1 8 1 7 1 11 3 26 8.67 3.098E-03 107 ✓ ✓ (23)(26) (17)
Q3UPL0-2 SC31A Isoform 2 of Protein transport protein Sec31A 1 8 2 8 0 10 3 26 8.67 9.640E-04 130 ✓ ✓ (5)(19)(26)
Q791V5 MTCH2 Mitochondrial carrier homolog 2 1 5 1 6 0 6 2 17 8.50 4.472E-04 34 ✓ ✓ (23)
P68254-2 1433T Isoform 2 of 14-3-3 protein theta 1 13 1 14 3 15 5 42 8.40 1.517E-04 28 ✓ ✓ ✓
(5)(8)(9)(23)(
26) (17)
Q6IRU2 TPM4 Tropomyosin alpha-4 chain 1 8 1 8 1 9 3 25 8.33 2.526E-05 29 ✓ ✓ (9)(23)(26)
Q8BGJ5 Q8BGJ5 MCG13402, isoform CRA_a 0 7 1 9 2 8 3 24 8.00 1.017E-03 57 ✓ ✓ (26)
A1BN54 A1BN54 Alpha actinin 1a 0 16 3 17 4 20 7 53 7.57 8.362E-04 103 ✓ ✓ (26)
448
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
Q62523 ZYX Zyxin 0 4 2 5 0 6 2 15 7.50 7.966E-03 61 ✓ ✓ ✓
(1)(9)(23)(26
)
Q61024 ASNS Asparagine synthetase [glutamine-hydrolyzing] 0 19 3 17 4 16 7 52 7.43 5.486E-04 64 ✓ ✓ (23)(26)
Q8BGD9 IF4B Eukaryotic translation initiation factor 4B 2 13 2 10 1 14 5 37 7.40 1.026E-03 69 ✓ ✓ (9)(26)
Q921F2 TADBP TAR DNA-binding protein 43 0 2 1 2 0 3 1 7 7.00 1.324E-02 45 ✓ ✓ (9)(14)(26)
P14733 LMNB1 Lamin-B1 0 2 1 2 0 3 1 7 7.00 1.324E-02 67 ✓ ✓ ✓ (23)
A2AL12 A2AL12 Heterogeneous nuclear ribonucleoprotein A3 4 19 3 24 3 22 10 65 6.50 2.511E-04 35 ✓ ✓ (5)(26)
P68510 1433F 14-3-3 protein eta 0 2 0 2 1 2 1 6 6.00 7.490E-03 28
P17751 TPIS Triosephosphate isomerase 2 11 3 10 1 14 6 35 5.83 1.921E-03 32 ✓ ✓
(5)(19)(23)(2
6) (17)
Q8VDM4 PSMD2 26S proteasome non-ATPase regulatory subunit 2 0 6 3 5 0 6 3 17 5.67 1.145E-02 100 ✓ ✓ (19)(23)
E9QAT0 E9QAT0 Fragile X mental retardation protein 1 homolog 1 4 1 3 0 4 2 11 5.50 3.126E-03 66
P18760 COF1 Cofilin-1 3 5 2 11 1 14 6 30 5.00 4.179E-02 19 ✓ ✓ ✓
(14)(19)(23)(
26)
P07742 RIR1 Ribonucleoside-diphosphate reductase large subunit 0 2 1 2 0 1 1 5 5.00 4.742E-02 90 ✓ ✓ (14)(23)(26)
J3QPE8 J3QPE8 MCG16555 4 11 1 10 2 12 7 33 4.71 1.193E-03 31 ✓ ✓ ✓
P61979-3 HNRPK Isoform 3 of Heterogeneous nuclear ribonucleoprotein K 5 15 3 11 1 15 9 41 4.56 3.772E-03 49 ✓ ✓
(5)(8)(14)(23
)(24)(26)
E9Q7H5 E9Q7H5 Uncharacterized protein 5 15 3 18 3 17 11 50 4.55 2.992E-04 33 ✓ (5)
G3UX26 G3UX26 Voltage-dependent anion-selective channel protein 2 (Fragment) 5 18 3 18 4 18 12 54 4.50 1.716E-05 30 ✓ ✓
Q9CQ65 MTAP S-methyl-5'-thioadenosine phosphorylase 2 12 3 13 3 11 8 36 4.50 1.510E-04 31 ✓ ✓ (23)(26) (17)
Q9WVA4 TAGL2 Transgelin-2 0 3 2 3 0 3 2 9 4.50 2.490E-02 22 ✓ ✓ (23)(26)
Q60749 KHDR1 KH domain-containing, RNA-binding, signal transduction-associated protein 1 1 6 1 4 1 3 3 13 4.33 1.944E-02 48 ✓ ✓ (9)(14)(26)
P10107 ANXA1 Annexin A1 5 21 5 21 6 23 16 65 4.06 2.566E-05 39 ✓ ✓ (9)(26) (17)
P08752 GNAI2 Guanine nucleotide-binding protein G(i) subunit alpha-2 1 3 1 4 1 4 3 11 3.67 1.324E-03 41 ✓ ✓ (20)(26)
P58252 EF2 Elongation factor 2 7 21 4 18 7 26 18 65 3.61 3.501E-03 95 ✓ ✓
(5)(8)(9)(14)(
23)(26) (17)
P10605 CATB Cathepsin B 1 4 1 5 2 5 4 14 3.50 2.111E-03 37 ✓ (26) (17)
P07356 ANXA2 Annexin A2 3 17 7 19 5 16 15 52 3.47 1.056E-03 39 ✓ ✓
(8)(9)(23)(26
) (17)
B7FAU9 B7FAU9 Filamin, alpha 2 14 8 15 5 21 15 50 3.33 1.388E-02 280 ✓ ✓ (9)(26)
P46935 NEDD4 E3 ubiquitin-protein ligase NEDD4 8 27 10 30 8 28 26 85 3.27 5.867E-05 103 ✓ ✓ (1)(20)(26)
449
No.
GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz GlcNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellu-
lar
Exclu-
sively
Extracel-
lular,
Lysoso-
mal,
Lumenal Both
6AzGl
cNAc
GalN
Az
Previous
O-GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
359 P16858 G3P Glyceraldehyde-3-phosphate dehydrogenase 20 63 21 62 20 61 61 186 3.05 3.925E-07 36 ✓ ✓
(1)(2)(4)(6)(8
)(9)(23)(24)(
26)
450
Table 6-3. Proteins identified using GalNAz enrichment. NIH 3T3 were treated in triplicate with either Ac4GalNAz
(200 µM, +) or Ac4GlcNAc (200 µM, -) for 16 hours. At this time the cell lysates were subjected to CuAAC with alkyne-biotin,
followed by enrichment with streptavidin beads and on-bead trypsinolysis. Labeled proteins were selected as those that were
represented by at least 1 unique-peptide in each Ac4GalNAz treated sample, a total of at least 3 spectral-counts from the same
three samples, and at least a total of 3 times more spectral counts in the Ac4GalNAz treated samples compared to Ac4GlcNAc.
Blue indicates proteins previously identified in O-GlcNAc proteomic studies, purple indicates proteins identified in both O-
GlcNAc and O-linked mucin proteomic studies and red indicates proteins identified in only O-linked mucin proteomic studies.
Novel proteins that were identified in this study are indicated in white.
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Q61191 HCFC1 Host cell factor 1 0 40 0 30 0 34 0 104 N/A 2.829E-04 210 ✓ ✓ ✓
(1)(2)(9)(13)(
19)(20)(21)(2
2)(23)(24)(26
)
Q80X50-5 UBP2L Isoform 5 of Ubiquitin-associated protein 2-like 0 31 0 39 0 33 0 103 N/A 1.396E-04 117 ✓ ✓
(5)(19)(23)(2
6)
Q80X50-2 UBP2L Isoform 2 of Ubiquitin-associated protein 2-like 0 30 0 38 0 32 0 100 N/A 1.568E-04 107 ✓ ✓
(5)(19)(23)(2
6)
Q8K4Z5 SF3A1 Splicing factor 3A subunit 1 0 27 0 31 0 32 0 90 N/A 3.964E-05 88.5 ✓ ✓ (9)(23)(26) (17)
Q6XLQ8 Q6XLQ8 Calumenin 0 25 0 29 0 28 0 82 N/A 2.214E-05 37.1 ✓ ✓
O35887 CALU Calumenin 0 25 0 24 0 24 0 73 N/A 2.110E-07 37.0 ✓ ✓ (9)(23) (16)(17)
Q7TQH0-2 ATX2L Isoform 2 of Ataxin-2-like protein 0 25 0 23 0 24 0 72 N/A 2.002E-06 113 ✓ ✓ ✓ (19)(23)
Q9DBR7 MYPT1 Protein phosphatase 1 regulatory subunit 12A 0 22 0 22 0 28 0 72 N/A 2.764E-04 115 ✓ ✓ ✓
(1)(5)(19)(20)
(26)
Q6PB44-2 PTN23 Isoform 2 of Tyrosine-protein phosphatase non-receptor type 23 0 25 0 25 0 22 0 72 N/A 1.788E-05 185 ✓ ✓
Q8CGF7 TCRG1 Transcription elongation regulator 1 0 24 0 23 0 24 0 71 N/A 2.358E-07 124 ✓ ✓ (19)(23)
Q60737 CSK21 Casein kinase II subunit alpha 0 23 0 22 0 25 0 70 N/A 1.213E-05 45.1 ✓ ✓ (5)
Q8CH18 CCAR1 Cell division cycle and apoptosis regulator protein 1 0 21 0 23 0 25 0 69 N/A 3.748E-05 132 ✓ ✓ (9)(19)(23)
Q3TLH4-5 PRC2C Isoform 5 of Protein PRRC2C 0 19 0 19 0 30 0 68 N/A 3.479E-03 302 ✓ ✓ (19)(23)(26)
G3X928 G3X928 SEC23-interacting protein 0 20 0 24 0 23 0 67 N/A 4.936E-05 111 ✓ ✓ ✓ (2)
O88532 ZFR Zinc finger RNA-binding protein 0 22 0 21 0 23 0 66 N/A 2.833E-06 117 ✓ ✓
(1)(5)(12)(13)
(19)(20)(26)
451
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
O70318 E41L2 Band 4.1-like protein 2 0 17 0 22 0 22 0 61 N/A 2.591E-04 110 ✓ ✓ ✓ (20)(23)
Q8R317-2 UBQL1 Isoform 2 of Ubiquilin-1 0 19 0 22 0 19 0 60 N/A 3.688E-05 58.6 ✓ ✓
Q9JKR6 HYOU1 Hypoxia up-regulated protein 1 0 23 0 22 0 14 0 59 N/A 2.307E-03 111 ✓ (26) (16)(17)
Q8R317 UBQL1 Ubiquilin-1 0 18 0 22 0 18 0 58 N/A 1.315E-04 61.9 ✓ ✓
P98078 DAB2 Disabled homolog 2 0 21 0 15 0 22 0 58 N/A 9.021E-04 82.3 ✓ ✓ ✓
Q7M6Y3-6 PICA Isoform 6 of Phosphatidylinositol-binding clathrin assembly protein 0 15 0 26 0 14 0 55 N/A 8.846E-03 70.5 ✓ ✓ (17)
Q80U93 NU214 Nuclear pore complex protein Nup214 0 18 0 13 0 22 0 53 N/A 2.462E-03 213 ✓ ✓
(1)(2)(19)(20)
(23)(26)
Q9QZM0 UBQL2 Ubiquilin-2 0 15 0 17 0 19 0 51 N/A 1.239E-04 67.3 ✓ ✓
Q91ZX7 LRP1 Prolow-density lipoprotein receptor-related protein 1 0 13 0 22 0 14 0 49 N/A 4.579E-03 504 ✓ (15)(16)(17)
Q80ZX0 Q80ZX0 Protein Sec24b 0 15 0 19 0 15 0 49 N/A 2.550E-04 136 ✓ ✓ (2)
Q3TN34 Q3TN34 JRAB 0 15 0 18 0 15 0 48 N/A 8.922E-05 108 ✓ ✓ (26)
E9Q4Q2 E9Q4Q2 Splicing factor 1 0 12 0 19 0 17 0 48 N/A 1.541E-03 59.7 ✓ ✓
F6TQN9 F6TQN9 Disabled homolog 2 (Fragment) 0 18 0 10 0 18 0 46 N/A 4.535E-03 67.8 ✓ ✓
A2AMY5 A2AMY5 Ubiquitin-associated protein 2 0 17 0 13 0 14 0 44 N/A 2.588E-04 118 ✓ ✓ ✓
Q8BFR4 GNS N-acetylglucosamine-6-sulfatase 0 15 0 14 0 14 0 43 N/A 1.749E-06 61.1 ✓ (9)(23) (17)
P70699 LYAG Lysosomal alpha-glucosidase 0 11 0 16 0 16 0 43 N/A 1.005E-03 106 ✓ (16)(17)
P10852 4F2 4F2 cell-surface antigen heavy chain 0 12 0 16 0 15 0 43 N/A 2.832E-04 58.3 ✓ ✓ (10)(17)
Q8VIJ6 SFPQ Splicing factor, proline- and glutamine-rich 0 10 0 18 0 14 0 42 N/A 3.738E-03 75.4 ✓ ✓
(5)(9)(19)(20)
(23)(24) (17)
Q63850 NUP62 Nuclear pore glycoprotein p62 0 12 0 15 0 15 0 42 N/A 1.510E-04 53.2 ✓ ✓
(2)(9)(19)(23)
(26) (17)
G3UWA6 G3UWA6 4F2 cell-surface antigen heavy chain 0 11 0 16 0 14 0 41 N/A 7.120E-04 62.2 ✓ ✓ (10)
H3BKM0 H3BKM0 AP-2 complex subunit beta 0 12 0 14 0 15 0 41 N/A 1.012E-04 101 ✓
Q5SUT0 Q5SUT0 Ewing sarcoma breakpoint region 1 0 12 0 15 0 13 0 40 N/A 1.116E-04 64.9 ✓ ✓ (26)
Q60865 CAPR1 Caprin-1 0 14 0 13 0 13 0 40 N/A 2.334E-06 78.1 ✓ ✓ ✓ (19)(23)(26)
B1ATZ0 B1ATZ0 HGF-regulated tyrosine kinase substrate 0 13 0 13 0 14 0 40 N/A 2.334E-06 85.7 ✓ ✓
E9Q3G8 E9Q3G8 Protein Nup153 0 13 0 12 0 14 0 39 N/A 2.304E-05 152 ✓ ✓ (2)(12)(20)
Q9DBG5 PLIN3 Perilipin-3 0 8 0 14 0 17 0 39 N/A 7.966E-03 47.2 ✓ ✓ ✓ (1)(9)(20)(23)
G3X972 G3X972 Protein Sec24c 0 12 0 14 0 12 0 38 N/A 4.520E-05 119 ✓ ✓ ✓ (2)
Q8CFQ9 Q8CFQ9 Fusion, derived from t(12;16) malignant liposarcoma (Human) 0 12 0 12 0 14 0 38 N/A 4.520E-05 52.6 ✓ ✓
452
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
Q8C2Q3 RBM14 RNA-binding protein 14 0 8 0 17 0 12 0 37 N/A 9.055E-03 69.4 ✓ ✓
(1)(9)(20)(23)
(24)
Q8VDM6 HNRL1 Heterogeneous nuclear ribonucleoprotein U-like protein 1 0 7 0 15 0 15 0 37 N/A 9.844E-03 95.9 ✓ ✓
Q9WVG6-2 CARM1 Isoform 2 of Histone-arginine methyltransferase CARM1 0 12 0 12 0 12 0 36 N/A 0.000E+00 63.4 ✓ ✓ ✓ (5)(9)(19)(23)
Q8BL80 RHG22 Rho GTPase-activating protein 22 0 12 0 10 0 14 0 36 N/A 4.841E-04 77.7 ✓
O70305-2 ATX2 Isoform 2 of Ataxin-2 0 10 0 15 0 11 0 36 N/A 1.419E-03 129 ✓ ✓ ✓
P54728 RD23B UV excision repair protein RAD23 homolog B 0 12 0 13 0 10 0 35 N/A 1.887E-04 43.5 ✓ ✓ (5)(23) (17)
Q9D824-4 FIP1 Isoform 4 of Pre-mRNA 3'-end-processing factor FIP1 0 10 0 13 0 11 0 34 N/A 2.114E-04 55.8 ✓ (23)
P59326 YTHD1 YTH domain family protein 1 0 12 0 10 0 11 0 33 N/A 4.471E-05 60.8 ✓ ✓ (19)
Q8BYK6 YTHD3 YTH domain family protein 3 0 9 0 14 0 10 0 33 N/A 1.971E-03 63.9 ✓ ✓ (1)(19)(20)
Q6PFD9 Q6PFD9 Nucleoporin 98 0 9 0 10 0 13 0 32 N/A 8.903E-04 125 ✓ ✓ (20)
Q9WV92-3 E41L3 Isoform 3 of Band 4.1-like protein 3 0 8 0 10 0 14 0 32 N/A 3.772E-03 102 ✓ ✓
Q6NXL1 Q6NXL1 Protein Sec24d 0 8 0 12 0 11 0 31 N/A 1.006E-03 113 ✓ ✓ (2)
Q99P91 GPNMB Transmembrane glycoprotein NMB 0 8 0 11 0 12 0 31 N/A 1.006E-03 63.6 ✓ ✓
Q8BH97 RCN3 Reticulocalbin-3 0 12 0 8 0 10 0 30 N/A 9.781E-04 38.0 ✓ ✓ (17)
P27546-2 MAP4 Isoform 2 of Microtubule-associated protein 4 0 10 0 10 0 10 0 30 N/A 0.000E+00 117 ✓ ✓ ✓
(9)(19)(23)(2
6)
Q9R0E1 PLOD3 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 3 0 12 0 12 0 6 0 30 N/A 7.490E-03 84.9 ✓
Q9Z1A1 Q9Z1A1 Protein Tfg 0 11 0 8 0 11 0 30 N/A 5.620E-04 43.0 ✓ ✓
Q9WU78 PDC6I Programmed cell death 6-interacting protein 0 6 0 11 0 13 0 30 N/A 8.624E-03 96.0 ✓ ✓ ✓
Q04857 CO6A1 Collagen alpha-1(VI) chain 0 7 0 11 0 11 0 29 N/A 1.921E-03 108 ✓ (17)
P40124 CAP1 Adenylyl cyclase-associated protein 1 0 11 0 9 0 9 0 29 N/A 1.315E-04 51.5 ✓ ✓ ✓ (9)(23)(26)
Q3UEB3-3 PUF60 Isoform 3 of Poly(U)-binding-splicing factor PUF60 0 7 0 12 0 10 0 29 N/A 2.651E-03 54.0 ✓ (9)(23)
Q8VCF0 MAVS Mitochondrial antiviral-signaling protein 0 9 0 11 0 9 0 29 N/A 1.315E-04 53.4 ✓ ✓ ✓ (5)(9)(20)(23)
Q8K2K6-3 AGFG1 Isoform 3 of Arf-GAP domain and FG repeat-containing protein 1 0 8 0 11 0 9 0 28 N/A 4.511E-04 55.0 ✓ (9)(19)(23)
Q91YT7 Q91YT7 Protein Ythdf2 0 9 0 9 0 10 0 28 N/A 9.679E-06 62.2 ✓ ✓
Q62165 DAG1 Dystroglycan 0 7 0 9 0 11 0 27 N/A 1.462E-03 96.8 ✓ (23) (15)(16)(17)
Q62351 TFR1 Transferrin receptor protein 1 0 12 0 11 0 4 0 27 N/A 2.325E-02 85.7 ✓ (10)(16)
Q8R4X3 RBM12 RNA-binding protein 12 0 7 0 10 0 10 0 27 N/A 8.438E-04 103 ✓ (9)(23)
Q8K4Q8 COL12 Collectin-12 0 9 0 9 0 9 0 27 N/A 0.000E+00 81.3 ✓
Q3UF95 Q3UF95 Large proline-rich protein BAG6 0 9 0 9 0 9 0 27 N/A 0.000E+00 119 ✓
453
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
Q02788 CO6A2 Collagen alpha-2(VI) chain 0 9 0 8 0 9 0 26 N/A 1.300E-05 110 ✓ (26)
Q5F2E7 NUFP2 Nuclear fragile X mental retardation-interacting protein 2 0 10 0 9 0 7 0 26 N/A 6.012E-04 75.6 ✓ (1)(20)
P01897 HA1L H-2 class I histocompatibility antigen, L-D alpha chain 0 10 0 8 0 8 0 26 N/A 2.020E-04 40.7 ✓ ✓
Q640N1 AEBP1 Adipocyte enhancer-binding protein 1 0 8 0 8 0 9 0 25 N/A 1.520E-05 128 ✓ (17)
P83741-4 WNK1 Isoform 4 of Serine/threonine-protein kinase WNK1 0 8 0 7 0 10 0 25 N/A 6.996E-04 225 ✓ ✓
(5)(9)(13)(19)
(23)
A2ATI9 A2ATI9 Golgi reassembly stacking protein 2 0 8 0 8 0 9 0 25 N/A 1.520E-05 45.0 ✓ ✓ (12)
F8VQJ3 F8VQJ3 Laminin subunit gamma-1 0 7 0 13 0 5 0 25 N/A 2.566E-02 177 ✓
P01899 HA11 H-2 class I histocompatibility antigen, D-B alpha chain 0 10 0 8 0 7 0 25 N/A 6.996E-04 40.8 ✓ ✓
Q9QX47-2 SON Isoform 2 of Protein SON 0 6 0 8 0 10 0 24 N/A 2.278E-03 230 ✓ (9)(23)
P04095 PR2C2 Prolactin-2C2 0 8 0 7 0 9 0 24 N/A 1.573E-04 25.4 ✓ ✓
Q5SUH7 Q5SUH7 Clathrin interactor 1 0 9 0 10 0 5 0 24 N/A 6.352E-03 67.7 ✓ ✓
P47930 FOSL2 Fos-related antigen 2 0 10 0 5 0 9 0 24 N/A 6.352E-03 35.3 ✓ ✓
D3YUW8 D3YUW8 Pogo transposable element with ZNF domain 0 6 0 10 0 8 0 24 N/A 2.278E-03 145 ✓ ✓
Q9R1Q9 VAS1 V-type proton ATPase subunit S1 0 7 0 9 0 7 0 23 N/A 3.264E-04 51.0 ✓ (17)
O08795 GLU2B Glucosidase 2 subunit beta 0 6 0 8 0 9 0 23 N/A 9.640E-04 58.8 ✓ (20) (16)
Q8BTS4 NUP54 Nuclear pore complex protein Nup54 0 6 0 11 0 6 0 23 N/A 1.003E-02 55.7 ✓ (2)(9)(23)(24)
Q91YD3 DCP1A mRNA-decapping enzyme 1A 0 8 0 7 0 8 0 23 N/A 2.117E-05 65.2 ✓ (1)(9)(23)
P29533 VCAM1 Vascular cell adhesion protein 1 0 5 0 11 0 7 0 23 N/A 1.219E-02 81.3 ✓
Q3UPH1 PRRC1 Protein PRRC1 0 6 0 7 0 9 0 22 N/A 1.143E-03 46.3 ✓ ✓ (19)(26) (17)
Q3U1M7 Q3U1M7 Protein Zmynd8 0 4 0 8 0 10 0 22 N/A 1.417E-02 130 ✓
P37889-2 FBLN2 Isoform 2 of Fibulin-2 0 7 0 6 0 8 0 21 N/A 2.655E-04 126 ✓ (16)
P11688 ITA5 Integrin alpha-5 0 7 0 7 0 7 0 21 N/A 0.000E+00 115 ✓ (10)(16)
Q9CZR2 NALD2 N-acetylated-alpha-linked acidic dipeptidase 2 0 7 0 6 0 8 0 21 N/A 2.655E-04 82.7 ✓
Q8BHN3 GANAB Neutral alpha-glucosidase AB 0 7 0 7 0 7 0 21 N/A 0.000E+00 107 ✓
Q6P4T3 Q6P4T3 Eyes absent 3 homolog (Drosophila) 0 7 0 7 0 7 0 21 N/A 0.000E+00 57.8 ✓ ✓
Q8K3Z9 PO121 Nuclear envelope pore membrane protein POM 121 0 9 0 3 0 8 0 20 N/A 2.292E-02 121 ✓ ✓ (1)
Q91VZ6 SMAP1 Stromal membrane-associated protein 1 0 6 0 8 0 6 0 20 N/A 5.620E-04 47.6 ✓
E9QAR6 E9QAR6 Protein Zfp384 0 7 0 5 0 8 0 20 N/A 1.641E-03 61.7 ✓
E9Q7W0 E9Q7W0 Recombining-binding protein suppressor of hairless 0 5 0 7 0 8 0 20 N/A 1.641E-03 54.3 ✓ ✓
Q9DBH5 LMAN2 Vesicular integral-membrane protein VIP36 0 4 0 8 0 7 0 19 N/A 6.214E-03 40.4 ✓ (17)
454
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
Q61187 TS101 Tumor susceptibility gene 101 protein 0 5 0 6 0 8 0 19 N/A 1.991E-03 44.1 ✓ (23)
A2AJ72 A2AJ72 Far upstream element (FUSE) binding protein 3 0 5 0 7 0 7 0 19 N/A 6.852E-04 61.4 ✓
Q5SFM8-2 RBM27 Isoform 2 of RNA-binding protein 27 0 7 0 4 0 7 0 18 N/A 3.883E-03 113 ✓ ✓ (9)(19)(23)
E9Q5E0 E9Q5E0 Myocyte-specific enhancer factor 2D 0 5 0 7 0 6 0 18 N/A 4.841E-04 54.1 ✓ ✓
P16675 PPGB Lysosomal protective protein 0 5 0 7 0 5 0 17 N/A 1.051E-03 53.8 ✓ (17)
Q61576 FKB10 Peptidyl-prolyl cis-trans isomerase FKBP10 0 6 0 8 0 3 0 17 N/A 1.754E-02 64.7 ✓ (16)
P09055 ITB1 Integrin beta-1 0 4 0 7 0 6 0 17 N/A 3.016E-03 88.2 ✓ (10)(16)
Q8CHY6 P66A Transcriptional repressor p66 alpha 0 4 0 8 0 5 0 17 N/A 9.206E-03 67.3 ✓ (1)(12)(20)
Q8R332-4 NUPL1 Isoform 4 of Nucleoporin p58/p45 0 5 0 8 0 4 0 17 N/A 9.206E-03 54.0 ✓
Q3UGN9 Q3UGN9 Signal transducing adapter molecule 1 0 6 0 7 0 4 0 17 N/A 3.016E-03 51.1 ✓
G3UWD2 G3UWD2 Runt related transcription factor 1, isoform CRA_c 0 4 0 4 0 9 0 17 N/A 2.728E-02 48.6 ✓
Q920A5 RISC Retinoid-inducible serine carboxypeptidase 0 3 0 8 0 5 0 16 N/A 2.138E-02 50.9 ✓ (17)
Q6NZN0-5 RBM26 Isoform 5 of RNA-binding protein 26 0 6 0 5 0 5 0 16 N/A 8.922E-05 111 ✓ ✓ (9)(19)(23)
Q501J7-2 PHAR4 Isoform 2 of Phosphatase and actin regulator 4 0 6 0 5 0 5 0 16 N/A 8.922E-05 73.4 ✓ (23)
P31230 AIMP1 Aminoacyl tRNA synthase complex-interacting multifunctional protein 1 0 3 0 8 0 5 0 16 N/A 2.138E-02 34.0 ✓ ✓ ✓ (1)
G5E8J9 G5E8J9 SCY1-like protein 2 0 5 0 7 0 4 0 16 N/A 3.772E-03 103 ✓
Q99NB8 UBQL4 Ubiquilin-4 0 5 0 6 0 5 0 16 N/A 8.922E-05 63.5 ✓ ✓ ✓
Q99LI5 ZN281 Zinc finger protein 281 0 3 0 7 0 6 0 16 N/A 1.135E-02 96.6 ✓
O70494-2 SP3 Isoform 2 of Transcription factor Sp3 0 5 0 5 0 6 0 16 N/A 8.922E-05 78.3 ✓ ✓
Q9JLV1 BAG3 BAG family molecular chaperone regulator 3 0 4 0 4 0 7 0 15 N/A 7.490E-03 61.8 ✓ ✓ ✓ (9)(23)(26) (17)
P27046 MA2A1 Alpha-mannosidase 2 0 5 0 6 0 4 0 15 N/A 9.781E-04 132 ✓ (16)(17)
Q6NXI6-2 RPRD2 Isoform 2 of Regulation of nuclear pre-mRNA domain-containing protein 2 0 5 0 5 0 5 0 15 N/A 0.000E+00 151 ✓ (9)(23)
P28659-2 CELF1 Isoform 2 of CUGBP Elav-like family member 1 0 4 0 6 0 5 0 15 N/A 9.781E-04 51.6 ✓ (9)(23)
Q8CHS8 VP37A Vacuolar protein sorting-associated protein 37A 0 4 0 4 0 7 0 15 N/A 7.490E-03 44.4 ✓
E9Q1T9 E9Q1T9 Exportin-2 0 6 0 3 0 6 0 15 N/A 7.490E-03 104 ✓
P10493 NID1 Nidogen-1 0 4 0 6 0 4 0 14 N/A 2.192E-03 137 ✓ (7)(15)(16)(17)
P11438 LAMP1 Lysosome-associated membrane glycoprotein 1 0 4 0 5 0 5 0 14 N/A 1.510E-04 43.8 ✓ ✓ (9)(23) (3)(15)(16)
Q91YQ5 RPN1 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 0 2 0 7 0 5 0 14 N/A 3.253E-02 68.5 ✓ (9)(23) (16)
P11087-2 CO1A1 Isoform 2 of Collagen alpha-1(I) chain 0 4 0 7 0 3 0 14 N/A 1.780E-02 118 ✓ (26) (16)
Q4PZA2-3 ECE1 Isoform C of Endothelin-converting enzyme 1 0 6 0 5 0 3 0 14 N/A 6.122E-03 85.4 ✓
455
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
K3W4Q8 K3W4Q8 Basigin 0 5 0 6 0 3 0 14 N/A 6.122E-03 24.1 ✓
P97863-3 NFIB Isoform 3 of Nuclear factor 1 B-type 0 4 0 3 0 7 0 14 N/A 1.780E-02 47.4 ✓
E9QKL6 E9QKL6 Interferon-activable protein 204 0 4 0 6 0 4 0 14 N/A 2.192E-03 69.4 ✓
Q9R045 ANGL2 Angiopoietin-related protein 2 0 3 0 7 0 3 0 13 N/A 3.138E-02 57.1 ✓
Q00493 CBPE Carboxypeptidase E 0 3 0 5 0 5 0 13 N/A 2.890E-03 53.2 ✓
Q99LA2 Q99LA2 Protein Zfp207 0 3 0 5 0 5 0 13 N/A 2.890E-03 49.8 ✓ ✓
E9Q7C1 E9Q7C1 Mediator of RNA polymerase II transcription subunit 15 0 6 0 2 0 5 0 13 N/A 2.265E-02 82.5 ✓ ✓
Q9Z1F9 SAE2 SUMO-activating enzyme subunit 2 0 5 0 4 0 3 0 12 N/A 2.278E-03 70.5 ✓ ✓ ✓ (26)
Q8R180 ERO1A ERO1-like protein alpha 0 3 0 4 0 5 0 12 N/A 2.278E-03 54.1 ✓ (23)
Q3UCQ1 FOXK2 Forkhead box protein K2 0 2 0 5 0 5 0 12 N/A 1.613E-02 68.4 ✓ (1)(9)(20)(23)
Q02614 S30BP SAP30-binding protein 0 4 0 4 0 4 0 12 N/A 0.000E+00 33.8 ✓ (1)(20)(23)
P97797-2 SHPS1 Isoform 2 of Tyrosine-protein phosphatase non-receptor type substrate 1 0 4 0 3 0 5 0 12 N/A 2.278E-03 56.0 ✓
P24668 MPRD Cation-dependent mannose-6-phosphate receptor 0 4 0 3 0 5 0 12 N/A 2.278E-03 31.2 ✓
Q8BIH0-2 SP130 Isoform 2 of Histone deacetylase complex subunit SAP130 0 5 0 3 0 4 0 12 N/A 2.278E-03 92.8 ✓ ✓
Q04887 SOX9 Transcription factor SOX-9 0 3 0 3 0 6 0 12 N/A 1.613E-02 56.0 ✓
F6TWX0 F6TWX0 Nuclear transcription factor Y subunit alpha (Fragment) 0 6 0 3 0 3 0 12 N/A 1.613E-02 22.7 ✓ ✓
P51569 AGAL Alpha-galactosidase A 0 2 0 4 0 5 0 11 N/A 1.417E-02 47.6 ✓ (17)
Q3TJD7 PDLI7 PDZ and LIM domain protein 7 0 3 0 4 0 4 0 11 N/A 3.882E-04 50.1 ✓ (16)
Q02819 NUCB1 Nucleobindin-1 0 2 0 4 0 5 0 11 N/A 1.417E-02 53.4 ✓ (9)(20)(23) (15)(16)(17)
Q62348 TSN Translin 0 4 0 4 0 3 0 11 N/A 3.882E-04 26.2 ✓ ✓ ✓ (9)(23)(26)
Q9JKP5 MBNL1 Muscleblind-like protein 1 0 2 0 6 0 3 0 11 N/A 3.800E-02 37.0 ✓ ✓ ✓ (9)(23)
Q91W59-2 RBMS1 Isoform 2 of RNA-binding motif, single-stranded-interacting protein 1 0 2 0 6 0 3 0 11 N/A 3.800E-02 40.0 ✓ (9)(23)
Q62203 SF3A2 Splicing factor 3A subunit 2 0 5 0 2 0 4 0 11 N/A 1.417E-02 49.9 ✓ (9)(23)
P05627 JUN Transcription factor AP-1 0 2 0 5 0 4 0 11 N/A 1.417E-02 35.9 ✓ ✓ (9)(23)
Q9R0P5 DEST Destrin 0 4 0 3 0 4 0 11 N/A 3.882E-04 18.5 ✓ ✓ ✓ (26)
Q8K3X4 I2BPL Interferon regulatory factor 2-binding protein-like 0 3 0 4 0 4 0 11 N/A 3.882E-04 80.5 ✓ (1)(20)(23)
E9PZ00 E9PZ00 Sulfated glycoprotein 1 0 3 0 4 0 4 0 11 N/A 3.882E-04 60.6 ✓
Q8BP71-5 RFOX2 Isoform 5 of RNA binding protein fox-1 homolog 2 0 3 0 3 0 5 0 11 N/A 5.328E-03 46.2 ✓
Q60929-2 MEF2A Isoform 2 of Myocyte-specific enhancer factor 2A 0 3 0 4 0 4 0 11 N/A 3.882E-04 52.6 ✓ ✓
Q5NCM6 Q5NCM6 Epsin 2 0 3 0 6 0 2 0 11 N/A 3.800E-02 61.6 ✓
456
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
Q3UMF0-4COBL1 Isoform 4 of Cordon-bleu protein-like 1 0 3 0 3 0 5 0 11 N/A 5.328E-03 130 ✓ ✓ ✓
Q9WV54 ASAH1 Acid ceramidase 0 4 0 4 0 2 0 10 N/A 7.490E-03 44.6 ✓ (17)
Q3TCN2-2 PLBL2 Isoform 2 of Putative phospholipase B-like 2 0 3 0 4 0 3 0 10 N/A 5.620E-04 49.9 ✓ (17)
P17439 GLCM Glucosylceramidase 0 2 0 5 0 3 0 10 N/A 1.944E-02 57.6 ✓ (17)
Q80W68-2 KIRR1 Isoform 2 of Kin of IRRE-like protein 1 0 2 0 3 0 5 0 10 N/A 1.944E-02 69.9 ✓ (16)
P15379-2 CD44 Isoform 13 of CD44 antigen 0 2 0 3 0 5 0 10 N/A 1.944E-02 40.0 ✓ ✓ (9)(23) (10)(16)
P42227-2 STAT3 Isoform Stat3B of Signal transducer and activator of transcription 3 0 4 0 3 0 3 0 10 N/A 5.620E-04 83.1 ✓ ✓ (9)(23)
Q99KP6 PRP19 Pre-mRNA-processing factor 19 0 3 0 4 0 3 0 10 N/A 5.620E-04 55.2 ✓ (23)
A2AA71 A2AA71 Protein transport protein Sec24A 0 3 0 3 0 4 0 10 N/A 5.620E-04 119 ✓ (2)
P57716 NICA Nicastrin 0 5 0 2 0 3 0 10 N/A 1.944E-02 78.4 ✓
P21956-2 MFGM Isoform 2 of Lactadherin 0 4 0 3 0 3 0 10 N/A 5.620E-04 47.1 ✓
E9Q6C7 E9Q6C7 Latrophilin-2 0 4 0 3 0 3 0 10 N/A 5.620E-04 167 ✓
D3YYT0 D3YYT0 Cadherin-2 0 3 0 3 0 4 0 10 N/A 5.620E-04 93.8 ✓
Q99LJ0 CT2NL CTTNBP2 N-terminal-like protein 0 4 0 3 0 3 0 10 N/A 5.620E-04 69.8 ✓
Q3UMF0-3 COBL1 Isoform 3 of Cordon-bleu protein-like 1 0 3 0 3 0 4 0 10 N/A 5.620E-04 129 ✓ ✓ ✓
Q3U4W8 Q3U4W8 Ubiquitin carboxyl-terminal hydrolase 0 4 0 4 0 2 0 10 N/A 7.490E-03 93.3 ✓ ✓
Q00422 GABPA GA-binding protein alpha chain 0 3 0 2 0 5 0 10 N/A 1.944E-02 51.3 ✓
D3Z191 D3Z191 Transmembrane protein 106B (Fragment) 0 4 0 2 0 4 0 10 N/A 7.490E-03 20.8 ✓
O35405 PLD3 Phospholipase D3 0 2 0 4 0 3 0 9 N/A 6.533E-03 54.4 ✓ (9)(23) (17)
O89023 TPP1 Tripeptidyl-peptidase 1 0 3 0 4 0 2 0 9 N/A 6.533E-03 61.3 ✓ (17)
Q99K90 TAB2 TGF-beta-activated kinase 1 and MAP3K7-binding protein 2 0 3 0 2 0 4 0 9 N/A 6.533E-03 76.4 ✓ (9)(23)
Q60838 DVL2 Segment polarity protein dishevelled homolog DVL-2 0 4 0 2 0 3 0 9 N/A 6.533E-03 78.8 ✓ (9)(23)
Q9QXS6-3 DREB Isoform E2 of Drebrin 0 5 0 2 0 2 0 9 N/A 3.994E-02 72.4 ✓ ✓ ✓ (23)
Q99KF1 TMED9 Transmembrane emp24 domain-containing protein 9 0 4 0 1 0 4 0 9 N/A 3.994E-02 27.1 ✓
Q91XX1 Q91XX1 Protein Pcdhga11 0 2 0 5 0 2 0 9 N/A 3.994E-02 101 ✓
Q3TW96 UAP1L UDP-N-acetylhexosamine pyrophosphorylase-like protein 1 0 3 0 4 0 2 0 9 N/A 6.533E-03 56.6 ✓ ✓ ✓
Q3TQ29 Q3TQ29 Pumilio homolog 2 0 4 0 3 0 2 0 9 N/A 6.533E-03 106 ✓
G3X8Q0 G3X8Q0 Trans-acting transcription factor 1 0 2 0 4 0 3 0 9 N/A 6.533E-03 80.4 ✓ ✓ ✓
E9QKG6 E9QKG6 Ankyrin repeat domain-containing protein 17 0 3 0 2 0 4 0 9 N/A 6.533E-03 247 ✓
P17047 LAMP2 Lysosome-associated membrane glycoprotein 2 0 2 0 3 0 3 0 8 N/A 1.324E-03 45.7 ✓ ✓ (9)(23) (3)(16)(17)
457
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
Q9Z247 FKBP9 Peptidyl-prolyl cis-trans isomerase FKBP9 0 2 0 3 0 3 0 8 N/A 1.324E-03 63.0 ✓ (17)
Q07797 LG3BP Galectin-3-binding protein 0 2 0 2 0 4 0 8 N/A 1.613E-02 64.4 ✓ (16)(17)
Q9EQQ9 NCOAT Bifunctional protein NCOAT 0 2 0 2 0 4 0 8 N/A 1.613E-02 103 ✓ (23) (16)
Q8K297 GT251 Procollagen galactosyltransferase 1 0 3 0 2 0 3 0 8 N/A 1.324E-03 71.0 ✓ (16)
Q05186 RCN1 Reticulocalbin-1 0 2 0 3 0 3 0 8 N/A 1.324E-03 38.1 ✓ (16)
Q08093 CNN2 Calponin-2 0 3 0 2 0 3 0 8 N/A 1.324E-03 33.1 ✓ ✓ ✓ (9)(23)(26)
P51859 HDGF Hepatoma-derived growth factor 0 4 0 2 0 2 0 8 N/A 1.613E-02 26.3 ✓ ✓ ✓ (9)(23)(26)
Q8BU11 TOX4 TOX high mobility group box family member 4 0 2 0 4 0 2 0 8 N/A 1.613E-02 65.9 ✓ (9)(23)
Q8R3V5-1 SHLB2 Isoform 1 of Endophilin-B2 0 3 0 2 0 3 0 8 N/A 1.324E-03 44.1 ✓ (5)
Q00560 IL6RB Interleukin-6 receptor subunit beta 0 2 0 4 0 2 0 8 N/A 1.613E-02 102 ✓ (20)
Q7TT18 MCAF1 Activating transcription factor 7-interacting protein 1 0 2 0 3 0 3 0 8 N/A 1.324E-03 139 ✓ (1)(23)
Q9R1J0 NSDHL Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating 0 3 0 2 0 3 0 8 N/A 1.324E-03 40.7 ✓ ✓
Q5SVW9 Q5SVW9 Transmembrane emp24 domain-containing protein 4 (Fragment) 0 2 0 4 0 2 0 8 N/A 1.613E-02 19.1 ✓
A2ACG7 A2ACG7 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 0 2 0 4 0 2 0 8 N/A 1.613E-02 67.5 ✓
Q9CY50 SSRA Translocon-associated protein subunit alpha 0 3 0 3 0 2 0 8 N/A 1.324E-03 32.0 ✓
Q64314 CD34 Hematopoietic progenitor cell antigen CD34 0 3 0 2 0 3 0 8 N/A 1.324E-03 41.0 ✓
Q02780-2 NFIA Isoform 1 of Nuclear factor 1 A-type 0 2 0 2 0 4 0 8 N/A 1.613E-02 49.9 ✓
E9Q1U8 E9Q1U8 Transcription intermediary factor 1-alpha 0 2 0 3 0 3 0 8 N/A 1.324E-03 110 ✓
D3Z0T0 D3Z0T0 Transcription initiation factor TFIID subunit 6 0 2 0 2 0 4 0 8 N/A 1.613E-02 69.8 ✓
Q61398 PCOC1 Procollagen C-endopeptidase enhancer 1 0 2 0 3 0 2 0 7 N/A 2.192E-03 50.1 ✓ (17)
Q61810-2 LTBP3 Isoform 2 of Latent-transforming growth factor beta-binding protein 3 0 3 0 2 0 2 0 7 N/A 2.192E-03 93.3 ✓ (16)
Q8VBZ3 CLPT1 Cleft lip and palate transmembrane protein 1 homolog 0 2 0 3 0 2 0 7 N/A 2.192E-03 75.2 ✓ (16)
F6VSK8 F6VSK8 Integrin alpha-6 (Fragment) 0 2 0 2 0 3 0 7 N/A 2.192E-03 75.0 ✓ (10)
Q9JKV1 ADRM1 Proteasomal ubiquitin receptor ADRM1 0 2 0 2 0 3 0 7 N/A 2.192E-03 42.0 ✓ ✓ (9)(20)(23)
Q99K48 NONO Non-POU domain-containing octamer-binding protein 0 2 0 2 0 3 0 7 N/A 2.192E-03 54.5 ✓ ✓ ✓
(9)(19)(23)(2
4)(26)
Q571G4 LIN54 Protein lin-54 homolog 0 2 0 3 0 2 0 7 N/A 2.192E-03 79.5 ✓ (9)(19)(23)
Q60710 SAMH1 SAM domain and HD domain-containing protein 1 0 3 0 2 0 2 0 7 N/A 2.192E-03 72.6 ✓ ✓ ✓ (23)
Q9WUU7 CATZ Cathepsin Z 0 3 0 1 0 3 0 7 N/A 2.490E-02 34.0 ✓
P14719-2 ILRL1 Isoform B of Interleukin-1 receptor-like 1 0 3 0 2 0 2 0 7 N/A 2.192E-03 38.5 ✓ ✓
P14428 HA1Q H-2 class I histocompatibility antigen, K-Q alpha chain (Fragment) 0 2 0 2 0 3 0 7 N/A 2.192E-03 36.8 ✓
458
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
Q9D032-2 SSBP3 Isoform 2 of Single-stranded DNA-binding protein 3 0 2 0 2 0 3 0 7 N/A 2.192E-03 37.7 ✓
Q7TSH6 Q7TSH6 Protein Scaf4 0 2 0 3 0 2 0 7 N/A 2.192E-03 129 ✓
Q62347 Q62347 Cyclic AMP-responsive element-binding protein 1 0 3 0 1 0 3 0 7 N/A 2.490E-02 30.9 ✓
E9Q7M2 E9Q7M2 Protein Tsc22d2 0 2 0 2 0 3 0 7 N/A 2.192E-03 78.1 ✓ ✓
A3KGT0 A3KGT0 CUG triplet repeat, RNA binding protein 2 0 3 0 2 0 2 0 7 N/A 2.192E-03 16.4 ✓
P28654 PGS2 Decorin 0 2 0 1 0 3 0 6 N/A 2.572E-02 39.8 ✓ (17)
P15307 REL Proto-oncogene c-Rel 0 1 0 3 0 2 0 6 N/A 2.572E-02 64.9 ✓ ✓ ✓ (9)(23)
Q7TSJ2-3 MAP6 Isoform 3 of Microtubule-associated protein 6 0 3 0 1 0 2 0 6 N/A 2.572E-02 32.8 ✓ ✓ ✓ (5)
Q5SXC4 Q5SXC4 Protein Vezf1 0 1 0 3 0 2 0 6 N/A 2.572E-02 56.5 ✓ (20)
P25425-10 PO2F1 Isoform 10 of POU domain, class 2, transcription factor 1 0 2 0 2 0 2 0 6 N/A 0.000E+00 56.2 ✓ (19)(23)
A2AUK7 A2AUK7 Erythrocyte protein band 4.1-like 1 0 1 0 2 0 3 0 6 N/A 2.572E-02 81.8 ✓ (13)
Q9R0I7 YLPM1 YLP motif-containing protein 1 0 1 0 3 0 2 0 6 N/A 2.572E-02 155 ✓ (1)(9)(19)(23)
P58871 TB182 182 kDa tankyrase-1-binding protein 0 2 0 3 0 1 0 6 N/A 2.572E-02 182 ✓ ✓ ✓
(1)(20)(23)(2
6)
Q80WC7 AGFG2 Arf-GAP domain and FG repeat-containing protein 2 0 1 0 2 0 3 0 6 N/A 2.572E-02 48.9 ✓ (1)(20)
Q689Z5 SBNO1 Protein strawberry notch homolog 1 0 1 0 2 0 3 0 6 N/A 2.572E-02 154 ✓ (1)(20)
P0C7T6 ATX1L Ataxin-1-like 0 2 0 1 0 3 0 6 N/A 2.572E-02 73.3 ✓ ✓ (1)
O88668 CREG1 Protein CREG1 0 2 0 2 0 2 0 6 N/A 0.000E+00 24.4 ✓
B1B0C7 B1B0C7 Basement membrane-specific heparan sulfate proteoglycan core protein 0 1 0 2 0 3 0 6 N/A 2.572E-02 469 ✓
P53986 MOT1 Monocarboxylate transporter 1 0 2 0 2 0 2 0 6 N/A 0.000E+00 53.2 ✓
Q920E5 FPPS Farnesyl pyrophosphate synthase 0 2 0 2 0 2 0 6 N/A 0.000E+00 40.6 ✓
Q08775-3 RUNX2 Isoform 3 of Runt-related transcription factor 2 0 1 0 3 0 2 0 6 N/A 2.572E-02 55.7 ✓
O88811-2 STAM2 Isoform 2 of Signal transducing adapter molecule 2 0 2 0 2 0 2 0 6 N/A 0.000E+00 46.2 ✓
H3BLR8 H3BLR8 Diphosphoinositol polyphosphate phosphohydrolase 1 0 2 0 2 0 2 0 6 N/A 0.000E+00 13.6 ✓
F6VCW7 F6VCW7 Nuclear factor of-activated T-cells 5 (Fragment) 0 2 0 2 0 2 0 6 N/A 0.000E+00 14.7 ✓
E9QME5 E9QME5 E3 ubiquitin-protein ligase TRIM33 0 2 0 1 0 3 0 6 N/A 2.572E-02 122 ✓
H9H9R4 H9H9R4 Protein 0610031J06Rik 0 1 0 2 0 3 0 6 N/A 2.572E-02 36.7
Q8BKC5 IPO5 Importin-5 0 2 0 2 0 1 0 5 N/A 7.490E-03 124 ✓ ✓ (9)(23) (17)
P97300-1 NPTN Isoform 1 of Neuroplastin 0 1 0 2 0 2 0 5 N/A 7.490E-03 31.3 ✓ (17)
Q91XA2 GOLM1 Golgi membrane protein 1 0 1 0 2 0 2 0 5 N/A 7.490E-03 44.3 ✓ (16)
O35375-5 NRP2 Isoform B0 of Neuropilin-2 0 1 0 2 0 2 0 5 N/A 7.490E-03 101 ✓ (16)
459
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
B2RXS4 PLXB2 Plexin-B2 0 2 0 1 0 2 0 5 N/A 7.490E-03 206 ✓ (10)
Q9WVH4 FOXO3 Forkhead box protein O3 0 2 0 1 0 2 0 5 N/A 7.490E-03 71.0 ✓ (9)(23)
Q9WU00-2 NRF1 Isoform Short of Nuclear respiratory factor 1 0 2 0 1 0 2 0 5 N/A 7.490E-03 46.1 ✓ (9)(23)
Q60793 KLF4 Krueppel-like factor 4 0 2 0 1 0 2 0 5 N/A 7.490E-03 51.8 ✓ (9)(23)
Q9Z1D1 EIF3G Eukaryotic translation initiation factor 3 subunit G 0 2 0 2 0 1 0 5 N/A 7.490E-03 35.6 ✓ ✓ ✓
(9)(19)(23)(2
6)
Q62261-2 SPTB2 Isoform 2 of Spectrin beta chain, non-erythrocytic 1 0 2 0 1 0 2 0 5 N/A 7.490E-03 251 ✓ (5)(21)
Q8VCQ8 Q8VCQ8 Caldesmon 1 0 2 0 1 0 2 0 5 N/A 7.490E-03 60.4 ✓ ✓ ✓ (26)
P97765 WBP2 WW domain-binding protein 2 0 2 0 2 0 1 0 5 N/A 7.490E-03 28.0 ✓ (20)
G5E899 G5E899 Plasminogen activator inhibitor 1 0 2 0 2 0 1 0 5 N/A 7.490E-03 45.0 ✓
Q8BXZ1 TMX3 Protein disulfide-isomerase TMX3 0 2 0 1 0 2 0 5 N/A 7.490E-03 51.8 ✓
Q6PD26 PIGS GPI transamidase component PIG-S 0 2 0 2 0 1 0 5 N/A 7.490E-03 61.7 ✓
P09450 JUNB Transcription factor jun-B 0 2 0 2 0 1 0 5 N/A 7.490E-03 35.7 ✓
O89032-3 SPD2A Isoform 3 of SH3 and PX domain-containing protein 2A 0 1 0 2 0 2 0 5 N/A 7.490E-03 119 ✓ ✓
H3BJ97 H3BJ97 Tubulointerstitial nephritis antigen-like 0 1 0 2 0 2 0 5 N/A 7.490E-03 49.2 ✓
E9QLT6 E9QLT6 Aryl hydrocarbon receptor nuclear translocator 0 2 0 2 0 1 0 5 N/A 7.490E-03 84.5 ✓
A2A9W7 A2A9W7 ADP-ribosylation factor-binding protein GGA3 0 2 0 2 0 1 0 5 N/A 7.490E-03 70.1 ✓
O88531 PPT1 Palmitoyl-protein thioesterase 1 0 1 0 2 0 1 0 4 N/A 1.613E-02 34.5 ✓ (17)
Q8BJS4-3 SUN2 Isoform 3 of SUN domain-containing protein 2 0 1 0 2 0 1 0 4 N/A 1.613E-02 78.1 ✓ (16)
P43406 ITAV Integrin alpha-V 0 1 0 2 0 1 0 4 N/A 1.613E-02 115 ✓ (10)
Q9WTK5 NFKB2 Nuclear factor NF-kappa-B p100 subunit 0 1 0 1 0 2 0 4 N/A 1.613E-02 96.8 ✓ ✓ ✓ (9)(23)(26)
Q9DBR0 AKAP8 A-kinase anchor protein 8 0 1 0 2 0 1 0 4 N/A 1.613E-02 76.2 ✓ ✓ (9)(23)
P60229 EIF3E Eukaryotic translation initiation factor 3 subunit E 0 1 0 1 0 2 0 4 N/A 1.613E-02 52.2 ✓ (9)(23)
B2RRE7 OTUD4 OTU domain-containing protein 4 0 2 0 1 0 1 0 4 N/A 1.613E-02 123 ✓ ✓ (9)(23)
P54254 ATX1 Ataxin-1 0 1 0 1 0 2 0 4 N/A 1.613E-02 83.7 ✓ (20)
B1AR09 B1AR09 Myeloid/lymphoid or mixed lineage-leukemia translocation to 6 homolog (Drosophila) 0 1 0 2 0 1 0 4 N/A 1.613E-02 111 ✓ ✓ (20)
Q9CZD3 SYG Glycine--tRNA ligase 0 2 0 1 0 1 0 4 N/A 1.613E-02 81.8 ✓ ✓ ✓ (14)(23)(26)
Q8VI36-2 PAXI Isoform Alpha of Paxillin 0 1 0 2 0 1 0 4 N/A 1.613E-02 60.8 ✓ ✓ (14)(23)
Q8C9B9 DIDO1 Death-inducer obliterator 1 0 2 0 1 0 1 0 4 N/A 1.613E-02 247 ✓
(1)(9)(19)(20)
(23)
Q3UHC0 TNR6C Trinucleotide repeat-containing gene 6C protein 0 1 0 2 0 1 0 4 N/A 1.613E-02 176 ✓ (1)(20)(23)
460
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
Q9R233-2 TPSN Isoform Short of Tapasin 0 1 0 2 0 1 0 4 N/A 1.613E-02 46.6 ✓
P50429-2 ARSB Isoform 2 of Arylsulfatase B 0 1 0 2 0 1 0 4 N/A 1.613E-02 47.9 ✓
O09159 MA2B1 Lysosomal alpha-mannosidase 0 1 0 2 0 1 0 4 N/A 1.613E-02 115 ✓
E9Q0W5 E9Q0W5 Protein FAM3C (Fragment) 0 1 0 2 0 1 0 4 N/A 1.613E-02 16.5 ✓
Q6NZD2 Q6NZD2 Sorting nexin 1 0 1 0 2 0 1 0 4 N/A 1.613E-02 58.8 ✓ ✓ ✓
P97496-2 SMRC1 Isoform 2 of SWI/SNF complex subunit SMARCC1 0 1 0 2 0 1 0 4 N/A 1.613E-02 120 ✓
G5E8E1 G5E8E1 Leucine rich repeat (In FLII) interacting protein 1, isoform CRA_e 0 1 0 1 0 2 0 4 N/A 1.613E-02 48.9 ✓ ✓ ✓
F6VXN4 F6VXN4 TSC22 domain family protein 4 (Fragment) ] 0 2 0 1 0 1 0 4 N/A 1.613E-02 10.3 ✓
E9Q6M7 E9Q6M7 Pumilio homolog 1 0 2 0 1 0 1 0 4 N/A 1.613E-02 100 ✓
D3YY34 D3YY34 Polyhomeotic-like protein 3 0 1 0 2 0 1 0 4 N/A 1.613E-02 102 ✓
D3YXK2 SAFB1 Scaffold attachment factor B1 0 1 0 2 0 1 0 4 N/A 1.613E-02 105 ✓ ✓ ✓
Q9EP69 SAC1 Phosphatidylinositide phosphatase SAC1 0 2 0 1 0 1 0 4 N/A 1.613E-02 66.9
P97298 PEDF Pigment epithelium-derived factor 0 1 0 1 0 1 0 3 N/A 0.000E+00 46.2 ✓ (17)
Q8BPB5 FBLN3 EGF-containing fibulin-like extracellular matrix protein 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 54.9 ✓ ✓ (16)
A8Y5F6 A8Y5F6 Podoplanin 0 1 0 1 0 1 0 3 N/A 0.000E+00 16.8 ✓ (26) (11)
Q62418-3 DBNL Isoform 3 of Drebrin-like protein 0 1 0 1 0 1 0 3 N/A 0.000E+00 48.3 ✓ ✓ ✓ (9)(23)(26)
Q8C0C0 ZHX2 Zinc fingers and homeoboxes protein 2 0 1 0 1 0 1 0 3 N/A 0.000E+00 92.2 ✓ (9)(23)
Q6P5G6 UBXN7 UBX domain-containing protein 7 0 1 0 1 0 1 0 3 N/A 0.000E+00 52.1 ✓ ✓ (9)(23)
P45377 ALD2 Aldose reductase-related protein 2 0 1 0 1 0 1 0 3 N/A 0.000E+00 36.1 ✓ ✓ ✓ (26)
Q9JIF7 COPB Coatomer subunit beta 0 1 0 1 0 1 0 3 N/A 0.000E+00 107 ✓ ✓ (14)(23)
Q9ESU7 Q9ESU7 Neutral amino acid transporter ASCT2 0 1 0 1 0 1 0 3 N/A 0.000E+00 58.4 ✓
Q01721 GAS1 Growth arrest-specific protein 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 35.7 ✓
P28653 PGS1 Biglycan 0 1 0 1 0 1 0 3 N/A 0.000E+00 41.6 ✓
P23780 BGAL Beta-galactosidase 0 1 0 1 0 1 0 3 N/A 0.000E+00 73.1 ✓
G3UWE1 G3UWE1 MCG11048, isoform CRA_c 0 1 0 1 0 1 0 3 N/A 0.000E+00 34.2 ✓
B7ZC19 B7ZC19 GPI transamidase component PIG-T 0 1 0 1 0 1 0 3 N/A 0.000E+00 49.1 ✓
Q9JLJ5 ELOV1 Elongation of very long chain fatty acids protein 1 0 1 0 1 0 1 0 3 N/A 0.000E+00 32.7 ✓ ✓ ✓
Q9Z1B5 MD2L1 Mitotic spindle assembly checkpoint protein MAD2A 0 1 0 1 0 1 0 3 N/A 0.000E+00 23.6 ✓ ✓
Q9QYH6 MAGD1 Melanoma-associated antigen D1 0 1 0 1 0 1 0 3 N/A 0.000E+00 85.6 ✓
Q9DBY0-2 FOXP4 Isoform 2 of Forkhead box protein P4 0 1 0 1 0 1 0 3 N/A 0.000E+00 72.7 ✓
461
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
Q8BKL1 Q8BKL1 Protein SSXT 0 1 0 1 0 1 0 3 N/A 0.000E+00 38.0 ✓
Q8BH93 MISSL MAPK-interacting and spindle-stabilizing protein-like 0 1 0 1 0 1 0 3 N/A 0.000E+00 23.9 ✓ ✓
Q61286-2 HTF4 Isoform ALF1A of Transcription factor 12 0 1 0 1 0 1 0 3 N/A 0.000E+00 72.9 ✓
P70445 4EBP2 Eukaryotic translation initiation factor 4E-binding protein 2 0 1 0 1 0 1 0 3 N/A 0.000E+00 12.9 ✓ ✓
F6T2Z7 F6T2Z7 Protein Cald1 (Fragment) 0 1 0 1 0 1 0 3 N/A 0.000E+00 41.4 ✓ ✓ ✓
D3Z0J5 D3Z0J5 Forkhead box protein P1 (Fragment) 0 1 0 1 0 1 0 3 N/A 0.000E+00 38.8 ✓
P08207 S10AA Protein S100-A10 0 1 0 1 0 1 0 3 N/A 0.000E+00 11.2 ✓
J3QNT2 J3QNT2 Uncharacterized protein 0 1 0 1 0 1 0 3 N/A 0.000E+00 41.1
P11276 FINC Fibronectin 1 48 1 65 0 57 2 170 85.00 3.402E-04 272 ✓ (16)(17)
E9PVC5 E9PVC5 Eukaryotic translation initiation factor 4 gamma 1 0 20 1 23 0 19 1 62 62.00 8.285E-05 175 ✓ ✓ ✓ (12)
Q8VHR5 P66B Transcriptional repressor p66-beta 0 12 1 15 0 18 1 45 45.00 1.143E-03 65.4 ✓ ✓ (1)(20)(23)
Q8BFW7 LPP Lipoma-preferred partner homolog 0 11 1 17 0 16 1 44 44.00 1.607E-03 65.8 ✓ ✓ ✓
(9)(20)(23)(2
6) (16)
Q60715 P4HA1 Prolyl 4-hydroxylase subunit alpha-1 0 5 1 8 0 10 1 23 23.00 7.933E-03 60.9 ✓ (16)
P60335 PCBP1 Poly(rC)-binding protein 1 0 8 1 9 0 6 1 23 23.00 1.473E-03 37.5 ✓ ✓ ✓
(9)(14)(23)(2
6)
Q3UPL0-2 SC31A Isoform 2 of Protein transport protein Sec31A 1 15 2 19 0 20 3 54 18.00 4.809E-04 130 ✓ ✓ (5)(19)(26)
O54931-2 AKAP2 Isoform 2 of A-kinase anchor protein 2 0 6 1 6 0 4 1 16 16.00 2.570E-03 97.1 ✓ ✓ ✓ (9)(23)(26)
Q62523 ZYX Zyxin 0 11 2 9 0 11 2 31 15.50 5.101E-04 60.5 ✓ ✓ ✓ (1)(9)(23)(26)
Q61029-3 LAP2B Isoform Epsilon of Lamina-associated polypeptide 2, isoforms beta/delta/epsilon/gamma 1 9 1 10 0 11 2 30 15.00 1.510E-04 46.0 ✓ ✓ ✓ (26)
Q61033 LAP2A Lamina-associated polypeptide 2, isoforms alpha/zeta 1 5 0 5 0 5 1 15 15.00 1.510E-04 75.1 ✓ ✓ ✓ (23)(26)
Q8CI51 PDLI5 PDZ and LIM domain protein 5 0 11 2 10 0 8 2 29 14.50 1.239E-03 63.3 ✓ ✓ ✓ (1)(23)(26)
P16092-6 FGFR1 Isoform 6 of Fibroblast growth factor receptor 1 0 3 1 6 0 3 1 12 12.00 2.539E-02 91.6 ✓
Q6DFW4 NOP58 Nucleolar protein 58 1 2 0 5 0 4 1 11 11.00 2.411E-02 60.3 ✓ ✓ ✓ (9)(23)(26)
P10605 CATB Cathepsin B 1 11 1 12 2 14 4 37 9.25 3.085E-04 37.3 ✓ ✓ (26) (17)
Q501J6 DDX17 Probable ATP-dependent RNA helicase DDX17 0 3 0 2 1 4 1 9 9.00 1.613E-02 72.4 ✓ ✓ ✓ (5)(9)(23)(26)
Q61990 PCBP2 Poly(rC)-binding protein 2 0 2 1 4 0 2 1 8 8.00 3.517E-02 38.2 ✓ ✓ ✓ (5)(14)(26) (17)
P06797 CATL1 Cathepsin L1 1 4 1 6 0 4 2 14 7.00 5.821E-03 37.5 ✓ (17)
P14733 LMNB1 Lamin-B1 0 2 1 3 0 2 1 7 7.00 1.324E-02 66.7 ✓ ✓ ✓ (9)(23)
Q922Q8 LRC59 Leucine-rich repeat-containing protein 59 0 2 1 2 0 3 1 7 7.00 1.324E-02 34.9 ✓
462
No.
GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz GalNAz
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Ex-
peri-
ment
1
Ex-
peri-
ment
1
Ex-
peri-
ment
2
Ex-
peri-
ment
2
Ex-
peri-
ment
3
Ex-
peri-
ment
3 Sum Sum Localization Localization Localization
Acces-
sion Gene Description - + - + - + - +
En-
rich-
ment
Ratio t-test
MW
[kDa]
Exclu-
sively
Intra-
cellular
Exclu-
sively
Extra-
cellular,
Lyso-
somal,
LumenalBoth
6AzGl
cNAc
GlcN
Az
Previous O-
GlcNAc
Proteomics
Identifica-
tion
Previous
Mucin
Pro-
teomics
Identifica-
tion
340
341
342
343
344
345
346
347
348
P06151 LDHA L-lactate dehydrogenase A chain 1 5 1 6 1 5 3 16 5.33 2.020E-04 36.5 ✓ ✓ ✓
(6)(9)(23)(24)
(26) (17)
E9Q1P8 I2BP2 Interferon regulatory factor 2-binding protein 2 1 1 0 2 0 2 1 5 5.00 4.742E-02 59.3 ✓ (23)
D6REF7 D6REF7 Sphingosine-1-phosphate lyase 1 1 6 2 5 1 7 4 18 4.50 2.192E-03 54.7 ✓
P18760 COF1 Cofilin-1 3 7 2 9 1 6 6 22 3.67 7.182E-03 18.5 ✓ ✓ ✓
(14)(19)(23)(
26)
P08752 GNAI2 Guanine nucleotide-binding protein G(i) subunit alpha-2 1 3 1 5 1 3 3 11 3.67 1.613E-02 40.5 ✓ ✓ (20)(26)
J3QPE8 J3QPE8 MCG16555 4 8 1 8 2 8 7 24 3.43 3.016E-03 30.7 ✓ ✓ ✓
P68254-2 1433T Isoform 2 of 14-3-3 protein theta 1 7 1 4 3 6 5 17 3.40 2.239E-02 27.7 ✓ ✓ ✓ (5)(8)(23)(26) (17)
P51150 RAB7A Ras-related protein Rab-7a 2 7 3 10 3 9 8 26 3.25 3.126E-03 23.5 ✓
P08113 ENPL Endoplasmin 6 24 9 29 11 27 26 80 3.08 9.361E-04 92.4 ✓ (16)(17)
463
Tables 6-1, 6-2, 6-3 References
1 Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012). Alfaro, J. F. et al. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109, 7280–7285 (2012).
2 Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011). Boyce, M. et al. Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108, 3141–3146 (2011).
3 Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993). Carlsson, S. R., Lycksell, P . O. & Fukuda, M. Assignment of O-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Arch. Biochem. Biophys. 304, 65–73 (1993).
4 Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004). Cieniewski-Bernard, C. Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry. Mol Cell Proteomics 3, 577–585 (2004).
5 Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008). Clark, P . M. et al. Direct In-Gel Fluorescence Detection and Cellular Imaging of O-GlcNAc-Modified Proteins. J Am Chem Soc 130, 11576–11577 (2008).
6 Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008). Dehennaut, V. et al. Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7, 2229–2245 (2008).
7 Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and Localization. Matrix Collagen and Related Research 13, 215–222 (1993). Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and Localization. Matrix Collagen and Related Research 13, 215–222 (1993). Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and Localization. Matrix Collagen and Related Research 13, 215–222 (1993). Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and Localization. Matrix Collagen and Related Research 13, 215–222 (1993). Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and Localization. Matrix Collagen and Related Research 13, 215–222 (1993). Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and Localization. Matrix Collagen and Related Research 13, 215–222 (1993). Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and Localization. Matrix Collagen and Related Research 13, 215–222 (1993). Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and Localization. Matrix Collagen and Related Research 13, 215–222 (1993). Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and Localization. Matrix Collagen and Related Research 13, 215–222 (1993). Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and Localization. Matrix Collagen and Related Research 13, 215–222 (1993).
8 Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008). Gurcel, C. et al. Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390, 2089–2097 (2008).
9 Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013). Hahne, H. et al. Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J Proteome Res 12, 927–936 (2013).
10 Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011). Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M. & Bertozzi, C. R. Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells. Bioorg. Med. Chem. Lett. 21, 4945–4950 (2011).
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20 Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012). Trinidad, J. C. et al. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229 (2012).
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22 Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010). Wang, Z. et al. Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Mol Cell Proteomics 9, 153–160 (2010).
23 Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010). Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010). Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010). Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010). Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010). Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010). Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010). Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010). Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010). Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010). Wang, Z. et al. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2 (2010).
24 Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007). Wang, Z., Pandey, A. & Hart, G. W. Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation. Mol Cell Proteomics 6, 1365–1379 (2007).
25 Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002). Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1, 791–804 (2002).
26 Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011). Zaro, B. W., Yang, Y.-Y., Hang, H. C. & Pratt, M. R. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108, 8146–8151 (2011).
464
Table 7-1. 1-deoxy-Ac3GlcNAlk Labeled Proteins. Mouse embryonic fibroblasts were treated in triplicate with either 1-
deoxy-Ac3GlcNAlk (200 µM, +) or Ac4GlcNAc (200 µM, -) for 16 hours. At this time the cell lysates were subjected to CuAAC
with alkyne-biotin, followed by enrichment with streptavidin beads and on-bead trypsinolysis. Labeled proteins were selected
as those that were represented by at least 1 unique-peptide in each 1-deoxy-Ac3GlcNAc treated sample, a total of at least 3
spectral-counts from the same three samples, and at least a total of 3 times more spectral counts in the 1-deoxy-Ac3GlcNAlk
treated samples compared to Ac 4GlcNAc. A t-test was also conducted with a maxmium value of 0.05.
N
o
.
1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
As-
ses-
sion
Gene
ID Description
- + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
GlcN
Alk
Known
Acetylated
Known O-
GlcNAcylat
ed
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Q9ERK4 XPO2 Exportin-2 OS=Mus musculus Cse1l PE=2 SV=1 - [XPO2] 0 12 0 4 0 5 0 21 N/A 4.974E-02 110 (20)(23)(24) (7)(10)
Q99P72RTN4 Reticulon-4 OS=Mus musculus Rtn4 PE=1 SV=2 - [RTN4] 0 9 0 5 0 5 0 19 N/A 8.971E-03 127 ✔ (20)(23)(24) (1)(13)(19)
Q99KP6 PRP19 Pre-mRNA-processing factor 19 OS=Mus musculus Prpf19 PE=2 SV=1 - [PRP19] 0 2 0 4 0 6 0 12 N/A 2.572E-02 55.2 (20)(23)(24) (7)
E9Q450 E9Q450 Tropomyosin alpha-1 chain OS=Mus musculus Tpm1 PE=3 SV=1 - [E9Q450] 0 4 0 4 0 3 0 11 N/A 3.882E-04 32.8 ✔
Q99KI0 ACONAconitate hydratase, mitochondrial OS=Mus musculus Aco2 PE=1 SV=1 - [ACON] 0 1 0 4 0 4 0 9 N/A 3.994E-02 85.4 (20)(21)(23)(24) (20)(21)(23)(24)
O09167RL21 60S ribosomal protein L21 OS=Mus musculus Rpl21 PE=2 SV=3 - [RL21] 0 2 0 4 0 3 0 9 N/A 6.533E-03 18.6 ✔ (20) (7)(12)
Q9CU62 SMC1AStructural maintenance of chromosomes protein 1A OS=Mus musculus Smc1a PE=1 SV=4 - [SMC1A] 0 3 0 4 0 2 0 9 N/A 6.533E-03 143 ✔ (20)(23)(24) (7)
Q64511 TOP2BDNA topoisomerase 2-beta OS=Mus musculus Top2b PE=1 SV=2 - [TOP2B] 0 3 0 4 0 2 0 9 N/A 6.533E-03 182 (20)(23)(24) (7)
P47738 ALDH2Aldehyde dehydrogenase, mitochondrial OS=Mus musculus Aldh2 PE=1 SV=1 - [ALDH2] 0 2 0 3 0 4 0 9 N/A 6.533E-03 56.5 (20)(21)(25)
Q8BJS4-3 SUN2 Isoform 3 of SUN domain-containing protein 2 OS=Mus musculus Sun2 - [SUN2] 0 3 0 3 0 3 0 9 N/A 0.000E+00 78.1
O35286 DHX15Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15 OS=Mus musculus Dhx15 PE=2 SV=2 - [DHX15] 0 1 0 4 0 3 0 8 N/A 3.902E-02 90.9 ✔ (20)(22)(23)(24) (7)(10)(19)
Q9EQH3 VPS35 Vacuolar protein sorting-associated protein 35 OS=Mus musculus Vps35 PE=1 SV=1 - [VPS35] 0 1 0 3 0 3 0 7 N/A 2.490E-02 91.7 ✔ (20)(23)(24) (7)
Q9ERG0-2 LIMA1 Isoform Alpha of LIM domain and actin-binding protein 1 OS=Mus musculus Lima1 - [LIMA1] 0 3 0 1 0 3 0 7 N/A 2.490E-02 66 ✔ (7)
Q9ERU9RBP2 E3 SUMO-protein ligase RanBP2 OS=Mus musculus Ranbp2 PE=1 SV=2 - [RBP2] 0 3 0 3 0 1 0 7 N/A 2.490E-02 341 ✔ (20)(23)(24) (1)(7)(13)
E9Q6J5 E9Q6J5 Protein Bod1l OS=Mus musculus Bod1l PE=4 SV=1 - [E9Q6J5] 0 2 0 2 0 3 0 7 N/A 2.192E-03 327 ✔ (7)
Q3V1V3ESF1 ESF1 homolog OS=Mus musculus Esf1 PE=1 SV=1 - [ESF1] 0 2 0 3 0 2 0 7 N/A 2.192E-03 98 (20) (7)
E9Q7B0 E9Q7B0 Prolyl 4-hydroxylase subunit alpha-1 OS=Mus musculus P4ha1 PE=4 SV=1 - [E9Q7B0] 0 2 0 3 0 2 0 7 N/A 2.192E-03 51.7
Q9CXF4 TBC15TBC1 domain family member 15 OS=Mus musculus Tbc1d15 PE=1 SV=1 - [TBC15] 0 3 0 1 0 2 0 6 N/A 2.572E-02 76.5 ✔ (7)
Q9CQI3 GMFB Glia maturation factor beta OS=Mus musculus Gmfb PE=1 SV=3 - [GMFB] 0 2 0 3 0 1 0 6 N/A 2.572E-02 16.7 (20)(23)(24)
Q9CY27TECR Very-long-chain enoyl-CoA reductase OS=Mus musculus Tecr PE=1 SV=1 - [TECR] 0 2 0 1 0 3 0 6 N/A 2.572E-02 36.1 (20)(21)(23)(24) (20)(21)(23)(24)
P10852 4F2 4F2 cell-surface antigen heavy chain OS=Mus musculus Slc3a2 PE=1 SV=1 - [4F2] 0 2 0 2 0 2 0 6 N/A 0.000E+00 58.3 ✔ (7)
E9Q066 E9Q066 La-related protein 4 OS=Mus musculus Larp4 PE=4 SV=1 - [E9Q066] 0 2 0 2 0 2 0 6 N/A 0.000E+00 79.6 ✔ (7)
Q8BFW7LPP Lipoma-preferred partner homolog OS=Mus musculus Lpp PE=1 SV=1 - [LPP] 0 1 0 2 0 2 0 5 N/A 7.490E-03 65.8 ✔ (20) (7)(13)(19)
Q60865 CAPR1Caprin-1 OS=Mus musculus Caprin1 PE=1 SV=2 - [CAPR1] 0 2 0 1 0 2 0 5 N/A 7.490E-03 78.1 ✔ (20) (7)(12)(19)
O55135 IF6 Eukaryotic translation initiation factor 6 OS=Mus musculus Eif6 PE=1 SV=2 - [IF6] 0 2 0 2 0 1 0 5 N/A 7.490E-03 26.5 ✔ (20) (7)
P31938 MP2K1Dual specificity mitogen-activated protein kinase kinase 1 OS=Mus musculus Map2k1 PE=1 SV=2 - [MP2K1] 0 1 0 2 0 2 0 5 N/A 7.490E-03 43.4 ✔ (7)
Q8K4Z3 NNRE NAD(P)H-hydrate epimerase OS=Mus musculus Apoa1bp PE=1 SV=1 - [NNRE] 0 1 0 2 0 2 0 5 N/A 7.490E-03 31 (20)
Q9CQW9 IFM3 Interferon-induced transmembrane protein 3 OS=Mus musculus Ifitm3 PE=1 SV=1 - [IFM3] 0 2 0 2 0 1 0 5 N/A 7.490E-03 14.9
465
N
o
.
1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
As-
ses-
sion
Gene
ID Description
- + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
GlcN
Alk
Known
Acetylated
Known O-
GlcNAcylat
ed
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
Q6P9J9 ANO6 Anoctamin-6 OS=Mus musculus Ano6 PE=1 SV=1 - [ANO6] 0 2 0 1 0 2 0 5 N/A 7.490E-03 106
P63037 DNJA1DnaJ homolog subfamily A member 1 OS=Mus musculus Dnaja1 PE=1 SV=1 - [DNJA1] 0 1 0 2 0 1 0 4 N/A 1.613E-02 44.8 ✔ (23)(24) (7)(12)(19)
Q8VIJ6SFPQ Splicing factor, proline- and glutamine-rich OS=Mus musculus Sfpq PE=1 SV=1 - [SFPQ] 0 2 0 1 0 1 0 4 N/A 1.613E-02 75.4 ✔ (20)(23)(24) (4)(7)(12)(13)(17)
P58871TB182 182 kDa tankyrase-1-binding protein OS=Mus musculus Tnks1bp1 PE=1 SV=2 - [TB182] 0 1 0 1 0 2 0 4 N/A 1.613E-02 182 ✔ (20) (1)(7)(13)(19)
P63276 RS17 40S ribosomal protein S17 OS=Mus musculus Rps17 PE=1 SV=2 - [RS17] 0 1 0 2 0 1 0 4 N/A 1.613E-02 15.5 ✔ (20)(23)(24)
B1AT03 B1AT03DNA ligase OS=Mus musculus Lig3 PE=3 SV=1 - [B1AT03] 0 1 0 2 0 1 0 4 N/A 1.613E-02 106 ✔
E9QP49 E9QP49 EH domain-binding protein 1-like protein 1 OS=Mus musculus Ehbp1l1 PE=4 SV=1 - [E9QP49] 0 2 0 1 0 1 0 4 N/A 1.613E-02 185
Q8BU30SYIC Isoleucine--tRNA ligase, cytoplasmic OS=Mus musculus Iars PE=2 SV=2 - [SYIC] 0 1 0 1 0 2 0 4 N/A 1.613E-02 144 (20)(23)(24) (7)(10)
Q9JLI8SART3Squamous cell carcinoma antigen recognized by T-cells 3 OS=Mus musculus Sart3 PE=2 SV=1 - [SART3] 0 2 0 1 0 1 0 4 N/A 1.613E-02 110 (20) (7)
E9QAI5 E9QAI5Protein Cad OS=Mus musculus Cad PE=3 SV=1 - [E9QAI5] 0 2 0 1 0 1 0 4 N/A 1.613E-02 236 (7)
D3YYD2 D3YYD2 Protein FAM111A OS=Mus musculus Fam111a PE=4 SV=1 - [D3YYD2] 0 2 0 1 0 1 0 4 N/A 1.613E-02 64.7
Q91V04 TRAM1Translocating chain-associated membrane protein 1 OS=Mus musculus Tram1 PE=1 SV=3 - [TRAM1] 0 1 0 2 0 1 0 4 N/A 1.613E-02 43
F6ZDT4 F6ZDT4 Ribonuclease H2 subunit C (Fragment) OS=Mus musculus Rnaseh2c PE=4 SV=1 - [F6ZDT4] 0 1 0 2 0 1 0 4 N/A 1.613E-02 9.3
D3Z0B9 D3Z0B9 Aldehyde dehydrogenase family 16 member A1 OS=Mus musculus Aldh16a1 PE=4 SV=1 - [D3Z0B9] 0 1 0 1 0 2 0 4 N/A 1.613E-02 79.4
P61804DAD1 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1 OS=Mus musculus Dad1 PE=2 SV=3 - [DAD1] 0 1 0 1 0 2 0 4 N/A 1.613E-02 12.5
P42125 ECI1 Enoyl-CoA delta isomerase 1, mitochondrial OS=Mus musculus Eci1 PE=2 SV=2 - [ECI1] 0 5 0 5 1 3 1 13 13.00 5.821E-03 32.2 (20)(23)(24)
Q61191 HCFC1Host cell factor 1 OS=Mus musculus Hcfc1 PE=1 SV=2 - [HCFC1] 1 4 0 5 0 4 1 13 13.00 1.058E-03 210 ✔ (20)(23)(24) (1)(2)(7)(9)(12)(13)(14)(15)(17)(18)(19)
Q6NV83-3 SR140 Isoform 3 of U2 snRNP-associated SURP motif-containing protein OS=Mus musculus U2surp - [SR140] 1 3 0 2 0 5 1 10 10.00 3.347E-02 113 (23)(24) (7)
O70194 EIF3D Eukaryotic translation initiation factor 3 subunit D OS=Mus musculus Eif3d PE=1 SV=2 - [EIF3D] 2 5 0 9 0 4 2 18 9.00 3.290E-02 63.9
P46978 STT3A Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A OS=Mus musculus Stt3a PE=1 SV=1 - [STT3A] 1 3 0 3 0 3 1 9 9.00 1.324E-03 80.5 ✔
P80317TCPZ T-complex protein 1 subunit zeta OS=Mus musculus Cct6a PE=1 SV=3 - [TCPZ] 2 7 0 6 0 4 2 17 8.50 1.064E-02 58 (20)(22)(23)(24) (7)(10)
P21107-2 TPM3 Isoform 2 of Tropomyosin alpha-3 chain OS=Mus musculus Tpm3 - [TPM3] 2 7 0 6 0 4 2 17 8.50 1.064E-02 29 ✔ (22)(23)(24) (19)
J3QNW0 J3QNW0 Cytosine-specific methyltransferase OS=Mus musculus Dnmt1 PE=3 SV=1 - [J3QNW0] 0 7 0 8 3 10 3 25 8.33 5.328E-03 170 (7)
E9QNN1 E9QNN1 ATP-dependent RNA helicase A OS=Mus musculus Dhx9 PE=4 SV=1 - [E9QNN1] 0 6 0 7 2 3 2 16 8.00 2.739E-02 150 (7)
Q9DBJ1 PGAM1Phosphoglycerate mutase 1 OS=Mus musculus Pgam1 PE=1 SV=3 - [PGAM1] 0 3 0 6 2 7 2 16 8.00 2.739E-02 28.8 (20)(22)(23)(24) (4)(7)(13)
Q8VCQ8 Q8VCQ8 Caldesmon 1 OS=Mus musculus Cald1 PE=2 SV=1 - [Q8VCQ8] 0 4 0 2 1 2 1 8 8.00 3.517E-02 60.4 ✔ (7)(19)
Q9ER72-2 SYCC Isoform 2 of Cysteine--tRNA ligase, cytoplasmic OS=Mus musculus Cars - [SYCC] 0 2 0 4 1 2 1 8 8.00 3.517E-02 85.5 ✔ (23)(24) (7)
P70399-3 TP53B Isoform 3 of Tumor suppressor p53-binding protein 1 OS=Mus musculus Tp53bp1 - [TP53B] 1 3 0 3 0 2 1 8 8.00 7.763E-03 207 ✔ (7)(10)
O70503 DHB12Estradiol 17-beta-dehydrogenase 12 OS=Mus musculus Hsd17b12 PE=2 SV=1 - [DHB12] 1 3 0 2 0 3 1 8 8.00 7.763E-03 34.7 ✔ (20)
A2AG46 A2AG46 Melanoma antigen, family D, 2 (Fragment) OS=Mus musculus Maged2 PE=4 SV=1 - [A2AG46] 0 2 0 3 1 3 1 8 8.00 7.763E-03 29.2
D3Z0A2 D3Z0A2 Protein arginine N-methyltransferase 1 OS=Mus musculus Prmt1 PE=4 SV=1 - [D3Z0A2] 4 9 0 10 0 12 4 31 7.75 4.897E-03 36.5 ✔ (7)
Q8BK67 RCC2 Protein RCC2 OS=Mus musculus Rcc2 PE=2 SV=1 - [RCC2] 2 5 0 5 0 4 2 14 7.00 5.821E-03 55.9 ✔ (20)(22)(23)(24) (7)
A2AGT5-2 CKAP5Isoform 2 of Cytoskeleton-associated protein 5 OS=Mus musculus Ckap5 - [CKAP5] 0 2 0 3 1 2 1 7 7.00 1.324E-02 219 (23)(24) (4)(7)
P57780 ACTN4Alpha-actinin-4 OS=Mus musculus Actn4 PE=1 SV=1 - [ACTN4] 2 7 0 4 1 9 3 20 6.67 2.227E-02 105 ✔ (20)(23)(24) (7)
Q9WTQ5-2 AKA12Isoform 2 of A-kinase anchor protein 12 OS=Mus musculus Akap12 - [AKA12] 2 6 0 9 1 5 3 20 6.67 1.316E-02 170 (7)
466
N
o
.
1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
As-
ses-
sion
Gene
ID Description
- + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
GlcN
Alk
Known
Acetylated
Known O-
GlcNAcylat
ed
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
F7CVJ5 F7CVJ5Protein Ahnak2 (Fragment) OS=Mus musculus Ahnak2 PE=4 SV=1 - [F7CVJ5] 1 3 0 4 1 5 2 12 6.00 7.490E-03 115
Q9R0P5DEST Destrin OS=Mus musculus Dstn PE=1 SV=3 - [DEST] 0 2 0 2 1 2 1 6 6.00 7.490E-03 18.5 ✔ (20)(23)(24) (7)(19)
Q8C156 CND2 Condensin complex subunit 2 OS=Mus musculus Ncaph PE=2 SV=1 - [CND2] 1 2 0 2 0 2 1 6 6.00 7.490E-03 82.3 ✔ (20)(23)(24) (7)
Q791V5 MTCH2Mitochondrial carrier homolog 2 OS=Mus musculus Mtch2 PE=1 SV=1 - [MTCH2] 0 2 0 2 1 2 1 6 6.00 7.490E-03 33.5 ✔ (20)(21) (7)
O35316 SC6A6Sodium- and chloride-dependent taurine transporter OS=Mus musculus Slc6a6 PE=1 SV=2 - [SC6A6] 0 2 0 2 1 2 1 6 6.00 7.490E-03 69.8 ✔
Q921M3-2 SF3B3 Isoform 2 of Splicing factor 3B subunit 3 OS=Mus musculus Sf3b3 - [SF3B3] 0 2 0 2 1 2 1 6 6.00 7.490E-03 125 (23)(24) (7)(10)
Q6ZQI3 MLEC Malectin OS=Mus musculus Mlec PE=2 SV=2 - [MLEC] 1 2 0 2 0 2 1 6 6.00 7.490E-03 32.3
Q99NF7 Q99NF7 Ppm1b protein OS=Mus musculus Ppm1b PE=2 SV=1 - [Q99NF7] 0 2 1 2 0 2 1 6 6.00 7.490E-03 52.1
F6THG2 F6THG2 Actin-related protein 2/3 complex subunit 1B (Fragment) OS=Mus musculus Arpc1b PE=4 SV=1 - [F6THG2] 0 2 0 2 1 2 1 6 6.00 7.490E-03 11.1
P68372 TBB4BTubulin beta-4B chain OS=Mus musculus Tubb4b PE=1 SV=1 - [TBB4B] 0 14 2 21 7 16 9 51 5.67 8.934E-03 49.8 (20)(22)(23)(24) (4)(10)(13)
E9Q7G0 E9Q7G0 Protein Numa1 OS=Mus musculus Numa1 PE=4 SV=1 - [E9Q7G0] 1 6 0 7 2 4 3 17 5.67 1.145E-02 236 ✔ (7)
Q9ET54-4 PALLD Isoform 4 of Palladin OS=Mus musculus Palld - [PALLD] 1 2 0 4 1 5 2 11 5.50 3.347E-02 108 ✔ (7)
Q8BL97-3 SRSF7 Isoform 3 of Serine/arginine-rich splicing factor 7 OS=Mus musculus Srsf7 - [SRSF7] 0 3 2 3 0 5 2 11 5.50 3.347E-02 17.9 (23)(24)
Q61656DDX5 Probable ATP-dependent RNA helicase DDX5 OS=Mus musculus Ddx5 PE=1 SV=2 - [DDX5] 1 3 0 3 1 5 2 11 5.50 1.580E-02 69.2 ✔ (20)(22)(23)(24) (4)(7)
P26039TLN1 Talin-1 OS=Mus musculus Tln1 PE=1 SV=2 - [TLN1] 0 4 1 4 1 3 2 11 5.50 3.126E-03 270 (20)(23)(24) (7)
Q9JHU4 DYHC1Cytoplasmic dynein 1 heavy chain 1 OS=Mus musculus Dync1h1 PE=1 SV=2 - [DYHC1] 1 3 0 4 1 4 2 11 5.50 3.126E-03 532 (20)(23)(24) (4)(7)
Q61171 PRDX2Peroxiredoxin-2 OS=Mus musculus Prdx2 PE=1 SV=3 - [PRDX2] 0 4 2 7 1 5 3 16 5.33 1.472E-02 21.8 (20) (7)(10)(12)
P60843IF4A1 Eukaryotic initiation factor 4A-I OS=Mus musculus Eif4a1 PE=2 SV=1 - [IF4A1] 3 6 0 6 1 9 4 21 5.25 1.316E-02 46.1 (20)(23)(24) (7)(10)(18)
P07742 RIR1 Ribonucleoside-diphosphate reductase large subunit OS=Mus musculus Rrm1 PE=1 SV=2 - [RIR1] 2 4 1 5 0 6 3 15 5.00 8.050E-03 90.2 ✔ (20)(23)(24) (7)(10)(19)
Q61553 FSCN1Fascin OS=Mus musculus Fscn1 PE=1 SV=4 - [FSCN1] 1 2 0 5 1 3 2 10 5.00 4.742E-02 54.5 ✔ (20)(23)(24) (4)(7)(10)
P08752 GNAI2Guanine nucleotide-binding protein G(i) subunit alpha-2 OS=Mus musculus Gnai2 PE=1 SV=5 - [GNAI2] 2 3 0 4 0 3 2 10 5.00 2.322E-02 40.5 ✔ (20)(22) (13)(19)
P40124CAP1 Adenylyl cyclase-associated protein 1 OS=Mus musculus Cap1 PE=1 SV=4 - [CAP1] 1 1 0 2 0 2 1 5 5.00 4.742E-02 51.5 ✔ (20)(23)(24) (7)
P62852 RS25 40S ribosomal protein S25 OS=Mus musculus Rps25 PE=2 SV=1 - [RS25] 1 2 0 2 0 1 1 5 5.00 4.742E-02 13.7 ✔ (20)(23)(24)
D3Z5M2 D3Z5M2 Protein Gm10110 OS=Mus musculus Gm10110 PE=4 SV=1 - [D3Z5M2] 1 2 0 2 0 1 1 5 5.00 4.742E-02 67.7
Q91X76 Q91X76 5'-nucleotidase domain containing 2 OS=Mus musculus Nt5dc2 PE=2 SV=1 - [Q91X76] 1 1 0 2 0 2 1 5 5.00 4.742E-02 46 ✔
Q99020 ROAA Heterogeneous nuclear ribonucleoprotein A/B OS=Mus musculus Hnrnpab PE=1 SV=1 - [ROAA] 0 2 0 1 1 2 1 5 5.00 4.742E-02 30.8 (20)(23)(24) (7)(17)
P14206RSSA 40S ribosomal protein SA OS=Mus musculus Rpsa PE=1 SV=4 - [RSSA] 0 2 1 1 0 2 1 5 5.00 4.742E-02 32.8 (20)(23)(24) (7)
Q99JY9ARP3 Actin-related protein 3 OS=Mus musculus Actr3 PE=1 SV=3 - [ARP3] 0 2 0 1 1 2 1 5 5.00 4.742E-02 47.3 (20)(23)(24)
Q6PFR5 TRA2ATransformer-2 protein homolog alpha OS=Mus musculus Tra2a PE=1 SV=1 - [TRA2A] 0 2 0 1 1 2 1 5 5.00 4.742E-02 32.3
P62821 RAB1ARas-related protein Rab-1A OS=Mus musculus Rab1A PE=1 SV=3 - [RAB1A] 0 9 1 6 3 4 4 19 4.75 4.232E-02 22.7 (22)
Q0P678 ZCH18Zinc finger CCCH domain-containing protein 18 OS=Mus musculus Zc3h18 PE=1 SV=1 - [ZCH18] 0 9 3 7 3 10 6 26 4.33 7.490E-03 106 (20)(23)(24) (7)
Q62261-2 SPTB2 Isoform 2 of Spectrin beta chain, non-erythrocytic 1 OS=Mus musculus Sptbn1 - [SPTB2] 1 3 0 5 2 5 3 13 4.33 1.944E-02 251 (23)(24) (4)(7)(9)(14)
P84104-2 SRSF3 Isoform Short of Serine/arginine-rich splicing factor 3 OS=Mus musculus Srsf3 - [SRSF3] 1 5 0 4 2 4 3 13 4.33 7.490E-03 14.2 ✔ (23)(24) (7)
Q569Z6 TR150 Thyroid hormone receptor-associated protein 3 OS=Mus musculus Thrap3 PE=1 SV=1 - [TR150] 1 9 0 6 5 9 6 24 4.00 3.032E-02 108 (20)(23)(24) (7)
H3BKR2 H3BKR2 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 (Fragment) OS=Mus musculus Gnb1 PE=4 SV=1 - [H3BKR2] 0 4 2 4 1 4 3 12 4.00 6.533E-03 30.3
467
N
o
.
1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk 1-deoxy-Ac 3GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
As-
ses-
sion
Gene
ID Description
- + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
GlcN
Alk
Known
Acetylated
Known O-
GlcNAcylat
ed
99Q501J6 DDX17Probable ATP-dependent RNA helicase DDX17 OS=Mus musculus Ddx17 PE=2 SV=1 - [DDX17] 1 2 0 3 1 3 2 8 4.00 1.324E-02 72.4 ✔ (20)(22)(23)(24) (4)(7)(19)
468
Table 7-2. Ac4GlcNAlk Labelled Proteins. Mouse embryonic fibroblasts were treated in triplicate with either Ac4GlcNAlk
(200 µM, +) or Ac4GlcNAc (200 µM, -) for 16 hours. At this time the cell lysates were subjected to CuAAC with alkyne-biotin,
followed by enrichment with streptavidin beads and on-bead trypsinolysis. Labeled proteins were selected as those that were
represented by at least 1 unique-peptide in each Ac4GlcNAlk treated sample, a total of at least 3 spectral-counts from the same
three samples, and at least a total of 3 times more spectral counts in the Ac4GlcNAlk treated samples compared to Ac4GlcNAc.
A t-test was also conducted with a maxmium value of 0.05 allowed.
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
P58871 TB182 182 kDa tankyrase-1-binding protein OS=Mus musculus Tnks1bp1 PE=1 SV=2 - [TB182] 0 29 0 28 0 27 0 84 N/A 1.082E-06 181.7 ✔ (20) (1)(7)(13)(16)(19)
O35286 DHX15 Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15 OS=Mus musculus Dhx15 PE=2 SV=2 - [DHX15] 0 21 0 20 0 24 0 65 N/A 5.566E-05 90.9 ✔ (20)(22)(23)(24) (7)(10)(16)(19)
Q60710 SAMH1 SAM domain and HD domain-containing protein 1 OS=Mus musculus Samhd1 PE=1 SV=2 - [SAMH1] 0 18 0 16 0 22 0 56 N/A 4.511E-04 72.6 (7)(16)
Q61024 ASNS Asparagine synthetase [glutamine-hydrolyzing] OS=Mus musculus Asns PE=2 SV=3 - [ASNS] 0 16 0 16 0 16 0 48 N/A 0.000E+00 64.2 (23)(24) (7)(19)
Q8BFW7 LPP Lipoma-preferred partner homolog OS=Mus musculus Lpp PE=1 SV=1 - [LPP] 0 13 0 19 0 13 0 45 N/A 1.691E-03 65.8 ✔ (20) (7)(8)(13)(16)(19)
Q99K48 NONO Non-POU domain-containing octamer-binding protein OS=Mus musculus Nono PE=1 SV=3 - [NONO] 0 13 0 16 0 15 0 44 N/A 7.658E-05 54.5 (22)(23)(24) (7)(12)(16)(17)(19)
Q3U4W8 Q3U4W8 Ubiquitin carboxyl-terminal hydrolase OS=Mus musculus Usp5 PE=2 SV=1 - [Q3U4W8] 0 14 0 14 0 13 0 41 N/A 2.115E-06 93.3 (7)
Q9R1C7-2 PR40A Isoform 2 of Pre-mRNA-processing factor 40 homolog A OS=Mus musculus Prpf40a - [PR40A] 0 12 0 17 0 10 0 39 N/A 3.351E-03 103.9 (23)(24) (7)(16)
Q9ERG0-2 LIMA1 Isoform Alpha of LIM domain and actin-binding protein 1 OS=Mus musculus Lima1 - [LIMA1] 0 12 0 14 0 12 0 38 N/A 4.520E-05 66 ✔ (7)(16)(19)
P61222 ABCE1 ATP-binding cassette sub-family E member 1 OS=Mus musculus Abce1 PE=2 SV=1 - [ABCE1] 0 14 0 9 0 13 0 36 N/A 1.419E-03 67.3 (23)(24) (7)(16)(19)
Q99P88 NU155 Nuclear pore complex protein Nup155 OS=Mus musculus Nup155 PE=2 SV=1 - [NU155] 0 11 0 13 0 12 0 36 N/A 3.166E-05 155 (7)(16)
Q9CPV4GLOD4 Glyoxalase domain-containing protein 4 OS=Mus musculus Glod4 PE=2 SV=1 - [GLOD4] 0 11 0 14 0 10 0 35 N/A 6.304E-04 33.3 (23)(24) (7)(16)(19)
O88291 ZN326 DBIRD complex subunit ZNF326 OS=Mus musculus Znf326 PE=1 SV=1 - [ZN326] 0 11 0 12 0 12 0 35 N/A 3.977E-06 65.2 (23)(24) (7)(16)
F7AA26 F7AA26 Protein Gm20459 (Fragment) OS=Mus musculus Gm20459 PE=4 SV=1 - [F7AA26] 0 12 0 12 0 11 0 35 N/A 3.977E-06 126
Q60865 CAPR1 Caprin-1 OS=Mus musculus Caprin1 PE=1 SV=2 - [CAPR1] 0 12 0 10 0 12 0 34 N/A 7.021E-05 78.1 ✔ (20) (7)(12)(16)(19)
O70318 E41L2 Band 4.1-like protein 2 OS=Mus musculus Epb41l2 PE=1 SV=2 - [E41L2] 0 12 0 11 0 11 0 34 N/A 4.464E-06 109.9 (23)(24) (7)(13)(16)
Q60854 SPB6 Serpin B6 OS=Mus musculus Serpinb6 PE=2 SV=1 - [SPB6] 0 12 0 11 0 11 0 34 N/A 4.464E-06 42.6 (19)
Q60598 SRC8 Src substrate cortactin OS=Mus musculus Cttn PE=1 SV=2 - [SRC8] 0 11 0 11 0 12 0 34 N/A 4.464E-06 61.2 (23)(24) (13)(19)
Q9Z1X4-2 ILF3 Isoform 2 of Interleukin enhancer-binding factor 3 OS=Mus musculus Ilf3 - [ILF3] 0 14 0 12 0 7 0 33 N/A 6.152E-03 78 (22)(23)(24) (7)(12)
P35235 PTN11 Tyrosine-protein phosphatase non-receptor type 11 OS=Mus musculus Ptpn11 PE=1 SV=2 - [PTN11] 0 13 0 12 0 7 0 32 N/A 4.543E-03 68.4 (23)(24) (7)(10)(16)
Q925B0 PAWR PRKC apoptosis WT1 regulator protein OS=Mus musculus Pawr PE=1 SV=2 - [PAWR] 0 13 0 11 0 8 0 32 N/A 1.833E-03 35.9 (7)(16)
P70698 PYRG1 CTP synthase 1 OS=Mus musculus Ctps1 PE=1 SV=2 - [PYRG1] 0 12 0 12 0 8 0 32 N/A 1.324E-03 66.6 (23)(24) (16)(19)
Q8BX02KANK2 KN motif and ankyrin repeat domain-containing protein 2 OS=Mus musculus Kank2 PE=1 SV=1 - [KANK2] 0 10 0 13 0 9 0 32 N/A 8.903E-04 90.2
Q80YR5 SAFB2 Scaffold attachment factor B2 OS=Mus musculus Safb2 PE=1 SV=2 - [SAFB2] 0 7 0 13 0 11 0 31 N/A 4.237E-03 111.8 (23)(24) (7)(16)
Q9QXD8 LIMD1 LIM domain-containing protein 1 OS=Mus musculus Limd1 PE=1 SV=2 - [LIMD1] 0 7 0 16 0 7 0 30 N/A 2.902E-02 71.4 (7)(16)
P70372 ELAV1 ELAV-like protein 1 OS=Mus musculus Elavl1 PE=1 SV=2 - [ELAV1] 0 8 0 13 0 9 0 30 N/A 2.814E-03 36.1 (7)(12)(19)
Q9R0X4ACOT9 Acyl-coenzyme A thioesterase 9, mitochondrial OS=Mus musculus Acot9 PE=1 SV=1 - [ACOT9] 0 11 0 12 0 7 0 30 N/A 2.814E-03 50.5 (23)(24)
G3X8Y3 G3X8Y3 N-alpha-acetyltransferase 15, NatA auxiliary subunit OS=Mus musculus Naa15 PE=4 SV=1 - [G3X8Y3] 0 10 0 11 0 8 0 29 N/A 3.936E-04 101 (7)
469
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
Q6NXL1Q6NXL1 Protein Sec24d OS=Mus musculus Sec24d PE=2 SV=1 - [Q6NXL1] 0 8 0 11 0 10 0 29 N/A 3.936E-04 112.6 (7)
Q9CT10RANB3 Ran-binding protein 3 OS=Mus musculus Ranbp3 PE=1 SV=2 - [RANB3] 0 8 0 10 0 10 0 28 N/A 1.510E-04 52.5 (23)(24) (7)
Q9JIK5 DDX21 Nucleolar RNA helicase 2 OS=Mus musculus Ddx21 PE=1 SV=3 - [DDX21] 0 7 0 10 0 10 0 27 N/A 8.438E-04 93.5 (22)(23)(24) (7)(10)(16)(19)
G5E896 G5E896 Enhancer of mRNA decapping 4, isoform CRA_b OS=Mus musculus Edc4 PE=4 SV=1 - [G5E896] 0 10 0 9 0 8 0 27 N/A 9.888E-05 152.4 (7)
Q91Z38 TTC1 Tetratricopeptide repeat protein 1 OS=Mus musculus Ttc1 PE=2 SV=1 - [TTC1] 0 5 0 9 0 12 0 26 N/A 1.291E-02 33.2 (7)(16)
Q923B1 DBR1 Lariat debranching enzyme OS=Mus musculus Dbr1 PE=1 SV=2 - [DBR1] 0 7 0 12 0 7 0 26 N/A 6.516E-03 62.3 (23)(24) (16)
A2AJ72 A2AJ72 Far upstream element (FUSE) binding protein 3 OS=Mus musculus Fubp3 PE=4 SV=1 - [A2AJ72] 0 9 0 6 0 11 0 26 N/A 3.967E-03 61.4 (7)
Q99P72 RTN4 Reticulon-4 OS=Mus musculus Rtn4 PE=1 SV=2 - [RTN4] 0 6 0 11 0 9 0 26 N/A 3.967E-03 126.5 ✔ (20)(23)(24) (1)(13)(19)
Q8K310 MATR3 Matrin-3 OS=Mus musculus Matr3 PE=1 SV=1 - [MATR3] 0 7 0 8 0 11 0 26 N/A 1.961E-03 94.6 (22)(23)(24) (7)(16)(19)
D3YXK2 SAFB1 Scaffold attachment factor B1 OS=Mus musculus Safb PE=1 SV=2 - [SAFB1] 0 9 0 9 0 8 0 26 N/A 1.300E-05 105 (23)(24) (7)
Q99KE1MAOM NAD-dependent malic enzyme, mitochondrial OS=Mus musculus Me2 PE=2 SV=1 - [MAOM] 0 9 0 10 0 6 0 25 N/A 2.272E-03 65.8 (23)(24)
O08915 AIP AH receptor-interacting protein OS=Mus musculus Aip PE=1 SV=1 - [AIP] 0 6 0 10 0 9 0 25 N/A 2.272E-03 37.6 (13)
Q8C1A5THOP1 Thimet oligopeptidase OS=Mus musculus Thop1 PE=1 SV=1 - [THOP1] 0 9 0 8 0 8 0 25 N/A 1.520E-05 78 (23)(24) (7)
Q8K298 ANLN Actin-binding protein anillin OS=Mus musculus Anln PE=1 SV=2 - [ANLN] 0 11 0 6 0 7 0 24 N/A 6.352E-03 122.7 (23)(24) (7)(19)
P51125-3 ICAL Isoform 3 of Calpastatin OS=Mus musculus Cast - [ICAL] 0 8 0 6 0 10 0 24 N/A 2.278E-03 79.6 (23)(24) (7)(19)
Q08093 CNN2 Calponin-2 OS=Mus musculus Cnn2 PE=2 SV=1 - [CNN2] 0 7 0 9 0 8 0 24 N/A 1.573E-04 33.1 (23)(24) (7)(19)
Q8K1M6-3 DNM1L Isoform 3 of Dynamin-1-like protein OS=Mus musculus Dnm1l - [DNM1L] 0 8 0 8 0 8 0 24 N/A 0.000E+00 78 (23)(24) (4)
Q91W50 CSDE1 Cold shock domain-containing protein E1 OS=Mus musculus Csde1 PE=2 SV=1 - [CSDE1] 0 8 0 6 0 9 0 23 N/A 9.640E-04 88.7 (23)(24) (7)(12)
Q9DBG5 PLIN3 Perilipin-3 OS=Mus musculus Plin3 PE=1 SV=1 - [PLIN3] 0 9 0 7 0 7 0 23 N/A 3.264E-04 47.2 (23)(24) (1)(7)(13)(16)
D3Z3F8 D3Z3F8 Spartin OS=Mus musculus Spg20 PE=4 SV=1 - [D3Z3F8] 0 8 0 4 0 10 0 22 N/A 1.417E-02 63
P49717 MCM4 DNA replication licensing factor MCM4 OS=Mus musculus Mcm4 PE=2 SV=1 - [MCM4] 0 5 0 10 0 7 0 22 N/A 7.246E-03 96.7 (23)(24)
Q80X50 UBP2L Ubiquitin-associated protein 2-like OS=Mus musculus Ubap2l PE=1 SV=1 - [UBP2L] 0 8 0 9 0 5 0 22 N/A 3.650E-03 116.7 (23)(24) (1)(4)(12)(13)(16)(19)
Q91WG2 RABE2 Rab GTPase-binding effector protein 2 OS=Mus musculus Rabep2 PE=2 SV=3 - [RABE2] 0 7 0 6 0 9 0 22 N/A 1.143E-03 62.1 (7)
Q80VB6Q80VB6 Dclk1 protein OS=Mus musculus Dclk1 PE=2 SV=1 - [Q80VB6] 0 6 0 9 0 7 0 22 N/A 1.143E-03 40.4
Q8BXQ2 PIGT GPI transamidase component PIG-T OS=Mus musculus Pigt PE=1 SV=2 - [PIGT] 0 7 0 6 0 9 0 22 N/A 1.143E-03 65.7
E9Q450 E9Q450 Tropomyosin alpha-1 chain OS=Mus musculus Tpm1 PE=3 SV=1 - [E9Q450] 0 6 0 8 0 8 0 22 N/A 3.882E-04 32.8 ✔
P51859 HDGF Hepatoma-derived growth factor OS=Mus musculus Hdgf PE=1 SV=2 - [HDGF] 0 7 0 7 0 8 0 22 N/A 2.526E-05 26.3 (23)(24) (7)(19)
Q7TMK9-2 HNRPQ Isoform 2 of Heterogeneous nuclear ribonucleoprotein Q OS=Mus musculus Syncrip - [HNRPQ] 0 8 0 7 0 7 0 22 N/A 2.526E-05 62.6 (23)(24) (7)(12)(19)
Q9Z0N1IF2G Eukaryotic translation initiation factor 2 subunit 3, X-linked OS=Mus musculus Eif2s3x PE=1 SV=2 - [IF2G] 0 7 0 7 0 8 0 22 N/A 2.526E-05 51 (10)(16)(19)
Q9JLV1 BAG3 BAG family molecular chaperone regulator 3 OS=Mus musculus Bag3 PE=1 SV=2 - [BAG3] 0 5 0 10 0 6 0 21 N/A 1.016E-02 61.8 (7)(16)(19)
Q8CIB5 FERM2 Fermitin family homolog 2 OS=Mus musculus Fermt2 PE=1 SV=1 - [FERM2] 0 5 0 8 0 8 0 21 N/A 2.192E-03 77.8
Q9CQX2 CYB5B Cytochrome b5 type B OS=Mus musculus Cyb5b PE=1 SV=1 - [CYB5B] 0 7 0 8 0 6 0 21 N/A 2.655E-04 16.3 (23)(24) (19)
Q9D4G5 Q9D4G5 Protein Pop1 OS=Mus musculus Pop1 PE=2 SV=1 - [Q9D4G5] 0 5 0 10 0 5 0 20 N/A 1.613E-02 114
D3Z6W2D3Z6W2 Tyrosine-protein phosphatase non-receptor type OS=Mus musculus Ptpn2 PE=3 SV=1 - [D3Z6W2] 0 4 0 9 0 7 0 20 N/A 1.012E-02 42.3
Q8CI11 GNL3 Guanine nucleotide-binding protein-like 3 OS=Mus musculus Gnl3 PE=1 SV=2 - [GNL3] 0 8 0 8 0 4 0 20 N/A 7.490E-03 60.7 (23)(24) (7)(16)(19)
470
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
P52479 UBP10 Ubiquitin carboxyl-terminal hydrolase 10 OS=Mus musculus Usp10 PE=1 SV=3 - [UBP10] 0 8 0 4 0 8 0 20 N/A 7.490E-03 87 (7)(16)(19)
Q9DBR1-2 XRN2 Isoform 2 of 5'-3' exoribonuclease 2 OS=Mus musculus Xrn2 - [XRN2] 0 8 0 8 0 4 0 20 N/A 7.490E-03 108 (23)(24) (7)
Q99JB2 STML2 Stomatin-like protein 2 OS=Mus musculus Stoml2 PE=1 SV=1 - [STML2] 0 9 0 6 0 5 0 20 N/A 5.167E-03 38.4 (23)(24) (7)(16)
Q62523 ZYX Zyxin OS=Mus musculus Zyx PE=1 SV=2 - [ZYX] 0 5 0 6 0 9 0 20 N/A 5.167E-03 60.5 (23)(24) (1)(7)(19)
Q6PGH2 HN1L Hematological and neurological expressed 1-like protein OS=Mus musculus Hn1l PE=2 SV=1 - [HN1L] 0 7 0 5 0 8 0 20 N/A 1.641E-03 20 (7)(16)(19)
G3X972 G3X972 Protein Sec24c OS=Mus musculus Sec24c PE=4 SV=1 - [G3X972] 0 7 0 8 0 5 0 20 N/A 1.641E-03 118.5
E9Q066 E9Q066 La-related protein 4 OS=Mus musculus Larp4 PE=4 SV=1 - [E9Q066] 0 7 0 7 0 6 0 20 N/A 3.688E-05 79.6 ✔ (7)
A2AMY5A2AMY5 Ubiquitin-associated protein 2 OS=Mus musculus Ubap2 PE=4 SV=1 - [A2AMY5] 0 7 0 7 0 6 0 20 N/A 3.688E-05 117.8 (7)
Q8CFD4SNX8 Sorting nexin-8 OS=Mus musculus Snx8 PE=2 SV=1 - [SNX8] 0 4 0 7 0 8 0 19 N/A 6.214E-03 52
Q8BMJ2SYLC Leucine--tRNA ligase, cytoplasmic OS=Mus musculus Lars PE=2 SV=2 - [SYLC] 0 6 0 8 0 5 0 19 N/A 1.991E-03 134.1 (7)(19)
Q99JF8 PSIP1 PC4 and SFRS1-interacting protein OS=Mus musculus Psip1 PE=1 SV=1 - [PSIP1] 0 7 0 5 0 7 0 19 N/A 6.852E-04 59.7 (22) (7)(16)
P25206 MCM3 DNA replication licensing factor MCM3 OS=Mus musculus Mcm3 PE=1 SV=2 - [MCM3] 0 7 0 6 0 6 0 19 N/A 4.520E-05 91.5 (22)(23)(24) (7)
Q9QWF0 CAF1A Chromatin assembly factor 1 subunit A OS=Mus musculus Chaf1a PE=1 SV=1 - [CAF1A] 0 9 0 4 0 5 0 18 N/A 1.713E-02 101.9 (7)(16)
Q9WUD1 CHIP STIP1 homology and U box-containing protein 1 OS=Mus musculus Stub1 PE=1 SV=1 - [CHIP] 0 5 0 9 0 4 0 18 N/A 1.713E-02 34.9 (7)(16)
Q8BI72 CARF CDKN2A-interacting protein OS=Mus musculus Cdkn2aip PE=2 SV=1 - [CARF] 0 7 0 6 0 5 0 18 N/A 4.841E-04 59.7 (7)(16)
B2RRE7 OTUD4 OTU domain-containing protein 4 OS=Mus musculus Otud4 PE=1 SV=1 - [OTUD4] 0 5 0 7 0 6 0 18 N/A 4.841E-04 123 (7)(16)
P42669 PURA Transcriptional activator protein Pur-alpha OS=Mus musculus Pura PE=1 SV=1 - [PURA] 0 7 0 6 0 5 0 18 N/A 4.841E-04 34.9 (23)(24) (7)(13)
Q924B0 Q924B0 Inositol (Myo)-1(Or 4)-monophosphatase 1 OS=Mus musculus Impa1 PE=2 SV=1 - [Q924B0] 0 6 0 6 0 6 0 18 N/A 0.000E+00 30.4 (19)
Q9CXF4TBC15 TBC1 domain family member 15 OS=Mus musculus Tbc1d15 PE=1 SV=1 - [TBC15] 0 7 0 8 0 2 0 17 N/A 3.791E-02 76.5 ✔ (7)(16)
Q80UG5-3 SEPT9 Isoform 3 of Septin-9 OS=Mus musculus Sept9 - [SEPT9] 0 7 0 3 0 7 0 17 N/A 1.316E-02 64.7
Q8WTY4 CPIN1 Anamorsin OS=Mus musculus Ciapin1 PE=1 SV=1 - [CPIN1] 0 4 0 6 0 7 0 17 N/A 3.016E-03 33.4 (7)(19)
Q91YS8 KCC1A Calcium/calmodulin-dependent protein kinase type 1 OS=Mus musculus Camk1 PE=1 SV=1 - [KCC1A] 0 5 0 5 0 7 0 17 N/A 1.051E-03 41.6 (7)
P35821 PTN1 Tyrosine-protein phosphatase non-receptor type 1 OS=Mus musculus Ptpn1 PE=1 SV=2 - [PTN1] 0 7 0 5 0 5 0 17 N/A 1.051E-03 49.6
E9PUH7 E9PUH7 Nuclear factor 1 OS=Mus musculus Nfix PE=3 SV=1 - [E9PUH7] 0 6 0 5 0 6 0 17 N/A 7.021E-05 54.1
Q60749 KHDR1 KH domain-containing, RNA-binding, signal transduction-associated protein 1 OS=Mus musculus Khdrbs1 PE=1 SV=2 - [KHDR1] 0 6 0 6 0 4 0 16 N/A 1.324E-03 48.3 (23)(24) (7)(10)(19)
Q6P5B5Q6P5B5 Fragile X mental retardation syndrome-related protein 2 OS=Mus musculus Fxr2 PE=2 SV=1 - [Q6P5B5] 0 6 0 5 0 5 0 16 N/A 8.922E-05 74.2 (7)
O70310 NMT1 Glycylpeptide N-tetradecanoyltransferase 1 OS=Mus musculus Nmt1 PE=1 SV=1 - [NMT1] 0 5 0 6 0 5 0 16 N/A 8.922E-05 56.9 (7)
G5E8E1 G5E8E1 Leucine rich repeat (In FLII) interacting protein 1, isoform CRA_e OS=Mus musculus Lrrfip1 PE=4 SV=1 - [G5E8E1] 0 5 0 6 0 5 0 16 N/A 8.922E-05 48.9 (7)
Q91VU7Q91VU7 Protein Pus7 OS=Mus musculus Pus7 PE=2 SV=2 - [Q91VU7] 0 5 0 8 0 2 0 15 N/A 4.471E-02 74.7 (7)
A2AJI0-2 MA7D1 Isoform 2 of MAP7 domain-containing protein 1 OS=Mus musculus Map7d1 - [MA7D1] 0 5 0 7 0 3 0 15 N/A 1.235E-02 89.3 (7)(16)
Q9Z1Z0-2 USO1 Isoform 2 of General vesicular transport factor p115 OS=Mus musculus Uso1 - [USO1] 0 6 0 3 0 6 0 15 N/A 7.490E-03 100.1 (23)(24) (7)(16)
Q99PG2OGFR Opioid growth factor receptor OS=Mus musculus Ogfr PE=2 SV=1 - [OGFR] 0 4 0 7 0 4 0 15 N/A 7.490E-03 70.6
Q99LP6 GRPE1 GrpE protein homolog 1, mitochondrial OS=Mus musculus Grpel1 PE=1 SV=1 - [GRPE1] 0 5 0 6 0 4 0 15 N/A 9.781E-04 24.3 (23)(24) (7)(16)
P63037 DNJA1 DnaJ homolog subfamily A member 1 OS=Mus musculus Dnaja1 PE=1 SV=1 - [DNJA1] 0 4 0 5 0 6 0 15 N/A 9.781E-04 44.8 ✔ (23)(24) (7)(12)(19)
P34022 RANG Ran-specific GTPase-activating protein OS=Mus musculus Ranbp1 PE=1 SV=2 - [RANG] 0 5 0 6 0 4 0 15 N/A 9.781E-04 23.6 (23)(24) (7)(12)(16)(19)
471
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
Q8VDG3 PARN Poly(A)-specific ribonuclease PARN OS=Mus musculus Parn PE=1 SV=1 - [PARN] 0 5 0 6 0 4 0 15 N/A 9.781E-04 71.5 (23)(24) (7)
B7ZP47 B7ZP47 Wapal protein OS=Mus musculus Wapal PE=2 SV=1 - [B7ZP47] 0 4 0 5 0 6 0 15 N/A 9.781E-04 133.3 (7)
Q8C3W1 CA198 Uncharacterized protein C1orf198 homolog OS=Mus musculus PE=1 SV=1 - [CA198] 0 6 0 4 0 5 0 15 N/A 9.781E-04 35.3 (19)
P60229 EIF3E Eukaryotic translation initiation factor 3 subunit E OS=Mus musculus Eif3e PE=1 SV=1 - [EIF3E] 0 5 0 7 0 2 0 14 N/A 3.253E-02 52.2 (23)(24) (7)
Q9CXW3 CYBP Calcyclin-binding protein OS=Mus musculus Cacybp PE=1 SV=1 - [CYBP] 0 2 0 6 0 6 0 14 N/A 2.490E-02 26.5 (23)(24) (7)(19)
O55222 ILK Integrin-linked protein kinase OS=Mus musculus Ilk PE=1 SV=2 - [ILK] 0 4 0 7 0 3 0 14 N/A 1.780E-02 51.3
O55135 IF6 Eukaryotic translation initiation factor 6 OS=Mus musculus Eif6 PE=1 SV=2 - [IF6] 0 3 0 6 0 5 0 14 N/A 6.122E-03 26.5 ✔ (20) (7)(16)
Q9ERU9RBP2 E3 SUMO-protein ligase RanBP2 OS=Mus musculus Ranbp2 PE=1 SV=2 - [RBP2] 0 3 0 5 0 6 0 14 N/A 6.122E-03 340.9 ✔ (20)(23)(24) (1)(7)(13)(16)
Q91VM9 IPYR2 Inorganic pyrophosphatase 2, mitochondrial OS=Mus musculus Ppa2 PE=2 SV=1 - [IPYR2] 0 6 0 4 0 4 0 14 N/A 2.192E-03 38.1 (23)(24) (7)(16)
Q8R4U7LUZP1 Leucine zipper protein 1 OS=Mus musculus Luzp1 PE=1 SV=2 - [LUZP1] 0 6 0 4 0 4 0 14 N/A 2.192E-03 119.2 (7)(13)(16)
Q6P9Q4FHOD1 FH1/FH2 domain-containing protein 1 OS=Mus musculus Fhod1 PE=2 SV=3 - [FHOD1] 0 4 0 6 0 4 0 14 N/A 2.192E-03 129.5
P18654 KS6A3 Ribosomal protein S6 kinase alpha-3 OS=Mus musculus Rps6ka3 PE=1 SV=2 - [KS6A3] 0 5 0 5 0 4 0 14 N/A 1.510E-04 83.6 (7)(16)
P53702 CCHL Cytochrome c-type heme lyase OS=Mus musculus Hccs PE=2 SV=2 - [CCHL] 0 5 0 4 0 5 0 14 N/A 1.510E-04 31 (7)
Q8R3C0MCMBP Mini-chromosome maintenance complex-binding protein OS=Mus musculus Mcmbp PE=2 SV=1 - [MCMBP] 0 7 0 2 0 4 0 13 N/A 4.064E-02 72.8 (23)(24) (7)(16)
Q8BV13CSN7B COP9 signalosome complex subunit 7b OS=Mus musculus Cops7b PE=1 SV=1 - [CSN7B] 0 3 0 3 0 7 0 13 N/A 3.138E-02 29.7 (7)(16)(19)
Q60855 RIPK1 Receptor-interacting serine/threonine-protein kinase 1 OS=Mus musculus Ripk1 PE=1 SV=1 - [RIPK1] 0 3 0 7 0 3 0 13 N/A 3.138E-02 74.8 (7)(16)
O70551 SRPK1 SRSF protein kinase 1 OS=Mus musculus Srpk1 PE=1 SV=2 - [SRPK1] 0 7 0 3 0 3 0 13 N/A 3.138E-02 73 (23)(24) (7)
Q8VE10NAA40 N-alpha-acetyltransferase 40 OS=Mus musculus Naa40 PE=2 SV=1 - [NAA40] 0 2 0 5 0 6 0 13 N/A 2.265E-02 27.2
E9Q9H2E9Q9H2 DnaJ homolog subfamily C member 2 OS=Mus musculus Dnajc2 PE=4 SV=1 - [E9Q9H2] 0 4 0 3 0 6 0 13 N/A 7.966E-03 63.4 (7)
J3QNB1J3QNB1 La-related protein 1 OS=Mus musculus Larp1 PE=4 SV=1 - [J3QNB1] 0 4 0 3 0 6 0 13 N/A 7.966E-03 121 (7)
Q9JHJ0 TMOD3 Tropomodulin-3 OS=Mus musculus Tmod3 PE=1 SV=1 - [TMOD3] 0 3 0 6 0 4 0 13 N/A 7.966E-03 39.5 (7)
G3X963 G3X963 ATPase family AAA domain-containing protein 2 OS=Mus musculus Atad2 PE=3 SV=1 - [G3X963] 0 4 0 3 0 6 0 13 N/A 7.966E-03 155.2
O09172 GSH0 Glutamate--cysteine ligase regulatory subunit OS=Mus musculus Gclm PE=2 SV=1 - [GSH0] 0 5 0 4 0 4 0 13 N/A 2.020E-04 30.5 (23)(24) (7)(16)(19)
Q61081 CDC37 Hsp90 co-chaperone Cdc37 OS=Mus musculus Cdc37 PE=2 SV=1 - [CDC37] 0 4 0 4 0 5 0 13 N/A 2.020E-04 44.6 (23)(24) (7)(16)(19)
Q9R1E0 FOXO1 Forkhead box protein O1 OS=Mus musculus Foxo1 PE=1 SV=2 - [FOXO1] 0 4 0 4 0 5 0 13 N/A 2.020E-04 69.5 (21)(26) (7)(16)
Q6A0A9F120A Constitutive coactivator of PPAR-gamma-like protein 1 OS=Mus musculus FAM120A PE=1 SV=2 - [F120A] 0 4 0 5 0 4 0 13 N/A 2.020E-04 121.6 (7)(16)
Q99KJ8 DCTN2 Dynactin subunit 2 OS=Mus musculus Dctn2 PE=1 SV=3 - [DCTN2] 0 4 0 4 0 5 0 13 N/A 2.020E-04 44.1 (7)(16)
P07607 TYSY Thymidylate synthase OS=Mus musculus Tyms PE=1 SV=1 - [TYSY] 0 4 0 4 0 5 0 13 N/A 2.020E-04 34.9 (7)(11)
Q8BGW1 FTO Alpha-ketoglutarate-dependent dioxygenase FTO OS=Mus musculus Fto PE=1 SV=1 - [FTO] 0 5 0 4 0 4 0 13 N/A 2.020E-04 58 (23)(24) (7)
A5A4Y9 A5A4Y9 Protein phosphatase 1 regulatory subunit 11 OS=Mus musculus Ppp1r11 PE=2 SV=1 - [A5A4Y9] 0 4 0 5 0 4 0 13 N/A 2.020E-04 14.5 (19)
Q7TS74-2 CKP2L Isoform 2 of Cytoskeleton-associated protein 2-like OS=Mus musculus Ckap2l - [CKP2L] 0 6 0 2 0 4 0 12 N/A 2.572E-02 68.9
Q9JIH2 NUP50 Nuclear pore complex protein Nup50 OS=Mus musculus Nup50 PE=1 SV=3 - [NUP50] 0 5 0 5 0 2 0 12 N/A 1.613E-02 49.5 (23)(24) (7)(16)
Q0VGB7PP4R2 Serine/threonine-protein phosphatase 4 regulatory subunit 2 OS=Mus musculus Ppp4r2 PE=1 SV=1 - [PP4R2] 0 3 0 3 0 6 0 12 N/A 1.613E-02 46.4 (23)(24) (7)(16)
P97452 BOP1 Ribosome biogenesis protein BOP1 OS=Mus musculus Bop1 PE=1 SV=1 - [BOP1] 0 3 0 6 0 3 0 12 N/A 1.613E-02 82.5 (7)(16)
Q8K3A9MEPCE 7SK snRNA methylphosphate capping enzyme OS=Mus musculus Mepce PE=1 SV=2 - [MEPCE] 0 5 0 2 0 5 0 12 N/A 1.613E-02 72
472
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
Q8R0K4CC137 Coiled-coil domain-containing protein 137 OS=Mus musculus Ccdc137 PE=2 SV=1 - [CC137] 0 3 0 3 0 6 0 12 N/A 1.613E-02 32.9
Q9WTK5 NFKB2 Nuclear factor NF-kappa-B p100 subunit OS=Mus musculus Nfkb2 PE=1 SV=1 - [NFKB2] 0 5 0 4 0 3 0 12 N/A 2.278E-03 96.8 (7)(16)(19)
Q9Z1Y4 TRIP6 Thyroid receptor-interacting protein 6 OS=Mus musculus Trip6 PE=1 SV=1 - [TRIP6] 0 5 0 3 0 4 0 12 N/A 2.278E-03 50.9 (7)(16)
E9Q4Q2E9Q4Q2 Splicing factor 1 OS=Mus musculus Sf1 PE=4 SV=1 - [E9Q4Q2] 0 3 0 4 0 5 0 12 N/A 2.278E-03 59.7 (7)
E9PWW9 E9PWW9 Protein Rsf1 OS=Mus musculus Rsf1 PE=4 SV=1 - [E9PWW9] 0 5 0 3 0 4 0 12 N/A 2.278E-03 160.5
Q9WTP6-2 KAD2 Isoform 2 of Adenylate kinase 2, mitochondrial OS=Mus musculus Ak2 - [KAD2] 0 4 0 4 0 4 0 12 N/A 0.000E+00 25.6 (21)(23)(24) (7)(10)(16)(19)
Q9CYN2 SPCS2 Signal peptidase complex subunit 2 OS=Mus musculus Spcs2 PE=2 SV=1 - [SPCS2] 0 4 0 4 0 4 0 12 N/A 0.000E+00 25 (23)(24)
Q8BK58HBAP1 HSPB1-associated protein 1 OS=Mus musculus Hspbap1 PE=1 SV=2 - [HBAP1] 0 4 0 4 0 4 0 12 N/A 0.000E+00 54.4
Q9DAW9 CNN3 Calponin-3 OS=Mus musculus Cnn3 PE=2 SV=1 - [CNN3] 0 2 0 3 0 6 0 11 N/A 3.800E-02 36.4 (7)
Q8VIJ6 SFPQ Splicing factor, proline- and glutamine-rich OS=Mus musculus Sfpq PE=1 SV=1 - [SFPQ] 0 3 0 2 0 6 0 11 N/A 3.800E-02 75.4 ✔ (20)(23)(24) (4)(7)(12)(13)(16)(17)
Q9CXT8MPPB Mitochondrial-processing peptidase subunit beta OS=Mus musculus Pmpcb PE=2 SV=1 - [MPPB] 0 2 0 4 0 5 0 11 N/A 1.417E-02 54.6 (7)
D3Z795 D3Z795 Proteasome assembly chaperone 1 OS=Mus musculus Psmg1 PE=4 SV=1 - [D3Z795] 0 2 0 4 0 5 0 11 N/A 1.417E-02 30.5 (7)
E9QPI5 E9QPI5 Sister chromatid cohesion protein PDS5 homolog A OS=Mus musculus Pds5a PE=4 SV=1 - [E9QPI5] 0 5 0 4 0 2 0 11 N/A 1.417E-02 150.1 (7)
Q8BH80Q8BH80 Vesicle-associated membrane protein, associated protein B and C OS=Mus musculus Vapb PE=2 SV=1 - [Q8BH80] 0 4 0 5 0 2 0 11 N/A 1.417E-02 26.9 (7)
Q811D0-2 DLG1 Isoform 2 of Disks large homolog 1 OS=Mus musculus Dlg1 - [DLG1] 0 5 0 4 0 2 0 11 N/A 1.417E-02 99.6
P31938 MP2K1 Dual specificity mitogen-activated protein kinase kinase 1 OS=Mus musculus Map2k1 PE=1 SV=2 - [MP2K1] 0 3 0 3 0 5 0 11 N/A 5.328E-03 43.4 ✔ (7)
Q8CCM0 Q8CCM0Calcium/calmodulin-dependent protein kinase type II subunit delta OS=Mus musculus Camk2d PE=2 SV=1 - [Q8CCM0] 0 3 0 3 0 5 0 11 N/A 5.328E-03 40.7 (7)
G3X928 G3X928 SEC23-interacting protein OS=Mus musculus Sec23ip PE=4 SV=1 - [G3X928] 0 5 0 3 0 3 0 11 N/A 5.328E-03 110.7 (2)(7)
Q99LR1 ABD12 Monoacylglycerol lipase ABHD12 OS=Mus musculus Abhd12 PE=1 SV=2 - [ABD12] 0 3 0 3 0 5 0 11 N/A 5.328E-03 45.2
P35831 PTN12 Tyrosine-protein phosphatase non-receptor type 12 OS=Mus musculus Ptpn12 PE=1 SV=3 - [PTN12] 0 5 0 3 0 3 0 11 N/A 5.328E-03 86.5
D3Z131 D3Z131 THO complex subunit 6 homolog OS=Mus musculus Thoc6 PE=4 SV=1 - [D3Z131] 0 3 0 5 0 3 0 11 N/A 5.328E-03 32.7
A2BE28-2 LAS1L Isoform 2 of Ribosomal biogenesis protein LAS1L OS=Mus musculus Las1l - [LAS1L] 0 4 0 4 0 3 0 11 N/A 3.882E-04 87.5 (7)(16)
Q8BIW1PRUNE Protein prune homolog OS=Mus musculus Prune PE=2 SV=1 - [PRUNE] 0 4 0 3 0 4 0 11 N/A 3.882E-04 50.2 (7)(16)
Q922D4-2 PP6R3 Isoform 2 of Serine/threonine-protein phosphatase 6 regulatory subunit 3 OS=Mus musculus Ppp6r3 - [PP6R3] 0 3 0 4 0 4 0 11 N/A 3.882E-04 92.6 (7)(16)
O09167 RL21 60S ribosomal protein L21 OS=Mus musculus Rpl21 PE=2 SV=3 - [RL21] 0 4 0 3 0 4 0 11 N/A 3.882E-04 18.6 ✔ (20) (6)(7)(12)
Q8BR92PALM2 Paralemmin-2 OS=Mus musculus Palm2 PE=1 SV=1 - [PALM2] 0 4 0 4 0 3 0 11 N/A 3.882E-04 42.1 (19)
Q8CES0NAA30 N-alpha-acetyltransferase 30 OS=Mus musculus Naa30 PE=2 SV=2 - [NAA30] 0 5 0 3 0 2 0 10 N/A 1.944E-02 39.4 (23)(24) (7)(16)
O08582 GTPB1 GTP-binding protein 1 OS=Mus musculus Gtpbp1 PE=1 SV=2 - [GTPB1] 0 3 0 2 0 5 0 10 N/A 1.944E-02 72.3 (7)(16)
Q11136 PEPD Xaa-Pro dipeptidase OS=Mus musculus Pepd PE=2 SV=3 - [PEPD] 0 2 0 5 0 3 0 10 N/A 1.944E-02 55 (7)
G3UZ44 G3UZ44 Paired mesoderm homeobox protein 1 OS=Mus musculus Prrx1 PE=3 SV=1 - [G3UZ44] 0 2 0 3 0 5 0 10 N/A 1.944E-02 22.5
G3X9V0 G3X9V0 MCG22048, isoform CRA_a OS=Mus musculus Psme2 PE=4 SV=1 - [G3X9V0] 0 4 0 2 0 4 0 10 N/A 7.490E-03 26.1 (7)
Q9DBC7 KAP0 cAMP-dependent protein kinase type I-alpha regulatory subunit OS=Mus musculus Prkar1a PE=1 SV=3 - [KAP0] 0 4 0 2 0 4 0 10 N/A 7.490E-03 43.2
P23242 CXA1 Gap junction alpha-1 protein OS=Mus musculus Gja1 PE=1 SV=2 - [CXA1] 0 4 0 4 0 2 0 10 N/A 7.490E-03 43
P97314 CSRP2 Cysteine and glycine-rich protein 2 OS=Mus musculus Csrp2 PE=1 SV=3 - [CSRP2] 0 3 0 4 0 3 0 10 N/A 5.620E-04 20.9 (7)(16)(19)
Q9CU62SMC1A Structural maintenance of chromosomes protein 1A OS=Mus musculus Smc1a PE=1 SV=4 - [SMC1A] 0 4 0 3 0 3 0 10 N/A 5.620E-04 143.1 ✔ (20)(23)(24) (7)(16)
473
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
Q9D0A3CO038 UPF0552 protein C15orf38 homolog OS=Mus musculus PE=2 SV=1 - [CO038] 0 4 0 3 0 3 0 10 N/A 5.620E-04 25.2 (16)
Q8C8U0LIPB1 Liprin-beta-1 OS=Mus musculus Ppfibp1 PE=1 SV=3 - [LIPB1] 0 3 0 4 0 3 0 10 N/A 5.620E-04 108.5 (23)(24)
Q8BK64AHSA1 Activator of 90 kDa heat shock protein ATPase homolog 1 OS=Mus musculus Ahsa1 PE=2 SV=2 - [AHSA1] 0 1 0 4 0 4 0 9 N/A 3.994E-02 38.1 (23)(24) (7)(16)
Q8C263SKA3 Spindle and kinetochore-associated protein 3 OS=Mus musculus Ska3 PE=1 SV=1 - [SKA3] 0 4 0 1 0 4 0 9 N/A 3.994E-02 45.3 (7)(16)
Q6ZPJ3 UBE2O Ubiquitin-conjugating enzyme E2 O OS=Mus musculus Ube2o PE=1 SV=3 - [UBE2O] 0 4 0 4 0 1 0 9 N/A 3.994E-02 140.7 (7)(16)
P70288 HDAC2 Histone deacetylase 2 OS=Mus musculus Hdac2 PE=1 SV=1 - [HDAC2] 0 2 0 5 0 2 0 9 N/A 3.994E-02 55.3 (20)(23)(24) (7)
Q80ZX0 Q80ZX0 Protein Sec24b OS=Mus musculus Sec24b PE=2 SV=1 - [Q80ZX0] 0 2 0 5 0 2 0 9 N/A 3.994E-02 135.5 (2)(7)
Q9JMB0GKAP1 G kinase-anchoring protein 1 OS=Mus musculus Gkap1 PE=1 SV=1 - [GKAP1] 0 2 0 5 0 2 0 9 N/A 3.994E-02 41.7
Q6ZQK5-2 ACAP2 Isoform 2 of Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 2 OS=Mus musculus Acap2 - [ACAP2] 0 2 0 5 0 2 0 9 N/A 3.994E-02 85.1
Q61239 FNTA Protein farnesyltransferase/geranylgeranyltransferase type-1 subunit alpha OS=Mus musculus Fnta PE=1 SV=1 - [FNTA] 0 4 0 1 0 4 0 9 N/A 3.994E-02 44
A2A6A1GPTC8 G patch domain-containing protein 8 OS=Mus musculus Gpatch8 PE=2 SV=1 - [GPTC8] 0 4 0 2 0 3 0 9 N/A 6.533E-03 164.9 (23)(24) (7)(16)
Q61187 TS101 Tumor susceptibility gene 101 protein OS=Mus musculus Tsg101 PE=1 SV=2 - [TS101] 0 2 0 3 0 4 0 9 N/A 6.533E-03 44.1 (7)(16)
Q9CYA6ZCHC8 Zinc finger CCHC domain-containing protein 8 OS=Mus musculus Zcchc8 PE=2 SV=3 - [ZCHC8] 0 3 0 2 0 4 0 9 N/A 6.533E-03 78 (7)(16)
Q6Y685 TACC1 Transforming acidic coiled-coil-containing protein 1 OS=Mus musculus Tacc1 PE=1 SV=1 - [TACC1] 0 3 0 2 0 4 0 9 N/A 6.533E-03 83.9 (7)(13)(16)
A2A4A6A2A4A6 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 OS=Mus musculus Plcg1 PE=4 SV=1 - [A2A4A6] 0 2 0 3 0 4 0 9 N/A 6.533E-03 148.4 (7)
P45377 ALD2 Aldose reductase-related protein 2 OS=Mus musculus Akr1b8 PE=1 SV=2 - [ALD2] 0 2 0 4 0 3 0 9 N/A 6.533E-03 36.1 (19)
O88532 ZFR Zinc finger RNA-binding protein OS=Mus musculus Zfr PE=1 SV=2 - [ZFR] 0 4 0 3 0 2 0 9 N/A 6.533E-03 116.8 (23)(24) (1)(4)(9)(12)(13)(19)
A2ACG7A2ACG7 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 OS=Mus musculus Rpn2 PE=4 SV=1 - [A2ACG7] 0 3 0 4 0 2 0 9 N/A 6.533E-03 67.5
F8VPU2 FARP1 Protein Farp1 OS=Mus musculus Farp1 PE=4 SV=1 - [F8VPU2] 0 4 0 3 0 2 0 9 N/A 6.533E-03 118.8
P83093 STIM2 Stromal interaction molecule 2 OS=Mus musculus Stim2 PE=1 SV=2 - [STIM2] 0 2 0 4 0 3 0 9 N/A 6.533E-03 83.9
Q9JI10 STK3 Serine/threonine-protein kinase 3 OS=Mus musculus Stk3 PE=1 SV=1 - [STK3] 0 3 0 2 0 4 0 9 N/A 6.533E-03 56.8
Q5NCR9 NSRP1 Nuclear speckle splicing regulatory protein 1 OS=Mus musculus Nsrp1 PE=1 SV=1 - [NSRP1] 0 3 0 3 0 3 0 9 N/A 0.000E+00 63.8 (7)
P24270 CATA Catalase OS=Mus musculus Cat PE=1 SV=4 - [CATA] 0 3 0 3 0 3 0 9 N/A 0.000E+00 59.8 (23)(24) (11)
Q5EG47AAPK1 5'-AMP-activated protein kinase catalytic subunit alpha-1 OS=Mus musculus Prkaa1 PE=1 SV=2 - [AAPK1] 0 3 0 3 0 3 0 9 N/A 0.000E+00 63.9 (23)(24)
E9PUT4 E9PUT4 Disks large-associated protein 5 OS=Mus musculus Dlgap5 PE=4 SV=1 - [E9PUT4] 0 3 0 3 0 3 0 9 N/A 0.000E+00 84.6
Q8R2M2 TDIF2 Deoxynucleotidyltransferase terminal-interacting protein 2 OS=Mus musculus Dnttip2 PE=1 SV=1 - [TDIF2] 0 1 0 4 0 3 0 8 N/A 3.902E-02 84.2 (7)(16)
Q5SUR0PUR4 Phosphoribosylformylglycinamidine synthase OS=Mus musculus Pfas PE=2 SV=1 - [PUR4] 0 3 0 4 0 1 0 8 N/A 3.902E-02 144.5 (7)
Q8R323 RFC3 Replication factor C subunit 3 OS=Mus musculus Rfc3 PE=2 SV=1 - [RFC3] 0 2 0 2 0 4 0 8 N/A 1.613E-02 40.5 (23)(24) (7)(16)
Q9JJY4 DDX20 Probable ATP-dependent RNA helicase DDX20 OS=Mus musculus Ddx20 PE=1 SV=2 - [DDX20] 0 2 0 2 0 4 0 8 N/A 1.613E-02 91.7 (7)
Q9EPJ9 ARFG1 ADP-ribosylation factor GTPase-activating protein 1 OS=Mus musculus Arfgap1 PE=1 SV=2 - [ARFG1] 0 2 0 2 0 4 0 8 N/A 1.613E-02 45.3 (23)(24)
Q9CWR0-2 ARHGP Isoform 2 of Rho guanine nucleotide exchange factor 25 OS=Mus musculus Arhgef25 - [ARHGP] 0 4 0 2 0 2 0 8 N/A 1.613E-02 67.4
Q9EPK6SIL1 Nucleotide exchange factor SIL1 OS=Mus musculus Sil1 PE=1 SV=2 - [SIL1]0 4 0 2 0 2 0 8 N/A 1.613E-02 52.4
Q6P5F9 XPO1 Exportin-1 OS=Mus musculus Xpo1 PE=1 SV=1 - [XPO1] 0 2 0 3 0 3 0 8 N/A 1.324E-03 123 (23)(24) (7)(16)
P63028 TCTP Translationally-controlled tumor protein OS=Mus musculus Tpt1 PE=1 SV=1 - [TCTP] 0 3 0 2 0 3 0 8 N/A 1.324E-03 19.4 (23)(24) (19)
P31230 AIMP1 Aminoacyl tRNA synthase complex-interacting multifunctional protein 1 OS=Mus musculus Aimp1 PE=1 SV=2 - [AIMP1] 0 3 0 3 0 2 0 8 N/A 1.324E-03 34 (23)(24) (1)
474
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
D3YX62 D3YX62 Heme oxygenase 2 (Fragment) OS=Mus musculus Hmox2 PE=4 SV=1 - [D3YX62] 0 3 0 3 0 2 0 8 N/A 1.324E-03 26.4
Q9D5T0ATAD1 ATPase family AAA domain-containing protein 1 OS=Mus musculus Atad1 PE=1 SV=1 - [ATAD1] 0 3 0 1 0 3 0 7 N/A 2.490E-02 40.7 (23)(24) (7)(16)
Q8VCH8 UBXN4 UBX domain-containing protein 4 OS=Mus musculus Ubxn4 PE=1 SV=1 - [UBXN4] 0 3 0 3 0 1 0 7 N/A 2.490E-02 56.4 (7)
E9QKG3E9QKG3 tRNA (guanine(10)-N2)-methyltransferase homolog OS=Mus musculus Trmt11 PE=4 SV=1 - [E9QKG3] 0 1 0 3 0 3 0 7 N/A 2.490E-02 52.9
Q3UYV9NCBP1 Nuclear cap-binding protein subunit 1 OS=Mus musculus Ncbp1 PE=1 SV=2 - [NCBP1] 0 3 0 2 0 2 0 7 N/A 2.192E-03 91.9 (23)(24) (7)(16)
Q7TNV0DEK Protein DEK OS=Mus musculus Dek PE=1 SV=1 - [DEK] 0 2 0 3 0 2 0 7 N/A 2.192E-03 43.1 (22)(23)(24) (7)(16)
Q9R060 NUBP1 Cytosolic Fe-S cluster assembly factor NUBP1 OS=Mus musculus Nubp1 PE=1 SV=1 - [NUBP1] 0 2 0 2 0 3 0 7 N/A 2.192E-03 34.1 (7)(16)
Q9Z2X8 KEAP1 Kelch-like ECH-associated protein 1 OS=Mus musculus Keap1 PE=1 SV=1 - [KEAP1] 0 2 0 3 0 2 0 7 N/A 2.192E-03 69.5 (7)(16)
Q9QYI3 DNJC7 DnaJ homolog subfamily C member 7 OS=Mus musculus Dnajc7 PE=1 SV=2 - [DNJC7] 0 2 0 3 0 2 0 7 N/A 2.192E-03 56.4 (7)(16)
Q9DBR0AKAP8 A-kinase anchor protein 8 OS=Mus musculus Akap8 PE=1 SV=1 - [AKAP8] 0 3 0 2 0 2 0 7 N/A 2.192E-03 76.2 (23)(24) (7)
A2A5R8 A2A5R8 Double-stranded RNA-binding protein Staufen homolog 1 OS=Mus musculus Stau1 PE=4 SV=1 - [A2A5R8] 0 2 0 3 0 2 0 7 N/A 2.192E-03 53.7 (7)
H3BJU7 H3BJU7 Rho guanine nucleotide exchange factor 2 OS=Mus musculus Arhgef2 PE=4 SV=1 - [H3BJU7] 0 2 0 3 0 2 0 7 N/A 2.192E-03 108.5 (7)
D3YWK1 D3YWK1 WD repeat domain phosphoinositide-interacting protein 2 OS=Mus musculus Wipi2 PE=4 SV=1 - [D3YWK1] 0 2 0 3 0 2 0 7 N/A 2.192E-03 46.4 (7)
P62835 RAP1A Ras-related protein Rap-1A OS=Mus musculus Rap1a PE=2 SV=1 - [RAP1A]0 2 0 3 0 2 0 7 N/A 2.192E-03 21 (7)
Q9ES97-3 RTN3 Isoform 3 of Reticulon-3 OS=Mus musculus Rtn3 - [RTN3] 0 2 0 2 0 3 0 7 N/A 2.192E-03 25.4 (19)
Q8C6B9AROS Active regulator of SIRT1 OS=Mus musculus Rps19bp1 PE=1 SV=1 - [AROS] 0 2 0 2 0 3 0 7 N/A 2.192E-03 16 (19)
Q9CPT5NOP16 Nucleolar protein 16 OS=Mus musculus Nop16 PE=2 SV=1 - [NOP16] 0 2 0 2 0 3 0 7 N/A 2.192E-03 21.1 (23)(24) (16)
Q922Y1 UBXN1 UBX domain-containing protein 1 OS=Mus musculus Ubxn1 PE=1 SV=1 - [UBXN1] 0 2 0 3 0 2 0 7 N/A 2.192E-03 33.6 (1)(7)(16)
Q9EP97 SENP3 Sentrin-specific protease 3 OS=Mus musculus Senp3 PE=1 SV=1 - [SENP3] 0 2 0 3 0 2 0 7 N/A 2.192E-03 64.4
Q9EP71 RAI14 Ankycorbin OS=Mus musculus Rai14 PE=1 SV=1 - [RAI14] 0 3 0 2 0 1 0 6 N/A 2.572E-02 108.8 (7)(16)
Q9CRC8LRC40 Leucine-rich repeat-containing protein 40 OS=Mus musculus Lrrc40 PE=2 SV=2 - [LRC40] 0 1 0 3 0 2 0 6 N/A 2.572E-02 68 (7)(16)
Q9EQM6 DGCR8 Microprocessor complex subunit DGCR8 OS=Mus musculus Dgcr8 PE=2 SV=2 - [DGCR8] 0 3 0 1 0 2 0 6 N/A 2.572E-02 86.3 (7)(16)
P10852 4F2 4F2 cell-surface antigen heavy chain OS=Mus musculus Slc3a2 PE=1 SV=1 - [4F2] 0 1 0 2 0 3 0 6 N/A 2.572E-02 58.3 ✔ (7)
Q9WV92-5 E41L3 Isoform 5 of Band 4.1-like protein 3 OS=Mus musculus Epb41l3 - [E41L3] 0 2 0 3 0 1 0 6 N/A 2.572E-02 100.7 (23)(24) (7)
D3YZP6 D3YZP6 Cysteine protease ATG4B (Fragment) OS=Mus musculus Atg4b PE=4 SV=1 - [D3YZP6] 0 3 0 2 0 1 0 6 N/A 2.572E-02 17.4 (7)
Q9D8M7-2 PHF10 Isoform 2 of PHD finger protein 10 OS=Mus musculus Phf10 - [PHF10] 0 2 0 3 0 1 0 6 N/A 2.572E-02 55.6 (7)
A6PWC3 A6PWC3 Nardilysin OS=Mus musculus Nrd1 PE=3 SV=1 - [A6PWC3] 0 3 0 1 0 2 0 6 N/A 2.572E-02 127.7 (7)
Q99K70 RRAGC Ras-related GTP-binding protein C OS=Mus musculus Rragc PE=2 SV=1 - [RRAGC] 0 3 0 1 0 2 0 6 N/A 2.572E-02 44.1 (7)
Q8CGY8 OGT1 UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit OS=Mus musculus Ogt PE=1 SV=2 - [OGT1] 0 3 0 1 0 2 0 6 N/A 2.572E-02 116.9 (1)(2)(4)(7)(12)(13)(16)(18)
Q9D735CS043 Uncharacterized protein C19orf43 homolog OS=Mus musculus PE=2 SV=1 - [CS043] 0 2 0 1 0 3 0 6 N/A 2.572E-02 18.4 (23)(24)
Q9WTJ4FIZ1 Flt3-interacting zinc finger protein 1 OS=Mus musculus Fiz1 PE=1 SV=1 - [FIZ1] 0 1 0 2 0 3 0 6 N/A 2.572E-02 52.7
P25085-2 IL1RA Isoform 2 of Interleukin-1 receptor antagonist protein OS=Mus musculus Il1rn - [IL1RA] 0 3 0 2 0 1 0 6 N/A 2.572E-02 18
Q8VEH6CBWD1 COBW domain-containing protein 1 OS=Mus musculus Cbwd1 PE=2 SV=1 - [CBWD1] 0 2 0 1 0 3 0 6 N/A 2.572E-02 43.7
P35951 LDLR Low-density lipoprotein receptor OS=Mus musculus Ldlr PE=1 SV=2 - [LDLR] 0 3 0 2 0 1 0 6 N/A 2.572E-02 94.9
Q9D8S4ORN Oligoribonuclease, mitochondrial OS=Mus musculus Rexo2 PE=1 SV=2 - [ORN] 0 2 0 1 0 3 0 6 N/A 2.572E-02 26.7
475
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
Q9D6U8F162A Protein FAM162A OS=Mus musculus Fam162a PE=2 SV=1 - [F162A] 0 2 0 3 0 1 0 6 N/A 2.572E-02 17.7
Q62219 TGFI1 Transforming growth factor beta-1-induced transcript 1 protein OS=Mus musculus Tgfb1i1 PE=1 SV=2 - [TGFI1] 0 3 0 1 0 2 0 6 N/A 2.572E-02 50.1
Q80XI4 PI42B Phosphatidylinositol 5-phosphate 4-kinase type-2 beta OS=Mus musculus Pip4k2b PE=1 SV=1 - [PI42B] 0 2 0 2 0 2 0 6 N/A 0.000E+00 47.3 (23)(24) (7)(16)
O08784 TCOF Treacle protein OS=Mus musculus Tcof1 PE=1 SV=1 - [TCOF] 0 2 0 2 0 2 0 6 N/A 0.000E+00 134.9 (23)(24) (7)(16)
P70217 HXD13 Homeobox protein Hox-D13 OS=Mus musculus Hoxd13 PE=2 SV=2 - [HXD13] 0 2 0 2 0 2 0 6 N/A 0.000E+00 35.9 (7)(16)
Q9QZE5COPG1 Coatomer subunit gamma-1 OS=Mus musculus Copg1 PE=2 SV=1 - [COPG1] 0 2 0 2 0 2 0 6 N/A 0.000E+00 97.5 (7)(16)
Q80YR4-2 ZN598 Isoform 2 of Zinc finger protein 598 OS=Mus musculus Znf598 - [ZN598] 0 2 0 2 0 2 0 6 N/A 0.000E+00 96.3 (7)(16)
Q9CR51VATG1 V-type proton ATPase subunit G 1 OS=Mus musculus Atp6v1g1 PE=2 SV=3 - [VATG1] 0 2 0 2 0 2 0 6 N/A 0.000E+00 13.7 (7)
Q9CR39WIPI3 WD repeat domain phosphoinositide-interacting protein 3 OS=Mus musculus Wdr45l PE=2 SV=2 - [WIPI3] 0 2 0 2 0 2 0 6 N/A 0.000E+00 38 (7)
B1AT03 B1AT03 DNA ligase OS=Mus musculus Lig3 PE=3 SV=1 - [B1AT03] 0 2 0 2 0 2 0 6 N/A 0.000E+00 106.1 ✔
Q91WS0 CISD1 CDGSH iron-sulfur domain-containing protein 1 OS=Mus musculus Cisd1 PE=1 SV=1 - [CISD1] 0 2 0 2 0 2 0 6 N/A 0.000E+00 12.1 (21)
P30658 CBX2 Chromobox protein homolog 2 OS=Mus musculus Cbx2 PE=1 SV=2 - [CBX2] 0 2 0 2 0 2 0 6 N/A 0.000E+00 54.9
Q9Z1B5 MD2L1 Mitotic spindle assembly checkpoint protein MAD2A OS=Mus musculus Mad2l1 PE=2 SV=2 - [MD2L1] 0 2 0 2 0 2 0 6 N/A 0.000E+00 23.6
Q8K136-3 SCNM1 Isoform 3 of Sodium channel modifier 1 OS=Mus musculus Scnm1 - [SCNM1] 0 2 0 2 0 2 0 6 N/A 0.000E+00 18.8
Q8R570 SNP47 Synaptosomal-associated protein 47 OS=Mus musculus Snap47 PE=1 SV=1 - [SNP47] 0 2 0 2 0 2 0 6 N/A 0.000E+00 46.5
B1AQJ2UBP36 Ubiquitin carboxyl-terminal hydrolase 36 OS=Mus musculus Usp36 PE=2 SV=1 - [UBP36] 0 2 0 2 0 2 0 6 N/A 0.000E+00 119.8
Q9EQH3 VPS35 Vacuolar protein sorting-associated protein 35 OS=Mus musculus Vps35 PE=1 SV=1 - [VPS35] 0 1 0 2 0 2 0 5 N/A 7.490E-03 91.7 ✔ (20)(23)(24) (7)(16)
Q8K327 CHAP1 Chromosome alignment-maintaining phosphoprotein 1 OS=Mus musculus Champ1 PE=1 SV=1 - [CHAP1] 0 2 0 2 0 1 0 5 N/A 7.490E-03 87.5 (23)(24) (7)(16)
Q6P9L6 KIF15 Kinesin-like protein KIF15 OS=Mus musculus Kif15 PE=1 SV=1 - [KIF15] 0 1 0 2 0 2 0 5 N/A 7.490E-03 160 (23)(24) (7)(16)
Q91WE2 F192A Protein FAM192A OS=Mus musculus Fam192a PE=2 SV=1 - [F192A] 0 2 0 1 0 2 0 5 N/A 7.490E-03 28.7 (23)(24) (7)(16)
P47791-2 GSHR Isoform Cytoplasmic of Glutathione reductase, mitochondrial OS=Mus musculus Gsr - [GSHR] 0 2 0 2 0 1 0 5 N/A 7.490E-03 51 (23)(24) (7)(16)
Q3TIU4 PDE12 2',5'-phosphodiesterase 12 OS=Mus musculus Pde12 PE=2 SV=2 - [PDE12]0 2 0 1 0 2 0 5 N/A 7.490E-03 67.5 (7)(16)
Q8BT60CPNE3 Copine-3 OS=Mus musculus Cpne3 PE=1 SV=2 - [CPNE3] 0 2 0 2 0 1 0 5 N/A 7.490E-03 59.5 (7)(16)
Q8BJL1 FBX30 F-box only protein 30 OS=Mus musculus Fbxo30 PE=2 SV=2 - [FBX30] 0 1 0 2 0 2 0 5 N/A 7.490E-03 82.6 (7)(16)
Q9QZ23NFU1 NFU1 iron-sulfur cluster scaffold homolog, mitochondrial OS=Mus musculus Nfu1 PE=1 SV=2 - [NFU1] 0 2 0 2 0 1 0 5 N/A 7.490E-03 28.5 (7)(16)
E9Q6J5 E9Q6J5 Protein Bod1l OS=Mus musculus Bod1l PE=4 SV=1 - [E9Q6J5] 0 1 0 2 0 2 0 5 N/A 7.490E-03 327.3 ✔ (7)
P97471 SMAD4 Mothers against decapentaplegic homolog 4 OS=Mus musculus Smad4 PE=1 SV=2 - [SMAD4] 0 2 0 1 0 2 0 5 N/A 7.490E-03 60.3 (23)(24) (7)
Q3TFP0 Q3TFP0 FUS interacting protein (Serine-arginine rich) 1 OS=Mus musculus Srsf10 PE=2 SV=1 - [Q3TFP0] 0 1 0 2 0 2 0 5 N/A 7.490E-03 22.1 (7)
H3BK31 H3BK31 Zinc fingers and homeoboxes protein 1 (Fragment) OS=Mus musculus Zhx1 PE=3 SV=1 - [H3BK31] 0 1 0 2 0 2 0 5 N/A 7.490E-03 68.6 (7)
D3YUW8 D3YUW8 Pogo transposable element with ZNF domain OS=Mus musculus Pogz PE=4 SV=1 - [D3YUW8] 0 1 0 2 0 2 0 5 N/A 7.490E-03 144.8 (7)
Q9R0P4 SMAP Small acidic protein OS=Mus musculus Smap PE=1 SV=1 - [SMAP] 0 2 0 2 0 1 0 5 N/A 7.490E-03 20 (23)(24) (16)
Q6NSQ7 LTV1 Protein LTV1 homolog OS=Mus musculus Ltv1 PE=2 SV=2 - [LTV1] 0 2 0 1 0 2 0 5 N/A 7.490E-03 54 (16)
P54071 IDHP Isocitrate dehydrogenase [NADP], mitochondrial OS=Mus musculus Idh2 PE=1 SV=3 - [IDHP] 0 1 0 2 0 2 0 5 N/A 7.490E-03 50.9 (21)(22)(23)(24)(25) (21)(22)(23)(24)(25)
Q3UG37Q3UG37 Histone deacetylase 6 (Fragment) OS=Mus musculus Hdac6 PE=2 SV=1 - [Q3UG37] 0 2 0 2 0 1 0 5 N/A 7.490E-03 110.2
Q8C650-2 SEP10 Isoform 2 of Septin-10 OS=Mus musculus Sept10 - [SEP10] 0 2 0 2 0 1 0 5 N/A 7.490E-03 49.8
476
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
Q80UU9PGRC2 Membrane-associated progesterone receptor component 2 OS=Mus musculus Pgrmc2 PE=1 SV=2 - [PGRC2] 0 1 0 2 0 2 0 5 N/A 7.490E-03 23.3
I1E4X0 I1E4X0 Disks large-associated protein 4 OS=Mus musculus Dlgap4 PE=4 SV=1 - [I1E4X0] 0 2 0 1 0 2 0 5 N/A 7.490E-03 26.9
Q9CQX4 PAF15 PCNA-associated factor OS=Mus musculus Paf PE=2 SV=1 - [PAF15] 0 2 0 1 0 2 0 5 N/A 7.490E-03 12
F7BQE4F7BQE4 Rho guanine nucleotide exchange factor 10 (Fragment) OS=Mus musculus Arhgef10 PE=4 SV=1 - [F7BQE4] 0 2 0 2 0 1 0 5 N/A 7.490E-03 109.3
Q9CR46SKA2 Spindle and kinetochore-associated protein 2 OS=Mus musculus Ska2 PE=2 SV=1 - [SKA2] 0 1 0 2 0 2 0 5 N/A 7.490E-03 13.7
Q8K1R7NEK9 Serine/threonine-protein kinase Nek9 OS=Mus musculus Nek9 PE=1 SV=2 - [NEK9] 0 1 0 1 0 2 0 4 N/A 1.613E-02 107.1 (23)(24) (7)(16)
Q9D8S9BOLA1 BolA-like protein 1 OS=Mus musculus Bola1 PE=1 SV=1 - [BOLA1] 0 1 0 2 0 1 0 4 N/A 1.613E-02 14.4 (7)(16)
Q505F5 LRC47 Leucine-rich repeat-containing protein 47 OS=Mus musculus Lrrc47 PE=1 SV=1 - [LRC47] 0 1 0 2 0 1 0 4 N/A 1.613E-02 63.6 (7)(16)
Q9WTX2 PRKRA Interferon-inducible double stranded RNA-dependent protein kinase activator A OS=Mus musculus Prkra PE=1 SV=1 - [PRKRA] 0 2 0 1 0 1 0 4 N/A 1.613E-02 34.3 (7)(16)
Q9JKP5 MBNL1 Muscleblind-like protein 1 OS=Mus musculus Mbnl1 PE=1 SV=1 - [MBNL1] 0 1 0 2 0 1 0 4 N/A 1.613E-02 37 (7)(16)
Q8BZW8 NHLC2 NHL repeat-containing protein 2 OS=Mus musculus Nhlrc2 PE=2 SV=1 - [NHLC2] 0 1 0 1 0 2 0 4 N/A 1.613E-02 78.4 (7)(16)
P00493 HPRT Hypoxanthine-guanine phosphoribosyltransferase OS=Mus musculus Hprt1 PE=1 SV=3 - [HPRT] 0 2 0 1 0 1 0 4 N/A 1.613E-02 24.6 (7)(12)
P35123 UBP4 Ubiquitin carboxyl-terminal hydrolase 4 OS=Mus musculus Usp4 PE=1 SV=3 - [UBP4] 0 2 0 1 0 1 0 4 N/A 1.613E-02 108.3 (7)
E9Q986 E9Q986 Catenin delta-1 OS=Mus musculus Ctnnd1 PE=4 SV=1 - [E9Q986] 0 1 0 1 0 2 0 4 N/A 1.613E-02 92.4 (7)
E9PY48 E9PY48 Condensin-2 complex subunit H2 OS=Mus musculus Ncaph2 PE=4 SV=1 - [E9PY48] 0 2 0 1 0 1 0 4 N/A 1.613E-02 65.3 (7)
P54823 DDX6 Probable ATP-dependent RNA helicase DDX6 OS=Mus musculus Ddx6 PE=2 SV=1 - [DDX6] 0 1 0 2 0 1 0 4 N/A 1.613E-02 54.2 (7)
P06745 G6PI Glucose-6-phosphate isomerase OS=Mus musculus Gpi PE=1 SV=4 - [G6PI] 0 1 0 1 0 2 0 4 N/A 1.613E-02 62.7 (22)(23)(24) (4)(7)(10)
Q8BJ71 NUP93 Nuclear pore complex protein Nup93 OS=Mus musculus Nup93 PE=2 SV=1 - [NUP93] 0 1 0 2 0 1 0 4 N/A 1.613E-02 93.2 (2)(7)(16)
E9Q3G8E9Q3G8 Protein Nup153 OS=Mus musculus Nup153 PE=4 SV=1 - [E9Q3G8] 0 1 0 2 0 1 0 4 N/A 1.613E-02 151.9 (2)(7)(13)
Q3UHX0 NOL8 Nucleolar protein 8 OS=Mus musculus Nol8 PE=1 SV=2 - [NOL8] 0 1 0 2 0 1 0 4 N/A 1.613E-02 128.6 (16)
O89090-2 SP1 Isoform 2 of Transcription factor Sp1 OS=Mus musculus Sp1 - [SP1] 0 2 0 1 0 1 0 4 N/A 1.613E-02 48.7 (10)(12)
P63276 RS17 40S ribosomal protein S17 OS=Mus musculus Rps17 PE=1 SV=2 - [RS17] 0 1 0 2 0 1 0 4 N/A 1.613E-02 15.5 ✔ (20)(23)(24) (20)(23)(24)
Q9WV84 NDKM Nucleoside diphosphate kinase, mitochondrial OS=Mus musculus Nme4 PE=1 SV=1 - [NDKM] 0 1 0 1 0 2 0 4 N/A 1.613E-02 20.5 (23)(24)
Q8VCW8 ACSF2 Acyl-CoA synthetase family member 2, mitochondrial OS=Mus musculus Acsf2 PE=2 SV=1 - [ACSF2] 0 1 0 2 0 1 0 4 N/A 1.613E-02 67.9 (22)(23)(24) (22)(23)(24)
P51480 CD2A1 Cyclin-dependent kinase inhibitor 2A, isoforms 1/2 OS=Mus musculus Cdkn2a PE=1 SV=2 - [CD2A1] 0 2 0 1 0 1 0 4 N/A 1.613E-02 17.9
Q5SSZ5-2 TENS3 Isoform 2 of Tensin-3 OS=Mus musculus Tns3 - [TENS3] 0 1 0 2 0 1 0 4 N/A 1.613E-02 58.7
Q99K01-3 PDXD1 Isoform 3 of Pyridoxal-dependent decarboxylase domain-containing protein 1 OS=Mus musculus Pdxdc1 - [PDXD1] 0 2 0 1 0 1 0 4 N/A 1.613E-02 78.5
Q8JZZ6 AMERL AMMECR1-like protein OS=Mus musculus Ammecr1l PE=2 SV=1 - [AMERL]0 2 0 1 0 1 0 4 N/A 1.613E-02 34.5
Q61235 SNTB2 Beta-2-syntrophin OS=Mus musculus Sntb2 PE=1 SV=2 - [SNTB2] 0 1 0 1 0 2 0 4 N/A 1.613E-02 56.3
P31324 KAP3 cAMP-dependent protein kinase type II-beta regulatory subunit OS=Mus musculus Prkar2b PE=1 SV=3 - [KAP3] 0 1 0 2 0 1 0 4 N/A 1.613E-02 46.1
Q6DFV1CNDG2 Condensin-2 complex subunit G2 OS=Mus musculus Ncapg2 PE=2 SV=2 - [CNDG2] 0 1 0 2 0 1 0 4 N/A 1.613E-02 130.8
P58854 GCP3 Gamma-tubulin complex component 3 OS=Mus musculus Tubgcp3 PE=2 SV=2 - [GCP3] 0 1 0 1 0 2 0 4 N/A 1.613E-02 103.4
E9PVM7E9PVM7 Glutathione S-transferase Mu 5 (Fragment) OS=Mus musculus Gstm5 PE=3 SV=1 - [E9PVM7] 0 2 0 1 0 1 0 4 N/A 1.613E-02 25.5
Q9D032-2 SSBP3 Isoform 2 of Single-stranded DNA-binding protein 3 OS=Mus musculus Ssbp3 - [SSBP3] 0 1 0 1 0 2 0 4 N/A 1.613E-02 37.7
Q9CWX9 DDX47 Probable ATP-dependent RNA helicase DDX47 OS=Mus musculus Ddx47 PE=2 SV=2 - [DDX47] 0 2 0 1 0 1 0 4 N/A 1.613E-02 50.6
477
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
J3QJY2 J3QJY2 Protein Erdr1 OS=Mus musculus Erdr1 PE=4 SV=1 - [J3QJY2] 0 1 0 1 0 2 0 4 N/A 1.613E-02 13.7
Q9JJU8 SH3L1 SH3 domain-binding glutamic acid-rich-like protein OS=Mus musculus Sh3bgrl PE=2 SV=1 - [SH3L1] 0 2 0 1 0 1 0 4 N/A 1.613E-02 12.8
Q91ZR2 SNX18 Sorting nexin-18 OS=Mus musculus Snx18 PE=2 SV=1 - [SNX18] 0 1 0 2 0 1 0 4 N/A 1.613E-02 67.9
Q99KC8VMA5A von Willebrand factor A domain-containing protein 5A OS=Mus musculus Vwa5a PE=1 SV=2 - [VMA5A] 0 1 0 1 0 2 0 4 N/A 1.613E-02 87.1
Q9QUR6 PPCE Prolyl endopeptidase OS=Mus musculus Prep PE=2 SV=1 - [PPCE] 1 20 0 19 0 25 1 64 64.00 3.699E-04 80.7 (20)(23)(24) (7)(19)
Q921K2 Q921K2 Poly (ADP-ribose) polymerase family, member 1 OS=Mus musculus Parp1 PE=2 SV=1 - [Q921K2] 1 17 0 18 0 14 1 49 49.00 2.128E-04 112.7 (7)
Q61584-4 FXR1 Isoform C of Fragile X mental retardation syndrome-related protein 1 OS=Mus musculus Fxr1 - [FXR1] 0 16 0 15 1 17 1 48 48.00 1.944E-05 72.8 (7)
Q61191 HCFC1 Host cell factor 1 OS=Mus musculus Hcfc1 PE=1 SV=2 - [HCFC1] 1 16 0 13 0 14 1 43 43.00 1.198E-04 210.3 ✔ (20)(23)(24) (1)(2)(7)(8)(9)(12)(13)(14)(15)(17)(18)(19)
P40124 CAP1 Adenylyl cyclase-associated protein 1 OS=Mus musculus Cap1 PE=1 SV=4 - [CAP1] 1 15 0 14 0 13 1 42 42.00 3.344E-05 51.5 ✔ (20)(23)(24) (7)(19)
Q91X76 Q91X76 5'-nucleotidase domain containing 2 OS=Mus musculus Nt5dc2 PE=2 SV=1 - [Q91X76] 1 13 0 12 0 16 1 41 41.00 4.338E-04 46 ✔
P80314 TCPB T-complex protein 1 subunit beta OS=Mus musculus Cct2 PE=1 SV=4 - [TCPB] 3 44 0 34 0 39 3 117 39.00 2.402E-04 57.4 (20)(22)(23)(24) (4)(7)(19)
E9Q2X6E9Q2X6 Structural maintenance of chromosomes protein OS=Mus musculus Smc4 PE=3 SV=1 - [E9Q2X6] 0 15 0 12 1 11 1 38 38.00 5.869E-04 144.1
Q8VCQ8 Q8VCQ8 Caldesmon 1 OS=Mus musculus Cald1 PE=2 SV=1 - [Q8VCQ8] 0 10 0 12 1 12 1 34 34.00 1.227E-04 60.4 ✔ (7)(19)
P21107-2 TPM3 Isoform 2 of Tropomyosin alpha-3 chain OS=Mus musculus Tpm3 - [TPM3] 2 22 0 24 0 21 2 67 33.50 3.997E-05 29 ✔ (22)(23)(24) (19)
Q62095 DDX3Y ATP-dependent RNA helicase DDX3Y OS=Mus musculus Ddx3y PE=1 SV=2 - [DDX3Y] 0 8 1 9 0 15 1 32 32.00 9.494E-03 73.4
Q8C156CND2 Condensin complex subunit 2 OS=Mus musculus Ncaph PE=2 SV=1 - [CND2] 1 8 0 13 0 10 1 31 31.00 2.570E-03 82.3 ✔ (20)(23)(24) (7)(16)
Q8BMK4 CKAP4 Cytoskeleton-associated protein 4 OS=Mus musculus Ckap4 PE=2 SV=2 - [CKAP4] 0 21 0 17 2 23 2 61 30.50 4.774E-04 63.7
Q9WU78 PDC6I Programmed cell death 6-interacting protein OS=Mus musculus Pdcd6ip PE=1 SV=3 - [PDC6I] 1 12 0 6 0 10 1 28 28.00 7.418E-03 96 (23)(24) (7)
P30416 FKBP4 Peptidyl-prolyl cis-trans isomerase FKBP4 OS=Mus musculus Fkbp4 PE=1 SV=5 - [FKBP4] 0 7 1 8 0 11 1 26 26.00 2.609E-03 51.5 (23)(24) (4)(7)(19)
Q791V5 MTCH2 Mitochondrial carrier homolog 2 OS=Mus musculus Mtch2 PE=1 SV=1 - [MTCH2] 0 4 0 8 1 13 1 25 25.00 3.810E-02 33.5 ✔ (20)(21) (7)
A2AFW6 A2AFW6 Mitochondrial carrier homolog 2 OS=Mus musculus Mtch2 PE=3 SV=1 - [A2AFW6] 0 8 0 5 1 12 1 25 25.00 1.764E-02 32.3 (7)
Q8BKC5IPO5 Importin-5 OS=Mus musculus Ipo5 PE=1 SV=3 - [IPO5] 1 6 0 10 0 8 1 24 24.00 3.098E-03 123.5 (22) (7)(16)
Q8BGJ5Q8BGJ5 MCG13402, isoform CRA_a OS=Mus musculus Ptbp1 PE=2 SV=1 - [Q8BGJ5] 0 18 0 19 2 10 2 47 23.50 6.847E-03 56.9 (7)(19)
Q9ET54-4 PALLD Isoform 4 of Palladin OS=Mus musculus Palld - [PALLD] 1 16 0 13 1 14 2 43 21.50 1.317E-04 108.2 ✔ (7)
Q9DCL9PUR6 Multifunctional protein ADE2 OS=Mus musculus Paics PE=1 SV=4 - [PUR6] 1 7 0 10 0 4 1 21 21.00 1.944E-02 47 (20)(22)(23)(24) (7)(11)(12)(16)(19)
Q8BGD9 IF4B Eukaryotic translation initiation factor 4B OS=Mus musculus Eif4b PE=1 SV=1 - [IF4B] 0 5 0 6 1 10 1 21 21.00 1.301E-02 68.8 (23)(24) (7)(19)
P30285 CDK4 Cyclin-dependent kinase 4 OS=Mus musculus Cdk4 PE=1 SV=1 - [CDK4] 1 4 0 10 0 5 1 19 19.00 3.347E-02 33.7 (7)(19)
Q9WVG6-2 CARM1 Isoform 2 of Histone-arginine methyltransferase CARM1 OS=Mus musculus Carm1 - [CARM1] 1 4 0 5 0 10 1 19 19.00 3.347E-02 63.4 (4)(7)(12)(16)
Q62418-3 DBNL Isoform 3 of Drebrin-like protein OS=Mus musculus Dbnl - [DBNL] 1 6 0 8 0 5 1 19 19.00 3.126E-03 48.3 (23)(24) (16)(19)
Q3UZ39LRRF1 Leucine-rich repeat flightless-interacting protein 1 OS=Mus musculus Lrrfip1 PE=1 SV=2 - [LRRF1] 1 16 0 19 2 21 3 56 18.67 3.496E-04 79.2 (7)(16)(19)
Q62167 DDX3X ATP-dependent RNA helicase DDX3X OS=Mus musculus Ddx3x PE=1 SV=3 - [DDX3X] 1 11 1 10 0 16 2 37 18.50 3.468E-03 73.1 (23)(24) (7)(19)
Q9Z1D1EIF3G Eukaryotic translation initiation factor 3 subunit G OS=Mus musculus Eif3g PE=1 SV=2 - [EIF3G] 0 6 0 7 1 5 1 18 18.00 1.051E-03 35.6 (7)(12)(16)(19)
Q9Z110-2 P5CS Isoform Short of Delta-1-pyrroline-5-carboxylate synthase OS=Mus musculus Aldh18a1 - [P5CS] 2 14 0 16 1 23 3 53 17.67 3.940E-03 87 (22) (7)(16)(19)
P57780 ACTN4 Alpha-actinin-4 OS=Mus musculus Actn4 PE=1 SV=1 - [ACTN4] 2 17 0 16 1 17 3 50 16.67 1.944E-05 104.9 ✔ (20)(23)(24) (7)(16)
Q3THK7GUAA GMP synthase [glutamine-hydrolyzing] OS=Mus musculus Gmps PE=1 SV=2 - [GUAA] 0 15 2 19 1 14 3 48 16.00 7.801E-04 76.7 (23)(24) (7)(19)
478
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
Q9CQ43 Q9CQ43 Deoxyuridine triphosphatase OS=Mus musculus Dut PE=2 SV=1 - [Q9CQ43] 1 6 0 5 0 5 1 16 16.00 4.472E-04 17.4 (7)(19)
Q60760-3 GRB10 Isoform 3 of Growth factor receptor-bound protein 10 OS=Mus musculus Grb10 - [GRB10] 1 6 0 5 0 5 1 16 16.00 4.472E-04 61.2
P07742 RIR1 Ribonucleoside-diphosphate reductase large subunit OS=Mus musculus Rrm1 PE=1 SV=2 - [RIR1] 2 16 1 18 0 13 3 47 15.67 7.194E-04 90.2 ✔ (20)(23)(24) (7)(10)(16)(19)
Q8BK67RCC2 Protein RCC2 OS=Mus musculus Rcc2 PE=2 SV=1 - [RCC2] 2 14 0 7 0 10 2 31 15.50 1.059E-02 55.9 ✔ (20)(22)(23)(24) (7)
Q9CQ65 MTAP S-methyl-5'-thioadenosine phosphorylase OS=Mus musculus Mtap PE=2 SV=1 - [MTAP] 1 9 0 12 1 10 2 31 15.50 5.101E-04 31 (20) (7)(19)
P42227-2 STAT3 Isoform Stat3B of Signal transducer and activator of transcription 3 OS=Mus musculus Stat3 - [STAT3] 1 13 0 12 2 21 3 46 15.33 7.859E-03 83.1 (26)
Q9JMH6-2 TRXR1 Isoform 2 of Thioredoxin reductase 1, cytoplasmic OS=Mus musculus Txnrd1 - [TRXR1] 1 10 1 14 1 19 3 43 14.33 6.879E-03 54.5 (23)(24) (7)(16)(19)
Q9R0P5 DEST Destrin OS=Mus musculus Dstn PE=1 SV=3 - [DEST] 0 2 0 5 1 7 1 14 14.00 4.381E-02 18.5 ✔ (20)(23)(24) (7)(19)
Q9CR00PSMD9 26S proteasome non-ATPase regulatory subunit 9 OS=Mus musculus Psmd9 PE=1 SV=1 - [PSMD9] 0 5 0 5 1 4 1 14 14.00 7.779E-04 24.7 (7)(16)
Q61553 FSCN1 Fascin OS=Mus musculus Fscn1 PE=1 SV=4 - [FSCN1] 1 8 0 7 1 12 2 27 13.50 5.965E-03 54.5 ✔ (20)(23)(24) (4)(7)(10)
P33174 KIF4 Chromosome-associated kinesin KIF4 OS=Mus musculus Kif4 PE=2 SV=3 - [KIF4] 1 10 0 7 1 9 2 26 13.00 1.058E-03 139.4
P37913 DNLI1 DNA ligase 1 OS=Mus musculus Lig1 PE=1 SV=2 - [DNLI1] 2 9 0 8 0 8 2 25 12.50 5.038E-04 102.2 (19)
Q8CI51 PDLI5 PDZ and LIM domain protein 5 OS=Mus musculus Pdlim5 PE=1 SV=4 - [PDLI5] 1 13 0 13 2 10 3 36 12.00 6.780E-04 63.3 (1)(7)(16)(19)
Q501J6 DDX17 Probable ATP-dependent RNA helicase DDX17 OS=Mus musculus Ddx17 PE=2 SV=1 - [DDX17] 1 9 0 7 1 8 2 24 12.00 3.882E-04 72.4 ✔ (20)(22)(23)(24) (4)(7)(19)
Q8BHK9ERC6L DNA excision repair protein ERCC-6-like OS=Mus musculus Ercc6l PE=1 SV=1 - [ERC6L] 0 4 0 5 1 3 1 12 12.00 5.328E-03 138.8 (7)(16)
Q3UPL0-2 SC31A Isoform 2 of Protein transport protein Sec31A OS=Mus musculus Sec31a - [SC31A] 1 4 0 3 0 5 1 12 12.00 5.328E-03 129.5 (4)(7)(12)(19)
P16858 G3P Glyceraldehyde-3-phosphate dehydrogenase OS=Mus musculus Gapdh PE=1 SV=2 - [G3P] 4 60 8 41 6 103 18 204 11.33 2.794E-02 35.8 (23)(24) (1)(2)(3)(5)(6)(7)(16)(17)(18)(19)
Q6DFW4 NOP58 Nucleolar protein 58 OS=Mus musculus Nop58 PE=1 SV=1 - [NOP58] 2 19 0 24 4 25 6 68 11.33 6.979E-04 60.3 (23)(24) (16)(19)
P24547 IMDH2 Inosine-5'-monophosphate dehydrogenase 2 OS=Mus musculus Impdh2 PE=1 SV=2 - [IMDH2] 2 14 0 17 2 14 4 45 11.25 3.411E-04 55.8 (22)(23)(24) (7)(10)(12)(16)(19)
Q05CL8LARP7 La-related protein 7 OS=Mus musculus Larp7 PE=1 SV=2 - [LARP7] 1 7 1 8 0 7 2 22 11.00 1.451E-04 64.8 (23)(24) (7)(16)
Q62426 CYTB Cystatin-B OS=Mus musculus Cstb PE=1 SV=1 - [CYTB] 0 4 0 3 1 4 1 11 11.00 2.111E-03 11 (23)(24)
Q7TQH0-3 ATX2L Isoform 3 of Ataxin-2-like protein OS=Mus musculus Atxn2l - [ATX2L] 1 10 0 9 2 13 3 32 10.67 1.921E-03 109.9 (23)(24) (7)(12)(16)
I7HIK9 I7HIK9 Cellular tumor antigen p53 OS=Mus musculus Trp53 PE=3 SV=1 - [I7HIK9] 0 9 0 14 3 8 3 31 10.33 1.145E-02 42.1
D3Z0A2 D3Z0A2 Protein arginine N-methyltransferase 1 OS=Mus musculus Prmt1 PE=4 SV=1 - [D3Z0A2] 4 13 0 14 0 14 4 41 10.25 8.533E-04 36.5 ✔ (7)
P61979-3 HNRPK Isoform 3 of Heterogeneous nuclear ribonucleoprotein K OS=Mus musculus Hnrnpk - [HNRPK] 4 17 1 15 0 18 5 50 10.00 5.486E-04 48.5 (22) (4)(6)(7)(10)(16)(17)(19)
O08553 DPYL2 Dihydropyrimidinase-related protein 2 OS=Mus musculus Dpysl2 PE=1 SV=2 - [DPYL2] 3 14 0 15 1 11 4 40 10.00 1.293E-03 62.2 (1)(13)
O09106 HDAC1 Histone deacetylase 1 OS=Mus musculus Hdac1 PE=1 SV=1 - [HDAC1] 2 4 0 9 0 7 2 20 10.00 1.989E-02 55 (20)(22)(23)(24) (7)(12)
P08752 GNAI2 Guanine nucleotide-binding protein G(i) subunit alpha-2 OS=Mus musculus Gnai2 PE=1 SV=5 - [GNAI2] 2 6 0 7 0 7 2 20 10.00 1.293E-03 40.5 ✔ (20)(22) (13)(19)
Q8VDM4 PSMD2 26S proteasome non-ATPase regulatory subunit 2 OS=Mus musculus Psmd2 PE=1 SV=1 - [PSMD2] 2 8 0 7 1 14 3 29 9.67 1.856E-02 100.1 (7)(12)
Q9CY58PAIRB Plasminogen activator inhibitor 1 RNA-binding protein OS=Mus musculus Serbp1 PE=1 SV=2 - [PAIRB] 2 8 0 6 0 5 2 19 9.50 6.859E-03 44.7 (22)(23)(24) (7)(12)(16)(19)
Q922Q8LRC59 Leucine-rich repeat-containing protein 59 OS=Mus musculus Lrrc59 PE=2 SV=1 - [LRC59] 2 6 0 5 0 7 2 18 9.00 3.772E-03 34.9 (23)(24)
Q9WV55 VAPA Vesicle-associated membrane protein-associated protein A OS=Mus musculus Vapa PE=1 SV=2 - [VAPA] 1 5 0 6 1 7 2 18 9.00 1.324E-03 27.8 (23)(24) (19)
Q3U308CTU2 Cytoplasmic tRNA 2-thiolation protein 2 OS=Mus musculus Ctu2 PE=2 SV=1 - [CTU2] 0 4 0 3 1 2 1 9 9.00 1.613E-02 56.1 (7)(16)
P70399-3 TP53B Isoform 3 of Tumor suppressor p53-binding protein 1 OS=Mus musculus Tp53bp1 - [TP53B] 1 4 0 3 0 2 1 9 9.00 1.613E-02 206.8 ✔ (7)(10)(16)
P32921-2 SYWC Isoform 2 of Tryptophan--tRNA ligase, cytoplasmic OS=Mus musculus Wars - [SYWC] 0 2 0 3 1 4 1 9 9.00 1.613E-02 53.6 (23)(24) (7)(10)(16)
479
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
Q7TPR4 ACTN1 Alpha-actinin-1 OS=Mus musculus Actn1 PE=1 SV=1 - [ACTN1] 6 41 0 46 9 44 15 131 8.73 2.140E-04 103 (23)(24) (7)(10)(19)
P63323 RS12 40S ribosomal protein S12 OS=Mus musculus Rps12 PE=1 SV=2 - [RS12] 1 6 0 4 1 7 2 17 8.50 6.074E-03 14.5 (7)(16)(19)
Q76MZ32AAA Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform OS=Mus musculus Ppp2r1a PE=1 SV=3 - [2AAA] 1 9 1 10 1 6 3 25 8.33 3.650E-03 65.3 (23)(24) (7)(10)
P84104-2 SRSF3 Isoform Short of Serine/arginine-rich splicing factor 3 OS=Mus musculus Srsf3 - [SRSF3] 1 8 0 9 2 8 3 25 8.33 3.882E-04 14.2 ✔ (23)(24) (7)
P18760 COF1 Cofilin-1 OS=Mus musculus Cfl1 PE=1 SV=3 - [COF1] 2 5 1 10 0 9 3 24 8.00 1.278E-02 18.5 (22)(23)(24) (7)(10)(12)(16)(18)(19)
Q9WUM4 COR1C Coronin-1C OS=Mus musculus Coro1c PE=1 SV=2 - [COR1C] 2 8 0 3 0 5 2 16 8.00 4.328E-02 53.1 (23)(24) (7)(10)(19)
Q91V41 RAB14 Ras-related protein Rab-14 OS=Mus musculus Rab14 PE=1 SV=3 - [RAB14] 1 6 1 6 0 4 2 16 8.00 3.320E-03 23.9
P09055 ITB1 Integrin beta-1 OS=Mus musculus Itgb1 PE=1 SV=1 - [ITB1] 1 6 0 6 1 4 2 16 8.00 3.320E-03 88.2 (23)(24)
Q921F4 HNRLL Heterogeneous nuclear ribonucleoprotein L-like OS=Mus musculus Hnrpll PE=1 SV=3 - [HNRLL] 1 2 0 4 0 2 1 8 8.00 3.517E-02 64.1 (7)
F8VQ93 F8VQ93 PH-interacting protein OS=Mus musculus Phip PE=4 SV=1 - [F8VQ93] 0 2 0 2 1 4 1 8 8.00 3.517E-02 206.6
B7ZCP4 B7ZCP4 Copine I OS=Mus musculus Cpne1 PE=4 SV=1 - [B7ZCP4] 1 3 0 2 0 3 1 8 8.00 7.763E-03 52.8 (7)
Q5SQB0Q5SQB0 Nucleophosmin OS=Mus musculus Npm1 PE=2 SV=1 - [Q5SQB0] 2 11 1 10 1 10 4 31 7.75 4.435E-05 29.5 (7)(18)(19)
Q8JZK9 HMCS1 Hydroxymethylglutaryl-CoA synthase, cytoplasmic OS=Mus musculus Hmgcs1 PE=1 SV=1 - [HMCS1] 1 4 0 6 1 5 2 15 7.50 2.890E-03 57.5 (23)(24) (4)(7)
Q8C1B7-3 SEP11 Isoform 3 of Septin-11 OS=Mus musculus Sept11 - [SEP11] 1 5 0 5 1 5 2 15 7.50 2.020E-04 48.9
P23198 CBX3 Chromobox protein homolog 3 OS=Mus musculus Cbx3 PE=1 SV=2 - [CBX3] 0 6 0 8 3 7 3 21 7.00 6.533E-03 20.8 (22)(23)(24) (19)
Q8C052MAP1S Microtubule-associated protein 1S OS=Mus musculus Map1s PE=1 SV=2 - [MAP1S] 1 2 0 3 0 2 1 7 7.00 1.324E-02 102.9 (7)(19)
Q8VCF0MAVS Mitochondrial antiviral-signaling protein OS=Mus musculus Mavs PE=1 SV=1 - [MAVS] 0 2 0 3 1 2 1 7 7.00 1.324E-02 53.4 (4)(13)(16)
P46978 STT3A Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A OS=Mus musculus Stt3a PE=1 SV=1 - [STT3A] 1 2 0 3 0 2 1 7 7.00 1.324E-02 80.5 ✔
P06151 LDHA L-lactate dehydrogenase A chain OS=Mus musculus Ldha PE=1 SV=3 - [LDHA] 5 17 0 12 2 17 7 46 6.57 4.182E-03 36.5 (23)(24) (5)(7)(16)(17)(19)
Q3U0V1FUBP2 Far upstream element-binding protein 2 OS=Mus musculus Khsrp PE=1 SV=2 - [FUBP2] 8 32 3 40 5 32 16 104 6.50 6.426E-04 76.7 (23)(24) (7)(10)(16)(19)
A2AR02 PPIG Peptidyl-prolyl cis-trans isomerase G OS=Mus musculus Ppig PE=1 SV=1 - [PPIG] 0 6 0 3 2 4 2 13 6.50 2.947E-02 88.3 (7)
Q6PAM1 TXLNA Alpha-taxilin OS=Mus musculus Txlna PE=2 SV=1 - [TXLNA] 0 10 0 8 4 7 4 25 6.25 1.189E-02 62.3 (7)(16)(19)
B1AU75 B1AU75 Nuclear autoantigenic sperm protein OS=Mus musculus Nasp PE=4 SV=1 - [B1AU75] 1 9 0 5 3 10 4 24 6.00 1.944E-02 84 (7)(19)
E9Q7G0E9Q7G0 Protein Numa1 OS=Mus musculus Numa1 PE=4 SV=1 - [E9Q7G0] 1 8 0 5 2 5 3 18 6.00 1.235E-02 235.5 ✔ (7)
Q8C7R4UBA6 Ubiquitin-like modifier-activating enzyme 6 OS=Mus musculus Uba6 PE=1 SV=1 - [UBA6] 1 6 0 3 1 3 2 12 6.00 3.411E-02 117.9 (23)(24) (7)(16)
Q9DC51GNAI3 Guanine nucleotide-binding protein G(k) subunit alpha OS=Mus musculus Gnai3 PE=1 SV=3 - [GNAI3] 2 3 0 4 0 5 2 12 6.00 1.944E-02 40.5 (20) (13)
O70503 DHB12 Estradiol 17-beta-dehydrogenase 12 OS=Mus musculus Hsd17b12 PE=2 SV=1 - [DHB12] 1 2 0 2 0 2 1 6 6.00 7.490E-03 34.7 ✔ (20)
O35316 SC6A6 Sodium- and chloride-dependent taurine transporter OS=Mus musculus Slc6a6 PE=1 SV=2 - [SC6A6] 0 2 0 2 1 2 1 6 6.00 7.490E-03 69.8 ✔
P14901 HMOX1 Heme oxygenase 1 OS=Mus musculus Hmox1 PE=1 SV=1 - [HMOX1] 1 2 0 2 0 2 1 6 6.00 7.490E-03 32.9 (23)(24)
G3X8R0 G3X8R0 Receptor accessory protein 5, isoform CRA_a OS=Mus musculus Reep5 PE=4 SV=1 - [G3X8R0] 1 2 0 2 0 2 1 6 6.00 7.490E-03 21.4
Q9D0I9 SYRC Arginine--tRNA ligase, cytoplasmic OS=Mus musculus Rars PE=2 SV=2 - [SYRC] 2 10 1 12 2 6 5 28 5.60 1.294E-02 75.6 (23)(24) (7)(16)
D3YWF6D3YWF6 Ubiquitin thioesterase OTUB1 OS=Mus musculus Otub1 PE=4 SV=1 - [D3YWF6] 3 8 1 10 1 10 5 28 5.60 1.244E-03 28 (7)
Q9D819IPYR Inorganic pyrophosphatase OS=Mus musculus Ppa1 PE=1 SV=1 - [IPYR] 2 6 0 5 2 11 4 22 5.50 3.831E-02 32.6 (23)(24) (7)(16)(19)
P62259 1433E 14-3-3 protein epsilon OS=Mus musculus Ywhae PE=1 SV=1 - [1433E] 3 9 0 10 2 8 5 27 5.40 2.243E-03 29.2 (22)(23)(24) (4)(7)(11)(16)(19)
Q61699-2 HS105 Isoform HSP105-beta of Heat shock protein 105 kDa OS=Mus musculus Hsph1 - [HS105] 5 17 2 16 2 13 9 46 5.11 1.396E-03 91.6 (23)(24) (4)(7)(16)
480
N
o
.
GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk GlcNAlk
Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts Spectral Counts
Experiment 1 Experiment 1 Experiment 2 Experiment 2 Experiment 3 Experiment 3 Sum Sum
Ac-
ces-
sion
Gene
ID Description - + - + - + - +
Fold
En-
rich-
ment t-test
MW
[kDa]
1-
Deoxy-
GlcNAlk
Known
Acety-
lated
Known O-
GlcNAcylated
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
P52480 KPYM Pyruvate kinase isozymes M1/M2 OS=Mus musculus Pkm PE=1 SV=4 - [KPYM] 9 27 1 24 6 30 16 81 5.06 1.729E-03 57.8 (22)(23)(24) (4)(6)(10)(12)(13)(16)(18)(19)
P10853 H2B1F Histone H2B type 1-F/J/L OS=Mus musculus Hist1h2bf PE=1 SV=2 - [H2B1F] 3 6 0 5 0 4 3 15 5.00 2.572E-02 13.9 (20)(21) (13)
Q64337-2 SQSTM Isoform 2 of Sequestosome-1 OS=Mus musculus Sqstm1 - [SQSTM] 1 1 0 2 0 2 1 5 5.00 4.742E-02 44.2 (7)(16)
Q9ER72-2 SYCC Isoform 2 of Cysteine--tRNA ligase, cytoplasmic OS=Mus musculus Cars - [SYCC] 0 1 0 2 1 2 1 5 5.00 4.742E-02 85.5 ✔ (23)(24) (7)
P62852 RS25 40S ribosomal protein S25 OS=Mus musculus Rps25 PE=2 SV=1 - [RS25] 1 2 0 2 0 1 1 5 5.00 4.742E-02 13.7 ✔ (20)(23)(24) (20)(23)(24)
A2AL12 A2AL12 Heterogeneous nuclear ribonucleoprotein A3 OS=Mus musculus Hnrnpa3 PE=4 SV=1 - [A2AL12] 4 14 5 23 3 22 12 59 4.92 5.725E-03 34.5 (4)(7)(19)
A2AMW0 A2AMW0Capping protein (Actin filament) muscle Z-line, beta OS=Mus musculus Capzb PE=4 SV=1 - [A2AMW0] 3 11 1 10 3 13 7 34 4.86 1.239E-03 29.3 (4)(19)
Q9D0E1-2 HNRPM Isoform 2 of Heterogeneous nuclear ribonucleoprotein M OS=Mus musculus Hnrnpm - [HNRPM] 8 17 1 21 2 15 11 53 4.82 7.573E-03 73.7 (22)(23)(24) (7)(16)
P63158 HMGB1 High mobility group protein B1 OS=Mus musculus Hmgb1 PE=1 SV=2 - [HMGB1] 3 9 0 4 1 6 4 19 4.75 4.232E-02 24.9 (21)(22)(23)(24) (7)(12)(16)(19)
Q8CGC7 SYEP Bifunctional glutamate/proline--tRNA ligase OS=Mus musculus Eprs PE=1 SV=4 - [SYEP] 5 10 0 21 5 15 10 46 4.60 2.877E-02 170 (23)(24) (7)(10)(12)(16)(19)
Q61033 LAP2A Lamina-associated polypeptide 2, isoforms alpha/zeta OS=Mus musculus Tmpo PE=1 SV=4 - [LAP2A] 1 10 1 7 3 6 5 23 4.60 1.201E-02 75.1 (23)(24) (16)(19)
Q61656 DDX5 Probable ATP-dependent RNA helicase DDX5 OS=Mus musculus Ddx5 PE=1 SV=2 - [DDX5] 1 4 0 2 1 3 2 9 4.50 2.490E-02 69.2 ✔ (20)(22)(23)(24) (4)(7)(16)
Q9Z2X1 HNRPF Heterogeneous nuclear ribonucleoprotein F OS=Mus musculus Hnrnpf PE=1 SV=3 - [HNRPF] 4 16 3 12 2 11 9 39 4.33 3.602E-03 45.7 (22)(23)(24) (7)(16)(19)
P46935 NEDD4 E3 ubiquitin-protein ligase NEDD4 OS=Mus musculus Nedd4 PE=1 SV=3 - [NEDD4] 13 33 3 37 9 38 25 108 4.32 1.086E-03 102.6 (1)(13)(19)
P20152 VIME Vimentin OS=Mus musculus Vim PE=1 SV=3 - [VIME] 7 23 5 25 5 24 17 72 4.24 3.164E-05 53.7 (23)(24) (7)(12)(13)(16)(17)(19)
E9PZM7E9PZM7 Protein Scaf11 OS=Mus musculus Scaf11 PE=4 SV=1 - [E9PZM7] 3 8 0 7 3 10 6 25 4.17 8.971E-03 162
Q9Z277 BAZ1B Tyrosine-protein kinase BAZ1B OS=Mus musculus Baz1b PE=1 SV=2 - [BAZ1B] 4 9 0 8 2 8 6 25 4.17 6.214E-03 170.5 (23)(24)
E9Q7H5E9Q7H5 Uncharacterized protein OS=Mus musculus Gm8991 PE=4 SV=1 - [E9Q7H5] 5 12 3 13 2 16 10 41 4.10 2.274E-03 32.6 (4)
J3QPE8 J3qpe8 MCG16555 OS=Mus musculus Vdac3-ps1 PE=4 SV=1 - [J3QPE8] 3 10 0 10 6 16 9 36 4.00 2.724E-02 30.7
Q9WVA4 TAGL2 Transgelin-2 OS=Mus musculus Tagln2 PE=1 SV=4 - [TAGL2] 1 3 0 4 2 5 3 12 4.00 2.131E-02 22.4 (23)(24) (7)(16)(19)
481
Tables 7-1 and 7-2 References
1 Alfaro, J. F., Gong, C.-X., Monroe, M. E., Aldrich, J. T., Clauss, T. R. W., Purvine, S. O., Wang, Z., Camp, D. G., Shabanowitz, J., Stanley, P ., Hart, G. W., Hunt, D. F., Yang, F., and Smith, R. D. (2012) Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc. Natl. Acad. Sci. U.S.A. 109, 7280–7285.
2 Boyce, M., Carrico, I. S., Ganguli, A. S., Yu, S.-H., Hangauer, M. J., Hubbard, S. C., Kohler, J. J., and Bertozzi, C. R. (2011) Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc. Natl. Acad. Sci. U.S.A.
3 Cieniewski-Bernard, C., Bastide, B., Lefebvre, T., Lemoine, J., Mounier, Y., and Michalski, J.-C. (2004) Identification of O-linked N-acetylglucosamine proteins in rat skeletal muscle using two-dimensional gel electrophoresis and mass spectrometry. Mol Cell Proteomics 3, 577–585.
4 Clark, P . M., Dweck, J. F., Mason, D. E., Hart, C. R., Buck, S. B., Peters, E. C., Agnew, B. J., and Hsieh-Wilson, L. C. (2008) Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. J. Am. Chem. Soc. 130, 11576–11577.
5 Dehennaut, V., Slomianny, M.-C., Page, A., Vercoutter-Edouart, A.-S., Jessus, C., Michalski, J.-C., Vilain, J.-P ., Bodart, J.-F., and Lefebvre, T. (2008) Identification of Structural and Functional O-Linked N-Acetylglucosamine-bearing Proteins in Xenopus laevis Oocyte. Molecular & Cellular Proteomics 7, 2229–2245.
6 Gurcel, C., Vercoutter-Edouart, A., Fonbonne, C., Mortuaire, M., Salvador, A., Michalski, J., and Lemoine, J. (2008) Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Analytical and Bioanalytical Chemistry 390, 2089–2097.
7 Hahne, H., Sobotzki, N., Nyberg, T., Helm, D., Borodkin, V. S., van Aalten, D. M. F., Agnew, B., and Kuster, B. (2013) Proteome Wide Purification and Identification of O-GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry. J. Proteome Res. 12, 927–936.
8 Khidekel, N., Ficarro, S. B., Clark, P . M., Bryan, M. C., Swaney, D. L., Rexach, J. E., Sun, Y. E., Coon, J. J., Peters, E. C., and Hsieh-Wilson, L. C. (2007) Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem Biol 3, 339–348.
9 Khidekel, N., Ficarro, S. B., Peters, E. C., and Hsieh-Wilson, L. C. (2004) Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc Natl Acad Sci USA 101, 13132–13137.
10 Nandi, A., Sprung, R., Barma, D. K., Zhao, Y., Kim, S. C., Falck, J. R., and Zhao, Y. (2006) Global identification of O-GlcNAc-modified proteins. Anal Chem 78, 452–458.
11 Sprung, R., Nandi, A., Chen, Y., Kim, S., Barma, D., Falck, J., and Zhao, Y. (2005) Tagging-via-substrate strategy for probing O-GlcNAc modified proteins. J. Proteome Res. 4, 950–957.
12 Teo, C. F., Ingale, S., Wolfert, M. A., Elsayed, G. A., Nöt, L. G., Chatham, J. C., Wells, L., and Boons, G.-J. (2010) Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc. Nat Chem Biol 6, 338–343.
13 Trinidad, J. C., Barkan, D. T., Gulledge, B. F., Thalhammer, A., Sali, A., Schoepfer, R., and Burlingame, A. L. (2012) Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics 11, 215–229.
14 Vosseller, K., Trinidad, J. C., Chalkley, R. J., Specht, C. G., Thalhammer, A., Lynn, A. J., Snedecor, J. O., Guan, S., Medzihradszky, K. F., Maltby, D. A., Schoepfer, R., and Burlingame, A. L. (2006) O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry. Mol Cell Proteomics 5, 923–934.
15 Wang, Z., Udeshi, N. D., O'malley, M., Shabanowitz, J., Hunt, D. F., and Hart, G. W. (2010) Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry. Molecular & Cellular Proteomics 9, 153–160.
16 Wang, Z., Udeshi, N. D., Slawson, C., Compton, P . D., Sakabe, K., Cheung, W. D., Shabanowitz, J., Hunt, D. F., and Hart, G. W. (2010) Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis. Science Signaling 3, ra2–ra2.
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19 Zaro, B. W., Yang, Y.-Y., Hang, H. C., and Pratt, M. R. (2011) Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc. Natl. Acad. Sci. U.S.A. 108, 8146–8151.
20 Hornbeck PV, Kornhauser JM, Tkachev S, Zhang B, Skrzypek E, Murray B, Latham V, Sullivan M (2012) PhosphoSite Plus. Nucleic Acids Res. 40(Database issue), D261–70.
21 Liu, Z., Cao, J., Gao, X., Zhou, Y., Wen, L., Yang, X., et al. (2010). CPLA 1.0: an integrated database of protein lysine acetylation. Nucleic Acids Research, 39(Database), D1029–D1034. doi:10.1093/nar/gkq939
22 Yang, Y.-Y., Ascano, J. M., & Hang, H. C. (2010). Bioorthogonal chemical reporters for monitoring protein acetylation. J Am Chem Soc, 132(11), 3640–3641. doi:10.1021/ja908871t
23 Choudhary, C., Kumar, C., Gnad, F., Nielsen, M. L., Rehman, M., Walther, T. C., et al. (2009). Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions. Science, 325(5942), 834–840. doi:10.1126/science.1175371
24 Lundby, A., Lage, K., Weinert, B. T., Bekker-Jensen, D. B., Secher, A., Skovgaard, T., Kelstrup, C. D., Dmytriyev, A., Choudhary, C., Lundby, C., and Olsen, J. V. (2012) Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificityand Subcellular Patterns. Cell Rep 2, 419–431.
25 Kim, S. C., Sprung, R., Chen, Y., Xu, Y., Ball, H., Pei, J., Cheng, T., Kho, Y., Xiao, H., Xiao, L., Grishin, N. V., White, M., Yang, X.-J., and Zhao, Y. (2006) Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol. Cell 23, 607–618.
26 Basu, A., Rose, K. L., Zhang, J., Beavis, R. C., Ueberheide, B., Garcia, B. A., Chait, B., Zhao, Y., Hunt, D. F., Segal, E., Allis, C. D., and Hake, S. B. (2009) Proteome-wide prediction of acetylation substrates. Proc. Natl. Acad. Sci. U.S.A. 106, 13785–13790.
482
Appendix B: NMR Spectra
483
O
AcO
NH
OAc
N
3
O
OAc
AcO
Compound 2.1 1,3,4,6-Tetra-O-Acetyl-N-azidoacetylglucosamine (Ac4GlcNAz).
484
Compound 2.2 N-(6-(diethylamino)-9-(2-(4-hept-6-ynoylpiperazine-1-
carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium (alk-rho).
O Et
2
N NEt
2
N
N
O
O
485
O
AcO
NH
OAc
O
OAc
AcO
Compound 2.3 1,3,4,6-Tetra-O-Acetyl-N-4-pentynylglucosamine (Ac4GlcNAlk).
486
O Et
2
N NEt
2
N
N
O
O
N
3
Compound 2.4 N-(9-(2-(4-(6-azidohexanoyl)piperazine-1-carbonyl)phenyl)- 6-
(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminium (az-rho).
487
Compound 2.5 1,3,4,6-Tetra-O-Acetyl-N-azidoacetylgalactosamine (Ac4GalNAz).
O
AcO
NH
OAc
N
3
O
OAc
AcO
488
O
AcO
NH
OAc
O
OAc
AcO
Compound 2.6 1,3,4,6-Tetra-O-Acetyl-N-4-pentynylgalactosamine (Ac4GalNAlk).
489
O
AcO
NH
OAc
O
OAc
AcO
Compound 2.6 1,3,4,6-Tetra-O-Acetyl-N-4-pentynylgalactosamine (Ac4GalNAlk).
490
Compound 2.7 4-(2-azidoethyl)phenol.
HO
N
3
491
HO
N
N
O
N
3
HO
Compound 2.8 (E)-4-((5-(2-azidoethyl)-2-hydroxyphenyl)azenyl)benzoic acid.
492
HO
N
N
O
N
3
O N
O
O
Compound 2.9 (E)-2,5-dioxopyrrolidin-1-yl4-((5-(2-azidoethyl)-2-hydroxy-
phenyl)azenyl) benzoate.
493
Compound 2.10 (E)-4-((5-(2-azidoethyl)-2-hydroxyphenyl)azenyl)- N-(15-oxo-19-
(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-4,7,10-trioxa-14-
azanonadecyl)benzamide (azido-azo biotin).
O
O
O
H
N
HO
N
N
HN
O
N
3
O
S
HN
NH
O
494
Compound 4.1 N-propargyloxycarbamate-1,3,4,6-tetra-O-acetyl-glucosamine
(Ac4GlcPoc).
O
AcO
NH
OAc
O
O
OAc
AcO
495
Compound 4.1 N-propargyloxycarbamate-1,3,4,6-tetra-O-acetyl-glucosamine
(Ac4GlcPoc).
O
AcO
NH
OAc
O
O
OAc
AcO
496
O
AcO
NH
OAc
O
O
OAc
AcO
Compound 4.2 N-propargyloxycarbamate-1,3,4,6-tetra-O-acetyl-galactosamine
(Ac4GalPoc).
497
O
AcO
NH
OAc
O
O
OAc
AcO
Compound 4.2 N-propargyloxycarbamate-1,3,4,6-tetra-O-acetyl-galactosamine
(Ac4GalPoc).
498
Compound 4.3 N-propargyloxycarbamate-1,3,4,6-tetra-O-acetyl-manosamine
(Ac4ManPoc).
O
AcO
AcO
OAc
AcO
HN O
O
499
Compound 4.3 N-propargyloxycarbamate-1,3,4,6-tetra-O-acetyl-manosamine
(Ac4ManPoc).
O
AcO
AcO
OAc
AcO
HN O
O
500
Compound 5.1 2-Acetylphenyl pent-4-ynoate (AspAlk)
O OH
O
O
501
Compound 5.1 2-Acetylphenyl pent-4-ynoate (AspAlk)
O OH
O
O
502
Compound 6.1 1,3,4-Tri-O-acetyl-2-acetamido-6-azido-2,6-dideoxy-D-
glucopyranose (6AzGlcNAc).
O
AcO
NH
O
N
3
AcO
OAc
503
Compound 6.2 4-(prop-2-yn-1-yl)phenol
OH
504
Compound 6.3 (E)-methyl 4-((2-hydroxy-5-(prop-2-yn-1-
yl)phenyl)diazenyl)benzoate.
OH
N
N
O
O
505
Compound 6.3 (E)-methyl 4-((2-hydroxy-5-(prop-2-yn-1-
yl)phenyl)diazenyl)benzoate.
OH
N
N
O
O
506
Compound 6.4 (E)-4-((2-hydroxy-5-(prop-2-yn-1-yl)phenyl)diazenyl)benzoic acid.
OH
N
N
OH
O
507
Compound 6.4 (E)-4-((2-hydroxy-5-(prop-2-yn-1-yl)phenyl)diazenyl)benzoic acid.
OH
N
N
OH
O
508
Compound 6.9 α-1-O-benzyl-N-acetyl-glucosamine.
O
AcHN
OH
HO
HO
OBn
509
Compound 6.10 α-1-O-benzyl-6-O-p-methylbenzenesulfonate-N-acetyl-glucosamine
O
AcHN
OTs
HO
HO
OBn
510
Compound 6.11 3,4-di-O-acetyl-α-1-O-benzyl-6-O-p-methylbenzenesulfonate-N-acetyl-glucosamine.
O
AcHN
OTs
AcO
AcO
OBn
511
O
AcHN
OTs
AcO
AcO
O
P
O
O
O
Compound 6.13 Diallyl(3,4-di-O-acetyl-6-O-p-methylbenzenesulfonate-N-acetyl-glucosamine)-α-1-phosphate.
512
O
AcHN
OTs
AcO
AcO
O
P
O
O
O
Compound 6.13 Diallyl(3,4-di-O-acetyl-6-O-p-methylbenzenesulfonate-N-acetyl-glucosamine)-α-1-phosphate.
513
O
AcHN
OTs
AcO
AcO
O
P
O
O
O
Compound 6.13 Diallyl(3,4-di-O-acetyl-6-O-p-methylbenzenesulfonate-N-acetyl-glucosamine)-α-1-phosphate.
514
Compound 6.14 Diallyl(6-O-p-methylbenzenesulfonate-N-acetyl-glucosamine)-α-1-phosphate.
O
AcHN
OTs
HO
HO
O
P
O
O
O
515
Compound 6.14 Diallyl(6-O-p-methylbenzenesulfonate-N-acetyl-glucosamine)-α-1-phosphate.
O
AcHN
OTs
HO
HO
O
P
O
O
O
516
O
AcHN
OTs
HO
HO
O
P
O
O
O
Compound 6.14 Diallyl(6-O-p-methylbenzenesulfonate-N-acetyl-glucosamine)-α-1-phosphate.
517
Compound 6.15 Diallyl(6-azido-6-deoxy-N-acetyl-glucosamine)-α-1-phosphate.
O
AcHN
N
3
HO
HO
O
P
O
O
O
518
Compound 6.15 Diallyl(6-azido-6-deoxy-N-acetyl-glucosamine)-α-1-phosphate.
O
AcHN
N
3
HO
HO
O
P
O
O
O
519
Compound 6.15 Diallyl(6-azido-6-deoxy-N-acetyl-glucosamine)-α-1-phosphate.
O
AcHN
N
3
HO
HO
O
P
O
O
O
520
Compound 6.16 6-azido-6-deoxy-N-acetyl-glucosamine-1-phosphate.
O
AcHN
N
3
HO
HO
OPO
3
-2
521
Compound 6.16 6-azido-6-deoxy-N-acetyl-glucosamine-1-phosphate.
O
AcHN
N
3
HO
HO
OPO
3
-2
522
Compound 6.16 6-azido-6-deoxy-N-acetyl-glucosamine-1-phosphate.
O
AcHN
N
3
HO
HO
OPO
3
-2
523
Compound 7.1 3,4,6-Tri-O-Acetyl-1-Chloro-1-Deoxy-N-Acetylglucosamine.
O
AcO
OAc
AcO
Cl
NH
O
524
Compound 6.16 6-azido-6-deoxy-N-acetyl-glucosamine-1-phosphate.
O
AcHN
N
3
HO
HO
OPO
3
-2
525
Compound 6.16 6-azido-6-deoxy-N-acetyl-glucosamine-1-phosphate.
O
AcHN
N
3
HO
HO
OPO
3
-2
526
Compound 7.3 1-Deoxyglucosamine hydrochloride.
O
HO
OH
HO
NH
3
+
Cl
-
527
Compound 7.4 3,4,6-Tri-O-Acetyl-1-Deoxy-N-4-pentynylglucosamine (1-deoxy-Ac3GlcNAlk).
O
AcO
NH
O
OAc
AcO
528
Compound 7.4 3,4,6-Tri-O-Acetyl-1-Deoxy-N-4-pentynylglucosamine (1-deoxy-Ac3GlcNAlk).
O
AcO
NH
O
OAc
AcO
529
Compound 7.5 1-Deoxy-N-4-pentynylglucosamine (1-deoxy-GlcNAlk).
O
HO
NH
O
OH
HO
530
Compound 7.5 1-Deoxy-N-4-pentynylglucosamine (1-deoxy-GlcNAlk).
O
HO
NH
O
OH
HO
531
Compound 8.1 2-deoxy-2-ethylthioureido-1,3,4,6-tetra-O-acetyl-β-D-glucopyranose.
O
AcO
NH
OAc
OAc
AcO
S
HN
532
Compound 8.2 3,4,6-Tri-O-acetyl-1,2-dideoxy-2’-ethylamino-a-D- glucopyranoso-
[2,1-d]-Δ2’-thiazoline.
O
AcO
OAc
AcO
S
N
NH
533
Compound 8.3 1,2-dideoxy-2’-ethylamino-a-D-glucopyranoso-[2,1-d]-Δ2’-
thiazoline (Thiamet-G).
O
HO
OH
HO
S
N
NH
534
Compound 9.1 2-Acetamido-2-deoxy-3,4,5,6-di-O-isopropylidine-aldehydo-D-
glucose dimethyl acetal.
O
O
O
O
NHAc
OMe MeO
535
Compound 9.2 2-Acetamido-2-deoxy-3,4-O-isopropylidine-aldehydo-D-glucose
dimethyl acetal.
OH
OH
O
O
NHAc
OMe MeO
536
Compound 9.3 2-Acetamido-6-O-benzoyl-2-deoxy-3,4-O-isopropylidene-aldehydo-
D-glucose dimethyl acetal.
OH
OBz
O
O
NHAc
OMe MeO
537
Compound 9.4 2-Acetamido-6-O-benzoyl-2-deoxy-3,4-O-isopropylidine-5-O-
mesyl-aldehydo-D-glucose dimethyl acetal.
OMs
OBz
O
O
NHAc
OMe MeO
538
Compound 9.6 2-Acetamido-2,5,6-trideoxy-5,6-epithio-3,4-O-isopropylidine-
aldehydo-D-glucose dimethyl acetal.
O
O
NHAc
OMe MeO
S
539
Compound 9.7 2-Acetamido-6-O-acetyl-5-S-acetyl-2-deoxy-3,4-O-
isopropylidine-5-thio-aldehydo-D-glucose dimethyl acetal.
O
O
NHAc
OMe MeO
SAc
OAc
540
Compound 9.8 2-Acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-α-D-
glucopyranose (Ac5SGlcNAc).
S
AcO
HN
OAc
AcO
OAc
O
541
Compound 9.9 1,3-Dibromoacetone.
O
Br Br
542
Compound 9.10 2-(1,3-dioxoisoindolin-2-yl)acetaldehyde.
N
O
O
O
543
N
O
O
O
Compound 9.11 2-(4-oxopent-2-enyl)isoindoline-1,3-dione.
544
Compound 9.13 2-(5-bromo-4-oxopent-2-enyl)isoindoline-1,3-dione.
N
O
O
O Br
545
N
O
O
Br O
O
Compound 9.14 2-(3-(2-(bromomethyl)-1,3-dioxolan-2-yl)allyl)isoindoline-1,3-dione.
546
Compound 9.15 3-(2-(bromomethyl)-1,3-dioxolan-2-yl)prop-2-en-1-amine.
H
2
N
O
O
Br
547
Compound 9.16 3,4,6-Tri-O-Acetyl-N-4-pentynylgalactosamine (Ac3GalNAlk).
O
OAc
AcO
AcO
NH
O
OH
548
Compound 9.16 3,4,6-Tri-O-Acetyl-N-4-pentynylgalactosamine (Ac3GalNAlk).
O
OAc
AcO
AcO
NH
O
OH
549
Compound 9.17 3,4,6-Tri-O-Acetyl-N-4-pentynylgalactosamine-1-Phosphate.
O
OAc
AcO
AcO
HN
OPO
3
-2
O
550
Compound 9.17 3,4,6-Tri-O-Acetyl-N-4-pentynylgalactosamine-1-Phosphate.
O
OAc
AcO
AcO
HN
OPO
3
-2
O
551
O
OH
HO
HO
HN
O
P
O
O
OH
P
O OH
O
O
O
HO OH
N
NH
O
O
Compound 9.18 Uridine-Diphosphate-N-4-pentynylgalactosamine (UDP-GalNAlk)
552
Compound 9.18 Uridine-Diphosphate-N-4-pentynylgalactosamine (UDP-GalNAlk)
O
OH
HO
HO
HN
O
P
O
O
OH
P
O OH
O
O
O
HO OH
N
NH
O
O
553
O
OH
HO
HO
HN
O
P
O
O
OH
P
O OH
O
O
O
HO OH
N
NH
O
O
Compound 9.18 Uridine-Diphosphate-N-4-pentynylgalactosamine (UDP-GalNAlk)
554

Abstract (if available)

Abstract Post-translational modifications (PTMs) are ancillary decorations that are transferred onto fully-synthesized proteins. These modifications have been shown to significantly alter the fate and function of their substrates, and they include protein acetylation, phosphorylation, ubquitination, lipidation and glycosylation, among others. PTMs have been shown to be vital for development and are often misregulated in human disease. Throughout my graduate research, I have been particularly focused on the nutrient-senstitive, intracellular-glycosylation PTM N-acetylglucosamine (O-GlcNAc) and, to lesser extents, acetylation and mucin-type O-linked glycosylation. O-GlcNAc modification is a dynamic, signaling PTM that has been implicated in diabetes, neurodegeneration and cancer and is known to be upregulated in response to cell stress as a cytoprotective mechanism. In order to study O-GlcNAc’s role in disease progression, I have developed and optimized chemical reporters of O-GlcNAc that behave similarly to endogenous O-GlcNAc but are equipped with bioorthogonal functionality (azides or alkynes) that can be reacted with corresponding alkyne- or azide-containing tags through Cu(I)-catalyzed Azide-Alkyne Cycloadditon (CuAAC) to visualize the modification and/or identify protein substrates. Importantly, these new chemical reporters of O-GlcNAc are significantly more selective than what was previously available. I employed these probes to identify novel O-GlcNAc substrates as well as characterize the specific biochemical role of O-GlcNAcylation on a particular protein of interest, NEDD₄₋₁. I have also utilized this strategy in developing chemical reporters of protein acetylation and cell surface glycosylation. In addition, I have exploited chemical reporters in order to read-out on metabolic cross-talk between biosynthetic pathways as a way to further understand how cells amend their metabolic machinery in order to adapt to changes in nutrient availability and cellular demand.

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Asset Metadata

Creator Zaro, Balyn Wood (author)

Core Title Optimization of chemical reporters of O-GlcNAc for improved specificity and metabolic mapping

Contributor Electronically uploaded by the author (provenance)

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 09/12/2014

Defense Date 06/13/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag acetylation,carbohydrates,chemical reporters,click chemistry,OAI-PMH Harvest,O-GlcNAc,post-translational modifications

Format application/pdf (imt)

Language English

Advisor Pratt, Matthew R. (committee chair), Chen, Lin (committee member), Zhang, Chao (committee member)

Creator Email balynzaro@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-476825

Unique identifier UC11287491

Identifier etd-ZaroBalynW-2921.pdf (filename),usctheses-c3-476825 (legacy record id)

Legacy Identifier etd-ZaroBalynW-2921.pdf

Dmrecord 476825

Document Type Dissertation

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Rights Zaro, Balyn Wood

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

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