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Using chemistry to reveal the consequences of post translational modifications in cancer

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Using chemistry to reveal the consequences of post translational modifications in cancer

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Content USING CHEMISTRY TO REVEAL THE CONSEQUENCES
OF POST TRANSLATIONAL MODIFICATIONS IN
CANCER
by
Leslie Anne Bateman
A Dissertation 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)
August 2014
Copyright 2014 Leslie Anne Bateman
Table of Contents
Chapter 1. Introduction - Roles of O-GlcNAc in metabolic
reprogramming of cancer cells
Introduction.......................................................................................................................................1
Hexosamine Biosynthetic Pathway...................................................................................................3
O-GlcNAc Transferase.........................................................................................................4
O-GlcNAcase.......................................................................................................................5
O-GlcNAc and metabolic reprogramming in cancer........................................................................5
O-GlcNAc promotes cell proliferation and survival.........................................................................8
Roles of O-GlcNAc in stress resistance............................................................................................9
Roles of O-GlcNAc in invasion, angiogenesis, and metastasis......................................................10
Methods of studying O-GlcNAcylation..........................................................................................12
Enzymatic Labeling...........................................................................................................14
Metabolic Labeling............................................................................................................15
β-elimination followed by Michael addition (BEMAD)...................................................16
Proteomic Analysis............................................................................................................16
Inhibitors of O-GlcNAc cycling enzymes.........................................................................17
Therapeutic potential......................................................................................................................19
Conclusions.....................................................................................................................................20
References.......................................................................................................................................20
Chapter 2. Robust in-gel fluorescence detection of mucin-type O-linked
glycosylation
Introduction.....................................................................................................................................33
Experimental Procedures................................................................................................................36
Results.............................................................................................................................................37
Conclusions.....................................................................................................................................43
References.......................................................................................................................................44
Chapter 3. Analysis of N-propargyloxycarbamate monosaccharides as
metabolic chemical reporters of carbohydrate salvage pathways and
protein glycosylation
Introduction.....................................................................................................................................48
Results.............................................................................................................................................50
Conclusions.....................................................................................................................................53
Experimental Procedures................................................................................................................57
References.......................................................................................................................................64
ii
Chapter 4. An Alkyne-Aspirin Chemical Reporter for the Detection of
Aspirin-Dependent Protein Modification in Living Cells
Introduction.....................................................................................................................................67
Results.............................................................................................................................................69
Conclusions.....................................................................................................................................69
Experimental Procedures................................................................................................................77
References.......................................................................................................................................83
Comprehensive References..............................................................................................89
Appendix.................................................................................................................................109
Table 4-1. Proteins identified using AspAlk enrichment..............................................................110
NMR spectra.................................................................................................................................115
iii
List of Figures
Figure 1-1. O-GlcNAc modification of proteins..............................................................................2
Figure 1-2. Known roles of O-GlcNAc modification in various hallmarks of cancer...................12
Figure 1-3. Chemoenzymatic chemical reporter............................................................................15
Figure 1-4. Metabolic chemical reporter........................................................................................16
Figure 1-5. O-GlcNAc cycling enzyme inhibitors.........................................................................19
Figure 2-1. The GalNAc salvage pathway.....................................................................................34
Figure 2-2. Fluorescent detection of mucin-type O-linked glycoproteins.....................................37
Figure 2-3. Characterization of Ac4GalNAz labeling....................................................................38
Figure 2-4. Characterizing the metabolic fate of Ac4GalNAz.......................................................40
Figure 2-5. GalNAz labels proteins in a variety of cell lines.........................................................41
Figure 3-1. N-propargylcarbamate containing metabolic chemical reporters are incorporated onto
proteins............................................................................................................................................50
Figure 3-2. Flow cytometry analysis of metabolic chemical reporter incorporation....................53
Figure 3-3. Incorporation of metabolic chemical reporters into the O-GlcNAcylation pathway..54
Figure 3-4. Characterization of N-propargyloxycarbamate (Poc) bearing metabolic chemical
reporters..........................................................................................................................................57
Figure 4-1. AspAlk is a chemical reporter of aspirin-dependent protein modification ................68
Figure 4-2. Characterization of AspAlk labeling in HCT-15 colorectal cancer cells.....................69
Figure 4-3. Kinetic analysis of AspAlk labeling............................................................................71
Figure 4-4. Identification AspAlk labelled proteins.......................................................................72
Figure 4-5. AspAlk labels proteins in a variety of cell lines..........................................................80
iv
Abstract
Post translational modifications (PTMs), including glycosylation and acetylation, have a wide
variety of implications in cells. My goal was to explore the molecular consequences of PTMs
using chemical approaches to reveal pathways that are important to human disease, including
cancer. This manuscript uncovers a variety of chemical probes that have been used to study mucin
O-linked glycosylation, O-GlcNAc modification, and aspirin-dependent acetylation.
v
Acknowledgements
I would like to thank my parents, Monica and Charlie, for their love and support throughout my
entire life. They have been behind me every step of the way, always there when I needed an extra
push or a listening ear. Through the ups and downs, I knew I could always count on their
unconditional love. Their unwavering support and guidance has allowed me to grow into the
person I am today. I hope to continue to make them proud throughout the rest of my career and
life. I’d also like to thank my grandparents, who haven’t been quiet about their distaste of Los
Angeles, but are nonetheless 100% behind me.
Of course, I would also not be the person I am today without the close relationships I have with
my brothers. Ryan has always been a source of love, support, advice and humor. He is one of the
rocks in my life and may not realize how much I’ve looked up to him. Jon has the ability to make
me smile more than anyone I’ve known and I can’t thank him enough for being a constant source
of joy throughout my life. Craig is one of the most genuine people I know and I respect his
perspective, which challenges me to work harder at being a better scientist and a better person. It
has brought me great happiness to gain two sisters, Dayna Bateman and Lindsay Blaszczak, who
have been perfect additions to the family.
I have a deep respect for Professor Matthew Pratt, who has guided my through my PhD. I’ve
learned so much from him in science and in life. I am amazed at his drive to succeed and his
patience in starting a lab in the first years of his career. I’ve grown a lot as a scientist while
working in Matt’s lab and I can attribute my successes to his guidance.
I’ve made some amazing friendships while at USC that have brought me great times. Brett Zirkle,
my roommate and friend has been an amazing friend who has infinite patience for my whining
vi
and provides excellent advice - in both biology and in life. Kevin Gaffney has been a source of
love and support from the very beginning and I couldn’t have gotten through grad school without
him. Frances Tran isn’t afraid to tell me honest hard truth and has been a great friend through
grad school. I appreciate her perspective and her constant support.
I would also like to express my appreciation for my little sister, Mildred Chavez. She has
challenged my perspective of the world and pushes me to be a better person. It has been my honor
to get to know her this past year and watch her develop into a talented, intelligent, sweet, and
funny young woman. I’m so proud of her achievements and I look forward to continuing to watch
her progress through high school, college, and beyond.
vii
Chapter 1. Introduction - Roles of O-GlcNAc in metabolic
reprogramming of cancer cells
O-linked β-N-acetylglucosamine (O-GlcNAc) modification is a posttranslational modification
(PTM) on serine and threonine residues of more than 3,000 nuclear, cytosolic and mitochondrial
proteins.
1-4
Modification by the monosaccharide, GlcNAc, occurs by way of the enzyme O-
GlcNAc transferase (OGT) from the high energy donor substrate, uridine diphosphate β-N-
acetylglucosamine (UDP-GlcNAc) and is not further extended to more complex glycans. The
removal of this modification is catalyzed by O-GlcNAcase (OGA) and the dynamic cycling of
addition and removal, which has been compared to phosphorylation, plays a key role in several
regulatory pathways including cell cycle progression, cellular stress response, transcription,
protein-protein interactions, protein degradation, protein localization, etc.
1,3,5-7
Before the 1980s,
glycosylation referred to N-linked and O-linked glycosylation of cell surface and secreted
proteins and often consisted of large complex sugar structures during the protein maturing
process in the ER and Golgi apparatus. Instead, O-GlcNAc modification, discovered in 1984, is
the addition of a monomer covalently linked to the serine and threonine residues through a β-C2
linkage without further addition to multimeric glycan chains.
8
O-GlcNAc modification has been
observed in some bacteria, fungi and all metazoans.
Figure 1-1. O-GlcNAc modification of proteins. O-GlcNAcylation is a dynamic modification of
proteins in the cytosol, nucleus, and mitochondria. It is added to protein substrates by the enzyme,
O-GlcNAc transferase (OGT) and removed by the enzyme O-GlcNAcase (OGA).
1
Approximately 5% of glucose that enters the cell, is metabolized through the hexosamine
biosynthetic pathway (HBP), which results in the production of UDP-GlcNAc.
9
OGT changes its
effective KM for protein substrates at varying UDP-GlcNAc concentrations, enhancing the role of
O-GlcNAc modification as a nutrient sensor.
10
Like phosphorylation, global levels of O-GlcNAc
respond to intracellular and extracellular stimuli including insulin, nutrient levels and cellular
stress - including heat shock, and oxidative stress.
6,11-16
O-GlcNAc levels on numerous proteins
are elevated in response various cell stresses and this has been shown to be protective by
promoting survival.
17
In support of this, when O-GlcNAc levels are suppressed, cells are
sensitized to death, suggesting that this modification is a key regulator of the cellular stress
response.
6
The importance of the HBP is highlighted in the gene disruption of OGT, which is
required for embryonic stem cell viability.
2,18-20
Additionally, tissue-specific mutations knocking
down OGT in mice causes hyperphosphorylation of tau in neurons followed by cell death as well
as T-cell apoptosis leading to growth arrest in fibroblasts.
2,18-20
In Caenorhabditis elegans, ogt-
and oga- knockdown induces defects in nutrient storage as well as Dauer formation.
4,19
This modification has been shown to have implications in a variety of diseases including diabetes
and hyperglycemia, neurodegeneration and cancer. Many groups have shown that increased
expression of OGT and elevated O-GlcNAc modification occurs in several human cancers
including colorectal, pancreatic, breast, prostate, lung and non-solid chronic lymphocytic
leukemia.
21-28
Additionally, reduction in this modification results in a decreased tumor growth and
invasion in vitro and in vivo.
21,23,27
The increased O-GlcNAcylation in cancers cells indicates that
this modification is a key contributor to the transformation.
29,30
In fact, several metabolic changes
in cancer cells have been linked to O-GlcNAc modification. The diverse regulatory roles of O-
GlcNAcylation have been proposed to contribute to the complex link between cancer cell
metabolism and oncogenic cellular processes. In this chapter, I will discuss the link between
2
metabolic reprogramming in cancer cells and their corresponding changes in O-GlcNAc
modification. I will discuss the advancements in studying this complex modification as well as
the potential for therapeutic targets within the HBP.
Hexosamine Biosynthetic Pathway
O-GlcNAc modification is modulated by two enzymes, UDP-N-acetyl-β-D-glucamine/peptide N-
acetylglucosaminyltransferase (OGT) and N-acetyl-β-glucosaminidase (O-GlcNAcase/OGA).
Regulation of this modification is affected by the expression of OGT and OGA, the concentration
of the sugar nucleotide donor of OGT (UDP-GlcNAc), the availability of protein substrates and
targeting of the enzymes to their substrates. Flux through glucose, amino acid, fatty acid, and
nucleotide metabolic pathways feed into the hexosamine biosynthetic pathway resulting in the
synthesis of UDP-GlcNAc. Glucose that enters the cell is converted to glucose-6-phosphate by
hexokinase (HK), followed by the conversion to fructose-6-phosphate by the enzyme glucose-6-
phosphate isomerase. Approximately 3-5% of glucose flux enters into the HBP through the first
and rate limiting step by glutamine/fructose-6-phosphate amidotransferase (GFAT) which
converts fructose-6-phosphate to glucosamine-6-phosphate using glutamine.
9
While little is
known about the regulation of GFAT, high concentrations of UDP-GlcNAc appear to
allosterically inhibit GFAT and free radicals promote GFAT activity.
31,32
The intersection of this
pathway with acetyl-CoA, a product of glycolysis, generates N-acetylglucosamine. The pentose
phosphate pathway processes ribose sugar moieties and is fed into pyrimidine biosynthesis and
ultimately results in the addition of uridine-diphosphate for the synthesis of the final product of
the HBP, UDP-GlcNAc. Mutations enzymes involved in this pathway (emeg32 and pgm3) lead to
decreases in O-GlcNAc levels and are embyronic lethal, suggesting the importance of the
production of UDP-GlcNAc and ultimately, O-GlcNAc modification.
18,33
3
O-GlcNAc Transferase
OGT is a soluble protein localized in the nucleus, cytoplasm, and mitochondria of all tissues.
4,7,34-36
The gene for all three variants of OGT - short, mitochondrial (mOGT) and nuclear/
cytoplasmic - is on the Xq13 gene in the mammalian X chromosome.
4,34-36
However, mOGT has
an alternative targeting sequence on its N-terminus and overexpression of this variant induces
apoptosis.
37
Deletion of OGT is lethal in mice, Drosophila, and Arabidopsis and has been shown
to be essential for embryonic stem cell viability.
2,38-41
OGT contains two functional domains including several repeats of the N-terminal tetrapeptide
repeat (TPR) domain and a C-terminal catalytic domain.
34,35,42
The TPR domains, whose structure
was recently solved, contains an extended alpha helix and mediates protein-protein interaction
and enzyme activity.
43-46
The C-terminal catalytic domain contains two Rossman folds.
43
While
the mechanism of OGT substrate specificity remains poorly understood, deletions in the TPR
domain alter the ability of OGT to modify protein substrates. The current hypothesis is that
protein-protein interactions of OGT are formed from a series of OGT complexes with different
specificity for substrates.
47
In support of this, it is known that there is no exact consensus
sequence for the substrates of OGT and OGT has been found to associate with a number of
proteins including phosphatidylinosital (3,4,5)-triphosphate which helps in OGT translocation to
the plasma membrane during insulin signaling.
48

UDP and UDP-GlcNAc levels change the effective KM of OGT, and OGT activity is also
regulated by post translational modifications including phosphorylation, O-GlcNAcylation and
S-nitrosylation.
10,34,49,50
Not only do increases in UDP-GlcNAc increase OGT activity due to the
presence of abundant substrate, it may also alter substrate specificity. This means that different
groups of proteins will be modified by OGT under varying concentrations of UDP-GlcNAc.
10

4
Monitoring of OGT activity has been accomplished by using titrated UDP-GlcNAc (
3
H-UDP-
GlcNAc) to a peptide substrate or the use of a displacement assay using a fluorescent substrate
analog.
51
O-GlcNAcase
The glycoside hydrolase, O-GlcNAcase (OGA), is the enzyme that catalyzes the cleavage O-
GlcNAc from proteins. While OGT is largely found in the nucleus and cytoplasm, OGA
predominately exists in the cytoplasm of all tissues.
52
OGA is encoded on the gene meningioma-
expressed antigen 5 (mgea5) and can exist in two isoforms including a full-length isoform with a
histone acetyltransferase (HAT) domain and short isoform that lacks this C-terminal HAT
domain.
53
Little is known about the functions and localization of each isoform and as well as the
overall regulation of OGA activity. However, it is known that OGA is phosphorylated and O-
GlcNAcylated
5,54
. Despite the lack of knowledge on the regulation of OGA, the enzymatic
mechanism has been established in which the N-terminal domain contains hydrolase activity with
two aspartic acid residues used as acid catalysts.
52
O-GlcNAc and metabolic reprogramming in cancer
The “Warburg effect” is the phenomenon that occurs when cancer cells switch from the highly
efficient energy generating process, oxidative phosphorylation, to aerobic glycolysis even in the
presence of high oxygen.
55
This metabolic reprogramming in addition to the increased glucose
and glutamine uptake, provides cancer cells an outlet for the increased demand of biomass needed
for rapid proliferation.
56-58
The major factors that contribute to this metabolic switch include
hypoxia, oncogenes and mutated tumor suppressors which promote Warburg pathways by
activation of HIF-1α, PI3K/Akt/mTOR, and c-Myc.
58
For example, HIF-1α and c-Myc up
regulate GLUT1 and other glucose transporters which promotes the increased flux of glucose in
cancer cells.
59
Increased uptake of glucose allows tumors to be monitored using
18
F-fluoro-2-
5
deoxy-glucose (FDG) paired with PET/CT imaging.
57
This strategy has been used to diagnose
and observe therapeutic responses to various treatments in cancer patients. The increased
abundance of glucose not only increases glycolytic flux, but also flux through other biosynthetic
routes including the pentose phosphate pathway (PPP) and hexosamine biosynthetic pathway
(HBP). PPP contributes to nucleotide synthesis, which accounts for increased nucleotide synthesis
found in cancer. This pathway also generates NADPH which is essential in the redox balance of
increased levels of reactive oxygen species (ROS) found in rapidly proliferating cancer cells.
Oncogenes such as Kras up regulate glycolytic enzymes as well as glutamine/fructose-6-
phosphate aminotransferase (GFAT), which is the rate-limiting step of the HBP.
60
The
combination of increased glucose flux in cancer with the increased expression of key HBP
enzymes point to increased flux through the HBP in cancer cells.
Another metabolic alteration in cancer is increased uptake of glutamine.
61,62
Oncogenes like c-
Myc up regulate expression of transporters of glutamine
61,63,64
. This metabolite has a variety of
fates including conversion to oxaloacetate (OAA) to resupply the mitochondrial TCA
intermediate, partial oxidation to generate lactate and NADPH for energy production
(glutaminolysis) and finally it is the donor substrate in the conversion of fructose-6-phosphate to
glucosamine-6-phosphate by GFAT in the HBP.
58
Therefore, increased uptake of glutamine
contributes to the increased flux through the HBP, leading to increases in O-GlcNAc modification
in cancer cells.
The metabolic alterations in cancer cells including the addiction of glucose and glutamine,
present the hypothesis that cancer cells have increased O-GlcNAc modification by way of
increased flux through the HBP. This hypothesis is supported by the findings that O-GlcNAc
levels are increased in all examined cancer types including breast, lung, liver, and pancreatic
cancers.
21-27

6
The metabolic switch in cancer cells promotes changes in expression of enzymes involved in the
HBP, which may contribute to the increases in O-GlcNAc modification found in cancer. The rate
limiting enzyme in the HBP, GFAT, is increased by EGF in breast cancer (MDA-MB-468) and
tumor hypoxia and subsequently HIF-1α transcriptionally induces expression of GFAT.
65
Down
regulation of oncogenes like Kras and c-Myc in pancreatic cancer decreases GFAT expression,
indicating that changes in regulation of oncogenes paired with increases of glucose and glutamine
uptake contribute to increased HBP flux.
60

In addition to changes in GFAT expression, both OGT and OGA have been shown to be
deregulated in cancers. OGT expression levels are elevated and OGA expression levels are
reduced in cancer cells including breast, prostate, and pancreatic and tumor tissue from lung and
colon cancer.
21,23,27
These changes in expression levels contribute to the hyper-O-GlcNAcylation
in cancer cells. The recent crystal structure of OGT indicated that the limiting factor for
enzymatic activity was UDP-GlcNAc rather than the polypeptide substrate.
43
Therefore, by
increasing concentrations of UDP-GlcNAc through increased flux through the HBP, there would
be a corresponding increase in global O-GlcNAc levels through enhancement of OGT activity.
10

The reduction of OGA expression would prevent the removal of this modification, further
enhancing hyper-O-GlcNAcylation in cancer.
Large increases in O-GlcNAc levels are buffered via a negative feedback mechanism in normal
cells. These cells can respond to abnormal levels of O-GlcNAc by increasing or decreasing OGT
and OGA expression in order to normalize the modification.
10,44,66
However, in cancer cells, this
mechanism is bypassed and O-GlcNAc levels are not normalized leading to a transformed
phenotype in cancer cells.
66
In fact, when OGT is knocked down in a variety of transformed
cancer cells caused selective death compared to the non transformed counterpart cells. This
7
opens a therapeutic window for the inhibition of OGT to decrease tumor growth by blocking the
hyper-O-GlcNAcylated state.
21,27

Several studies have highlighted the importance of O-GlcNAc modification in the metabolic
reprogramming of cancer cells. It has been reported that many glycolytic enzymes are O-GlcNAc
modified including glucose-6-phosphate isomerase (GPI), phosphofructokinase (PFK1), fructose-
bisphosphate, aldolase A (ALDOA), alpha-enolase (Eno1) and pyruvate kinase (PK) and lactate
dehydrogenase (LDHA).
67-69
Specifically, PFK1 is O-GlcNAc modified at serine 529, which
inhibits its kinase activity in cancer cells.
70
This inhibition leads to build up of upstream
metabolic intermediates that are shunted into the PPP. Increased flux through the PPP generates
nucleotides for cancer cell proliferation and aids in combating oxidative stress through the
generation of NADPH.
71
Similarly, we have found that pyruvate kinase isoform 2 is O-GlcNAc
modified. Pyruvate kinase catalyzes the conversion of phophoenolpyruvate (PEP) to pyruvate and
has four isoforms, L, R, M1 and M2. M1 and M2 are splice variants of the PKM gene where M1
is expressed in adult muscle tissue, where M2 is expressed in proliferating cells and cancer cells.
While M1 is expressed as a constitutively active tetramer with high catalytic activity, M2 can be
expressed as either an active tetramer or a low-active dimer. The ability of cancer cells to adjust
their metabolic needs is dependent on the dimer-tetramer ratio of this key glycolytic enzyme.
O-GlcNAc promotes cell proliferation, and survival
In order to achieve infinite replication, cancer cells must acquire the ability to maintain
proliferative signaling as well as evade tumor suppressors.
29
These qualities are well known
hallmarks of cancer that promote the survival and growth of tumor cells. There are many changes
that occurs in order to achieve this, including oncogenic changes that promote cell cycle
progression while evading key checkpoints. The role of O-GlcNAc in the promotion of cell
proliferation and survival can be observed in the study that shows FOXM1 stability being
8
impacted by O-GlcNAc modification and therefore the reduction of O-GlcNAcylation in breast
cancer leads to a reduction in FOXM1. FOXM1 is a transcription factor responsible for the up-
regulation G1/S and G2/S transition cell cycle genes.
72
Other studies have shown that cell cycle
progression has been decreased in prostate and breast cancer due to the reduction of O-
GlcNAcylation. It has also been observed that Cyclin D1, a positive regulator of the G1/S
transition, is inhibited due to a reduction in O-GlcNAc modification.
27
These studies have shown
the direct impact of O-GlcNAc modification on the regulation of cell cycle proteins, which play a
key role in the promotion of cell proliferation.
Roles of O-GlcNAc in stress resistance
With rapid proliferation, many obstacles arise for cancer cells. Remarkably, transformed cancer
cells are able to deal with many physiological stresses and evade death
30
. These stresses include
nutrient stress, proteotoxic stress, oxidative stress and hypoxia. Many studies have shown that
hyper-O-GlcNAcylation combats stress and promotes pro-oncogenic cancers cells and cancer cell
survival. One major stress that cancer cells must deal with is reactive oxygen species (ROS). ROS
is the result of aerobic metabolism and can also be generated via extracellular milieu.
73
Over
production of ROS can be harmful and lead to oxidative stress when levels exceed the capability
of antioxidants. Oxidative stress results in damage to several cellular components including
proteins, nuclear and mitochondrial DNA, and intracellular lipids.
74
It is expected that cancer
cells must combat increased oxidative stress from production of excess ROS from increased
metabolism, rapid growth, and impaired mitochondrial function.
73
One key hallmark of cancer is
the adaptive ability to avoid cell death resulting from oxidative stress. Studies have shown that O-
GlcNAc modification of the rate limiting enzyme of glycolysis, phosphofructokinase-1 (PFK1) is
a link between the protective role of O-GlcNAc in cancer. When PFK1 is O-GlcNAc modified, its
activity is inhibited, resulting in increasing flux of fructose-6-phosphate into the pentose
phosphate pathway (PPP).
71
The PPP generates NADPH, which is a co-factor that is required for
9
the maintenance of reduced glutathione (GSH) pools. Reduced GSH is an essential molecule for
the reduction of oxidative stress and avoidance of ROS-induced cell death. Another study has
shown that FOXO4 is O-GlcNAc modified and this modification increases its transcriptional
activity.
75
FOXO4 is involved in the oxidative stress response, providing another link of increases
in O-GlcNAcylation to the reduction of oxidative stress.
Another type of stress that cancer cell must bypass is proteotoxic stress. In order for cells to
maintain homeostasis, they must balance proper protein folding, translocation, and aggregation.
76

There are several mechanisms by which cells can maintain proteostasis, including heat shock
response (HSR) proteins. The transcription factor heat shock transcription factor (HSF1)
regulates the expression of six groups of proteins: HSP100, HSP90, HSP70, HSP60, HSP40 and
small HSPs.
76,77
It has been shown that in response to proteotoxic stress, cancer cells up-regulate
these proteins.
30
The importance of this strategic mechanism to promote cancer survival has been
shown by targeting the HSR with small molecule inhibition and RNAi knockdown of various
HSPs, resulting suppression of tumor growth and increases in cancer cell apoptosis.
78
O-GlcNAc
has been demonstrated to increase the levels and activity of HSPs and plays a role in the
protection of cancer cells against proteotoxic stress.
6
In fact, treating cells with glutamine and
increasing flux to the HBP results in the increased expression of HSF1 in vivo.
13
Additionally, O-
GlcNAc modification of proteins that aggregate in diseases states, including tau, TAK1-binding
protein and α-synuclein, actually reduces the phenotype.
71,79,80
Therefore, O-GlcNAc plays key
roles in combating proteotoxic stress through several mechanisms like transcriptional induction of
HSP proteins and increasing stability of proteins that aggregate.
Roles of O-GlcNAc in invasion, angiogenesis, and metastasis
Studies have shown that hyper-O-GlcNAcylation contributes to several other hallmarks of cancer
including invasion, metastasis, and angiogenesis. Tumor invasion and metastasis have been
10
suggested to be critical functions of tumor progression and contribute to about 90% of cancer
mortality.
81
Increasing O-GlcNAc modification elevates in the invasion of both breast and liver
cancer cells and decreasing O-GlcNAc modification via knockdown of OGT has the opposite
effect in vivo and in vitro in breast and prostate cancer.
21-23,82
This effect is highlighted in the
study of epithelial to mesenchymal transition (EMT), which involves the reduction of epithelial
markers (E-cadherin) and induction of mesenchymal markers (Vimentin and N-cadherin) by way
of several transcription factors (Zeb1, Zeb2, Twist, Slug, and Snail).
83-85
Increases in O-
GlcNAcylation support EMT through corresponding decreases in E-cadherin expression and
increases in Vimentin in breast and liver cancer.
22,27,82

In order to maintain oxygen and nutrient delivery to the interior of tumors, as well as promote
invasion, angiogenesis must occur.
29,86
This is accomplished through pro-angiogenic factors,
including vascular endothelial growth factors (VEGF) and fibroblast growth factors (FGF), to
increase proliferation of endothelial cells and grow tumor blood vessels.
87
This hallmark of
cancer is also supported by increases in O-GlcNAcylation.
23
Not only does knockdown of OGT,
thereby reduction of O-GlcNAc levels, inhibit VEGF and angiogenesis, but it also suppresses
vital expression of matrix metalloproteinases MM-2 and MM-9 which are involved in tumor
stroma remodeling.
88,89

11
Figure 1-2. Known roles of O-GlcNAc modification of proteins in various hallmarks of cancer.
Hallmarks of cancer: metabolic, oxidative and proteotoxic stress, excessive cell proliferation,
invasion and metastasis, angiogenesis, infinite replication, and resistance of cell death. Proteins
that are O-GlcNAc modified are indicated by a pink circle. Proteins in blue have increases in
expression, stability or activity as a result of O-GlcNAcylation and proteins in purple denote
decreases.
Methods of studying O-GlcNAcylation
This chapter has highlighted the important role of O-GlcNAc modification in cancer by showing
the various contributions to several hallmarks of cancer. One major obstacle in studying this post-
translational modification was the lack of robust methods for visualization and identification. O-
GlcNAc modification is highly dynamic, chemically and enzymatically labile, sub-stoichiometric,
12
compartment specific, and dependent on cell cycle state and cell type. Therefore, traditional
biochemical methods have presented a variety of problems. O-GlcNAc lacks a formal charge and
does not cause a migrational changes in gel electrophoresis techniques like SDS PAGE. The
linkage of the sugar to residues is easily broken during most mass spectrometry (MS) analysis
during collision-induced dissociated (CID).
90
In addition to the MS limitations, O-GlcNAc
modification sites are difficult to identify due to the lack of consensus sequence, unlike other
post-translational modifications like N-linked glycosylation. Previous studies have used methods
involving detection of O-GlcNAc through its interaction with lectins. Lectin affinity columns
contain lectin proteins, like succinylated wheat germ agglutinin (sWGA), that have varying
binding specificity to sugars. sWGA has shown some success in the enrichment of protein
fragments containing O-GlcNAc modifications, including several transcription factors.
6,91,92

sWGA is more specific to O-GlcNAc than WGA , which binds to both sialic acid and O-GlcNAc,
due to the negative charge of sWGA at physiological pH.
93
However, this method has also shown
to enrich for other sugar moieties and is thus not specific of O-GlcNAc modification.
91
Another
method used to observe O-GlcNAcylation is pan specific antibodies. The most widely used
antibodies for O-GlcNAc modification detection by Western blotting are RL2 and CTD 110.1.
94,95
These antibodies are aimed toward peptide sequences or secondary structures that mimic O-
GlcNAc modification, but are not truly specific for O-GlcNAc as they have been shown to have
affinity for other types of glycosylation.
96
Both lectin and antibodies have been used for site
mapping and involve HPLC purification followed by Edman degradation to determine sites of
modification.
69,91
Low stoichiometries of O-GlcNAc modification and the complexity and
duration of these methods has proven arduous. Many new methods have arisen to circumvent
these obstacles including chemical probes used in metabolic and enzymatic labeling.
13
Enzymatic Labeling
One major development in studying O-GlcNAc modification was the use of chemoenzymatic
labeling. The enzyme, β-1,4-galactosyltransferase (GalT), catalyzes the addition of galactose to
sugar structures that contain a GlcNAc terminal residue from the donor substrate, UDP-galactose.
Originally, the Hart lab used this enzyme to label nuclear and cytoplasmic terminal glucosamine.
However, in 2003, Khidekel et al engineered a mutant version of this enzyme that tolerated more
substrates including a keto-derivative of UDP-galactose and the azide-bearing UDP-GalNAz that
was able to label O-GlcNAc modifications.
97
Both of these biologically inert moieties allow for
specific reactions with their biologically inert tag counterparts including an amino-biotin probe
for enrichment, or alkyne-rhodamine and alkyne-azo-biotin for fluorescent visualization or
enrichment and identification, respectively. The reaction of the keto group with an amino-probe
and the azide with the alkyne with either copper catalyzed or strain promoted azide alkyne
cycloaddition, provide a chemical linkage to a tag that can be used for direct detection and
identification of O-GlcNAc modification.
98
Specifically, tags containing biotin can be enriched
and detected using streptavadin bead enrichment and immunoprecipitation. Enriched proteins can
also be trypsonized and the peptides can be subjected to MS analysis for identification of O-
GlcNAc modified proteins. Fluorescent tags, like alkyne-rhodamine, allow for visualization of O-
GlcNAc modified proteins on SDS-PAGE using in-gel fluorescence microscopy. These methods
have been used by Tai et al to identify several O-GlcNAc modified proteins including CREB and
a variety of transcription factors and the UDP-GalNAz has been used to label proteins within live
cells by Clark et al.
98,99

One recent modification of this protocol was the installment of the keto-galactose derivative and
enrichment with a polyethylene glycol (PEG) tag. This long PEG chain (2 kDa) causes a gel
separation of multiply O-GlcNAcylated proteins, enabling the researcher to observe the
stoichiometry of O-GlcNAcylated proteins as well and quantities.
100
These chemoenzymatic
14
methods have provided great advancements in studies of O-GlcNAc modification dynamics,
regulation, and cross-talk with other post-translational modifications like phosphorylation. This
method can be coupled with mass spectrometry analysis to identify important O-GlcNAc
modified proteins under various stresses and conditions of cells.
Figure 1-3. Chemoenzymatic chemical reporter. Cell lystates are modified using UDP-GalNAz
and the mutant glycosyltransferase GalTY289L). Modified proteins can be visualized using
CuAAC with an alkyne rhodamine tag via in-gel fluorescence on SDS-PAGE.
Metabolic Labeling
An alternative method to the chemoenzymatic labeling method, metabolic labeling, takes
advantage of the promiscuity of salvage pathways and biosynthetic machinery to incorporate
analogs of biomolecules. Substrate analogs have been developed to selectively tag lipids,
proteins, oligosaccharides, lipids, nucleotide bases and ATP. These analogs generally display a
biologically inert chemical handle, like an azide or alkyne, used in bio-orthogonal reactions. The
first report of an O-GlcNAc chemical reporter was by V ocadlo et al, who developed
azidoacetylglucosamine (Ac4GlcNAz).
101
They showed that this O-GlcNAc analog could be
accepted by the GlcNAc salvage pathway and incorporated onto O-GlcNAc modified proteins.
The azide moiety could be reacted with a triphenyl phosphine tag via Staudinger ligation or an
alkyne tag with copper-catalyzed azide-alkyne cycloaddition (CuAAC) or “click” chemistry.
These tags can be fluorescent probes for visualization or affinity probes for enrichment and
identification. This method has proven to be non-invasive, robust and efficient to label O-
GlcNAcylation. Several modifications have been made to this technique including work in our
15
lab showing that reversing the orientation of the azide and alkyne reduces background and
making small chemical perturbations, like GlcNAlk, actually increases the probes selectivity for
O-GlcNAcylation over other forms of glycosylation like O-linked mucin type and N-linked
glycans.
102,103
This technique was also modified for in vivo studies where copper has shown to be
toxic. The use of copper can be avoided by using a cyclooctyne tag and the “click” reaction is
driven by strain.
Figure 1-4. Metabolic chemical reporter. Alkyne-modified proteins can be reacted with azide-tags
using CuAAC. Azide-rhodamine or azido-azo-biotin tags can be used for in-gel fluorescent
detection or affinity purification and identification of proteins, respectively.
β-Elimination followed by Michael addition (BEMAD)
Site identification of O-GlcNAc modification has been accomplished through the technique,
BEMAD, or β-elimination followed by Michael addition. This method involves the β-Elimination
of a hydrogen atom on the serine backbone carbon, generating an alkene intermediate. This is
followed by a Michael addition with dithiothreitol (DTT) or biotin pentamine to generate a mass
tag. When this method was used before proteomic analysis with MS, both the presence of O-
GlcNAc modifications and sites of modification were observed.
104
This method can be extended
in quantitative methods with the use of heavy and light DTT and allowed for differentiation of
phosphates, GlcNAc and nonspecific interactions.
105

16
Proteomic Analysis
Many of the mentioned techniques can be used to enrich for O-GlcNAc modified proteins and
these can be analyzed with MS to obtain a full glycoproteomics list. The chemoenzymatic and
metabolic labeling approaches isolate only O-GlcNAc modified proteins, reducing the complexity
of the subsequent MS/MS analysis. Some groups have also paired these techniques with
quantitative techniques such as SILAC and ICAT to elucidate peptide modification
stoichiometries.
Specifically, Kidekel et al have used the chemoenzymatic strategy with the keto UDP-galactose
tag to observe the O-GlcNAcylation dynamics in cortical neurons.
106
In this case, CAD and ETD
techniques were used for site identification. Similarly, Hart and co-workers studied biomarkser in
erythrocyte proteins from diabetic and normal patients using the UDP-GalNAz tag with an UV-
cleavable alkyne-biotin probe for enrichment followed by ETD based MS. This group identified
approximately 120 O-GlcNAcylated proteins.
107

Many other groups have utilized the metabolic labeling strategy to add to the growing list of the
O-GlcNAcylated proteome using MS.
67,103,108-110
Recently, our lab identified over 250 new O-
GlcNAc modified proteins through the comparison of selectivity and efficiency of several
chemical reporters including GlcNAz, GlcNAlk, GalNAz, and GalNAlk.
103
This study was
followed up with the development of 6Az-GlcNAc, which have shown to be more selective and
reliable for exclusively O-GlcNAc modification. This study found several new O-GlcNAc
modified proteins.
Inhibitors of O-GlcNAc cycling enzymes
One of the key ways in study the implications of post-translational modification is to be able to
manipulate the cycling enzymes. Unfortunately, OGT and OGA knockouts are not viable, so
17
alternative approaches have been made in order to manipulate O-GlcNAc levels and how these
changes effect various cell functions. One major advancement is the development of potent small
molecule inhibitors of both OGT and OGA.
Inhibitors of OGT have been difficult to discover due to the competition against endogenous
UDP-GlcNAc, which has nanomolar affinity. Another major problem is the chemical structures
and charges of these inhibitors that make them insoluble and unable to be transported into the
cell. The Walker lab gained some success through the development of a ligand displacement
assay that interrogated OGT. From this assay, they found a family of molecules with an
oxobenzo[d]oxazole core through modification of the catalytic base within the active site.
111,112

However, these compounds had poor solubility and the irreversible inhibition of OGT at this site
kills the enzyme, which may effect metabolism through back up in the HBP. The V ocadlo lab
synthesized an analog of O-GlcNAc, 5-thioglucosamine (5S-GlcNAc) that has been a successful
competitive inhibitor for OGT.
113
Further studies are required to detail the off-target effects.
The interest in OGA inhibitors grew due to the reciprocal relationship between O-GlcNAc and
phosphorylation in neurodegenerative diseases. It was shown by the V ocadlo lab that increases in
O-GlcNAc modification block aggregation of proteins like tau
114
. In these cases, increases in O-
GlcNAcylation and reduction in phosphorylation has a protective effect in brains suffering from
Alzheimer’s or Parkinson’s disease. Several inhibitors of OGA have been found with nano-molar
potencies, but suffer from selectivity problems in eukaryotes, limited chemical stability, and have
a difficult synthetic route. One such inhibitor, GlcNAcstatin, has potency against the bacterial
homolog for OGA, but has not been tested in eukaryotic cells. Similarly, gluco-nagstatin, was
active against OGA with a Ki of 420 nM. In vitro studies have shown that O-(2-acetamido-2-
deoxy-D-glycopyranosylidene) amino-N-phenylcarbamate (PUGNAc), has been shown to
increase O-GlcNAcylation through inhibition of OGA, and thereby decrease phosphorylation of
tau.
115,116
However, PUGNAc is not selective and is not readily bio-available. In order to improve
18
upon these inhibitors, the V ocadlo lab synthesized thiamet-G. This analog was devised through
analysis of the active site of OGA, whose mechanism involves substrate assistance from the 2-
acetamido group and formation of the intermediate, oxazoline. Additionally, an important
carboxyl residue in OGA catalytic site acts as a base by interacting with the proton of the amide.
This inhibitor has been shown to be selective and potent and has been used to increase O-GlcNAc
levels in several studies.
79,117

Figure 1-5. O-GlcNAc cycling enzyme inhibitors. Structure of some of the frequently used OGT
and OGA inhibitors.
Therapeutic potential
This chapter highlights a variety of pathways that O-GlcNAc plays both well understood and less
understood roles. Altered metabolism in cancer has direct implications in this modification, and
O-GlcNAcylation also seems to play a role in promoting the switch in cancer metabolism. This
19
sets up the hexosamine biosynthetic pathway, the synthetic pathway that results in the generation
of the donor substrate, UDP-GlcNAc, as an interesting therapeutic target. While OGT and OGA
may seem like an obvious first target, these enzymes have proven to be difficult to synthesize bio-
available, potent inhibitors. A more plausible target may be directed towards the rate limiting
enzyme of the HBP, glutamine/fructose-6-phosphate amidotransferase (GFAT). Our lab has
shown that cells treated with the irreversible inhibitor for glutamine utilizing enzymes, 6-diazo-5-
oxo-norleucine (DON), has been shown to induce apoptosis in cancer cell under oxidative stress.
While DON is not specific for GFAT, these results have inspired the synthesis of specific GFAT
inhibitors to determine the therapeutic potential of inducing apoptosis in oxidative stressed cancer
cells.
Conclusions
Here I have summarized the hallmarks of cancer and reviewed the link between O-GlcNAc
modification and these complex changes required for cancer cell survival. While much is still
unknown about the role of hyper-O-GlcNAcylation in cancer, many studies have shown strong
links to many hallmarks of cancer including combating stresses (oxidative, proteotoxic, and
metabolic), promotion of invasion and metastasis, promotion of excessive proliferation and
thereby promotion of cancer cell survival. I have also reviewed the technology developed to study
this modification. These methods continue to grow to enhance our understanding of this
important post translational modification so that the links to disease can be better understood.
20
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32
Chapter 2. Robust in-gel fluorescence detection of mucin-type
O-linked glycosylation
*
Mucin-type O-linked glycosylation is an abundant post-translational modification that occurs in
the secretory pathway of higher eukaryotes.
1
The modification 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 oligosaccharide structures that impact several biological processes including
protein structure, trafficking and cell-cell communication.
3
Accordingly, changes in mucin-type
glycosylation have been implicated in human diseases ranging from immunodeficiency to cancer.
2
Because of the importance of mucin-type O-linked glycosylation, several methods have been
developed for its visualization and identification.
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 for complex O-linked
glycans, which instead require the enlistment of several glycosidases that can unveil the core
GalNAc residue for recognition by Helix pomatia agglutinin.
7
Furthermore unlike N-linked
glycans, which can be selectively liberated by the enzyme Peptide N-Glycosidase F (PNGase-F),
there is no available 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 selective and can cleave other
post-translational modifications as well as degrade the parent protein.
9-13
33
*
Balyn W. Zaro contributed to the work presented in this chapter.
Figure 2-1. The GalNAc salvage pathway. UDP-GalNAc is synthesized from GalNAc by
GalNAc 1-kinase and UDP-GalNAc pyrophosphorylase. The donor sugar is then transported 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.
34
Bioorthogonal labeling reactions have emerged as an alternative to these approaches. Notably, the
Bertozzi laboratory has developed a metabolic labeling strategy which utilizes the fully-protected,
azide-bearing GalNAc chemical reporter Ac4GalNAz (Figure 2-1). Ac4GalNAz can enter cells,
where it is deacetylated, and converted to UDP-GalNAz by the enzymes of the GalNAc salvage
pathway, GalNAc 1-kinase and UDP-GalNAc phosphorylase.
14
This azide-bearing UDP sugar
donor is a good substrate for ppGalNAcTs resulting in addition of GalNAz onto serine and
threonine residues. GalNAz-modified proteins were first labeled with a FLAG-tagged phosphine
probe via Staudinger ligation.
15
This approach selectively labels azide-modified proteins for
visualization via immunoblotting techniques. 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
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 Staudinger
ligation for the identification of 18 glycoproteins via mass spectroscopy-based proteomics.
21
Recent studies have suggested that Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition (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 conditions 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
35
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.
21

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 hours, after which time cells were washed and lysed in 1% NP-40 buffer (1% NP40, 150 mM
NaCl, 50 mM TEA, pH 7.4). 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 hour [Alkynl
rhodamine (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•H2O (1 mM, 50 mM freshly prepared stock solution in water)]. At this time, the reaction
was quenched with 1 mL ice-cold methanol and placed at -80 ℃ for 2 hours to precipitate
proteins. The precipitated proteins were resuspended in 4% SDS buffer (4% SDS, 150 mM NaCl,
50 mM TEA, pH 7.4) and 2x SDS-free loading buffer (20% glycerol, 0.2% bromophenol blue,
14% 2-mercaptoethanol) was added. Samples were boiled for 5 min, and proteins were resolved
by SDS-PAGE. In-gel fluorescence scanning revealed robust labeling at all concentrations tested
(Figure 2-2B).
36
Figure 2-2. Fluorescent detection of mucin-type O-linked glycoproteins (A) The fluorescent 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 hours and
analyzed by in-gel fluorescent scanning. *The band visible at ~20 kDa is a contaminating
fluorescent molecular weight marker.
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
1-5,11
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
37
µM) for 0-12 hours. Following CuAAC, in-gel fluorescence demonstrated incorporation of the
chemical reporter in as little as 4 hours and significant labeling after 8 hours (Figure 2-3A). 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 fluorescence signal from GalNAz-modified proteins over time (Figure
2-3B). HEK293T cells were labeled with Ac4GalNAz (200 µM) for 16 hours after which time
cells were washed with PBS and media containing Ac4GalNAc (200 µM) was added. Cells were
treated for 0 - 72 hours, lysed, and subjected to CuAAC. In-gel fluorescence exhibited a steady
decrease in labeling over 3 days. Importantly this corresponds well with global cell surface
turnover experiments that were performed with radioactive methionine pulse-chase.
31
Figure 2-3. Characterization of Ac4GalNAz labeling. (A) Incorporation of Ac4GalNAz.
HEK-293T cells were labeled with 200 µM Ac4GalNAz for 0-12 hours and analyzed by in-gel
fluorescence scanning. (B) Pulse-chase analysis of Ac4GalNAz. HEK-293T cells were labeled
with 200 µM Ac4GalNAz for 16 hours after which time the media was replaced with media
containing 200 µM Ac4GalNAc for 0-72 hours. In-gel fluorescence scanning was then conducted.
38
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 fluorescent 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 through O-GlcNAc on serine and threonine residues.
21

Because of this interconversion, cellular fractionation would be required to selectively visualize
mucin O-linked glycoproteins or O-GlcNAc modified proteins. To determine if our conditions
also result in a mixture of azide labeled mucin and O-GlcNAcylated proteins, specific proteins
with known glycosylation patterns were analyzed. Specifically, we first visualized GalNAz
labeling of GlyCAM-IgG, a fusion protein containing both N-linked and mucin O-linked glycans.
GlyCAM-IgG was expressed in COS-7 cells treated with GalNAz or DMSO vehicle for 24 hours,
resulting in secretion 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 glycoprotein by GalNAz, consistent with
previous results (Figure 2-4A).
14
To determine 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 glycosylation at the
cell surface (Figure 2-4A).
14
39
Figure 2-4. 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 hours. In-gel fluorescence analysis followed. (B) Incorporation of Ac4GalNAz onto
FoxO1A. COS-7 cells expressing FoxO1A were treated with 200 µM Ac4GalNAz for 24 hours
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 at four sites.
33
FLAG-tagged FoxO1A was expressed in COS-7 cells
treated with Ac4GalNAz or DMSO vehicle for 24 hours. Cells were then lysed and FoxO1A
immunoprecipitated 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 2-4B). This result is contradictory to the report by the
Bertozzi lab; however, our conditions differ in labeling time (24 hours vs 3 days).
21
In addition,
we performed GalNAz labeling in media containing lower levels of glucose, and thus presumably
lower endogenous levels of UDP-GlcNAc, and observed conversion to GlcNAz and FoxO1A
labeling (Figure 2-4B).
34
Under these lower glucose conditions, we were able to visualize
40
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 other known modifications of FoxO1A including ubiquitination
35
and/
or phosphorylation.
36
Figure 2-5. 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 hours 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. Accordingly, NIH3T3, CHO,
COS-7, HEK-293T, HeLa, Mcf-7 and SH-SY5Y cells were treated with Ac4GalNAz (200 µM) for
41
16 hours. Following lysis and CuAAC, in-gel fluorescence scanning revealed distinct labeling
patterns and intensities for all cell lines tested (Figure 2-5). These data demonstrate that GalNAz
in combination with our CuAAC conditions and fluorescent tag can monitor changes in mucin-
type glycosylation that are key to understanding cellular processes.
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 GalNAz to robustly visualize mucin-type O-linked glycoproteins. In contrast to previous
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 hours. In addition, at higher concentrations
(200 µM) GalNAz labeling can be visualized in as little as 4 hours, potentially enabling the
analysis of dynamic changes in mucin glycoproteins on short timescales. We next conducted a
pulse-chase experiment in order 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
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 metabolic fate of GalNAz under our
conditions, we used known glycoproteins. We first exploited the chimeric fusion protein
GlyCAM-IgG to confirm preferential labeling of mucin type O-linked glycans with GalNAz.
Consistent with previous reports, GalNAz treatment resulted in robust labeling of GlyCAM-IgG
and pretreatment of the sample with PNGase-F did not result in a significant decrease in labeling,
42
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 selectivity to
short treatment times (16 to 24 hours) when compared to previous results (3 days).
14
While it is
possible that GalNAz may label other O-GlcNAc modified proteins under our labeling
conditions, we suspect that this is unlikely, as FoxO1A is a mutant protein designed to be
constitutively and heavily glycosylated when compared to other O-GlcNAc substrates. Finally,
we explored the generality of our in-gel fluorescence detection. 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.
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 modification levels and patterns, which are not readily
determined using published immunoblotting 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.
43
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32. Daude, N. et al. Molecular cloning, characterization, and mapping of a full-length cDNA
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47
Chapter 3. Analysis of N-propargyloxycarbamate
monosaccharides as metabolic chemical reporters of
carbohydrate salvage pathways and protein glycosylation
*
Metabolic chemical reporters of glycosylation allow for the visualization and identification of a
variety of glycoconjugates by taking advantage of the promiscuity of carbohydrate metabolism.
Here, we describe the synthesis and characterization of metabolic chemical reporters bearing an
N-propargyloxycarbonyl (Poc) group that creates discrimination between glycosylation pathways.
An ever expanding repertoire of bioorthogonal reactions has enabled the specific labeling of
reporter molecules in a range of biological contexts.
1-3
In many applications, 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 anolog of N-acetyl-mannosamine (ManNAc) termed N-azidoacetyl-mannosamine
(ManNAz), which was developed for the visualization of sialic-acid-containing carbohydrates
upon reaction with an immuno-tag using the Staudinger ligation.
4
Inspired by this result, a variety
of other metabolic chemical reporters have been developed to target sialic acid modification
5-7
,
mucin O-linked glycosylation,
8,9
fucosylation,
7,10
and intracellular O-GlcNAc modification (O-
GlcNAcylation).
11,12
These chemical reporters take advantage of carbohydrate scavenging
pathways that convert them into the corresponding nucleotide sugar-donors for utilization by
glycosyltransferases. Until recently, these 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).
13

48
*
Balyn W. Zaro (USC) and Kelly N. Chuh (USC) contributed to the work presented in this chapter.
However, cells are armed with metabolic pathways that can enzymatically interconvert
monosaccharides and uridine diphosphate (UDP) sugar donors. For example, N-acetyl-
glucosamine (GlcNAc) can be reversibly converted to both N-acetyl-galactosamine (GlcNAc)
14

and N-acetyl-mannosamine (ManNAc).
15
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 labeling
of a combination of mucin O-linked, O-GlcNAc, and some N-linked glycans.
12,16,17
While these
different types of glycosylation can be separated using biochemical methods, a more ideal
metabolic chemical reporter would specifically read-out on only one type of glycosylation.
Because each of the carbohydrate 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 identification of O-GlcNAc modified proteins.
12
Herein, we continue
to examine the chemical tolerance of mammalian glycosylation pathways through the synthesis
and characterization of the N-propargyloxycarbonyl (Poc) containing chemical reporters
GlcPoc
18
, GalPoc, and ManPoc
19
(Figure 3-1A). Interestingly, we find that each chemical reporter
displays unique labelling efficiencies and that qualitatively GlcPoc and GalPoc are incorporated
into the same proteins while ManPoc labels a different pattern. Finally, 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.
49
Figure 3-1. N-propargylcarbamate containing metabolic chemical reporters are incorporated onto
proteins. (A) Metabolic chemical reporters Ac4GlcPoc: N-propargylcarbamate-1,3,4,6-tetra-O-
acetyl-glucosamine, Ac4GalPoc: N-propargylcarbamate-1,3,4,6-tetra-O-acetyl-galactosamine,
Ac4ManPoc: 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.
To test the ability of Poc-modified monosaccharides to transit carbohydrate salvage pathways and
serve as substrates for glycosyltransferases, the hydrochloride salts of glucosamine,
galactosamine, and mannosamine were reacted with propargylchloroformate. The remaining free
hydroxyl groups were subsequently acetylated to give Ac4GlcPoc, Ac4GalPoc, and Ac4ManPoc
(Fig. 3-1A). NIH3T3 cells were metabolically labeled with each compound at 150 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 the Cu(I)-
50
catalyzed azide-alkyne cycloaddition (CuAAC). In-gel fluorescent scanning showed labeling of a
variety of proteins with all three chemical reporters (Fig. 4-1B). ManPoc labels cells at the
highest level followed by GlcPoc and finally GalPoc. Interestingly, GlcPoc and GalPoc
qualitatively label 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 the 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 (Fig. 3-4A).
All three reporters labeled proteins at concentrations as low as 50 µM.
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 (150 µM), or Ac4ManPoc (150 µM) for
different amounts of time. Fluorescent modification using CuAAC and in-gel fluorescence
demonstrated incorporation of all three chemical reporters in as little as 4 h (Fig. 3-4B),
consistent with labeling times of other reporters including GlcNAz and GlcNAlk. Additionally,
metabolic chemical reporters can be used to determine the stability of a modification or protein in
a pulse-chase format by measuring the decrease in fluorescence signal over time. Accordingly,
NIH3T3 cells were treated with Ac4GlcPoc (150 µM), Ac4GalPoc (150 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 or Ac4ManNAc) at 150 µM
was added. Cells were treated for 0-72 h before analysis by in-gel fluorescence as above (Fig.
3-4C). Interestingly, GlcPoc and GalPoc displayed similar signal decay rates, again suggesting
51
that they label the same protein substrates. In contrast, loss of ManPoc was somewhat slower,
supporting its incorporation into a different type of glycosylation.
We further analyzed the efficiency of the Poc metabolic chemical reporters using flow-cytometry.
Accordingly, NIH3T3 cells were treated with each chemical reporter at 150 µM 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 3-2). Again this demonstrates that the Poc group is differentially tolerated by the different
carbohydrate salvage pathways and/or glycosyltransferases.
52
Figure 3-2. Flow cytometry analysis of metabolic chemical reporter incorporation. NIH3T3 cells
were treated with each chemical reporter (150 µM) for 16 hours. Cells were then fixed with 3.7%
PFA and permeabilized with 0.1% Triton X-100 before reaction with azido-rhodamine under
CuAAC conditions and analysis by flow cytometry. Error bars represent S.E.M. of three
experiments.
Finally, we used each chemical reporter to enrich and identify specific proteins with known
glycans, thereby unambiguously determining which type(s) of glycosylation, O-GlcNAcylation or
cell-surface (e.g. N-linked and mucin O-linked) each reporter labels. To examine the O-GlcNAc
modification pathway, NIH3T3 cells were treated with Ac4GlcPoc (150 µM), Ac4GalPoc (150
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).
Labelled proteins were enriched with streptavidin beads, washed, and eluted with sodium
53
dithionite. Western blot analysis of these enriched proteomes was then performed using an
antibody against a known O-GlcNAc substrate NEDD4 (Fig. 3-3).
12
Interestingly, NEDD4 was
enriched by all three chemical reporters, with GlcPoc and ManPoc displaying approximately 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 3-3. Incorporation of metabolic chemical reporters into the O-GlcNAcylation pathway.
NIH3T3 cells were treated with each chemical reporter (150 µM) for 16 hours. Labelled proteins
were reacted with azido-azo-biotin using CuAAC before streptavadin enrichment and analysis of
O-GlcNAc modification by anti-NEDD4 Western blotting.
The continually expanding range of metabolic chemical reporters of glycosylation have enabled
the visualization and identification of many types of glycans and underlying protein substrates.
We and others have demonstrated that many chemical reporters can be metabolically transformed
and therefore label multiple glycosylation 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 developed 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 pattern of GlcPoc and GalPoc labeled proteins are approximately identical,
suggesting that they are incorporated into the same type of glycosylation. In contrast to the azide-
bearing chemical reporter GalNAz when compared to GlcNAz,
16,20
GalPoc is utilized at a much
54
lower efficiency than GlcPoc, consistent with our previous analysis of the structurally similar
alkyne-containing derivative GalNAlk.
12
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,
12,21
demonstrating 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 overall levels of ManPoc
incorporation and its distinct labeling pattern, we believe that only a minority of ManPoc enters
the O-GlcNAcylation pathway, while the majority is transformed to sialic acid, supporting
previous in vitro experiments.
19
55
Fig. 3-4. 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 fluorescence 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.
56
Materials and Methods
All reagents used for chemical synthesis were purchased from Sigma-Aldrich unless otherwise
specified and used without further purification. All anhydrous reactions were performed under
argon atmosphere. Analytical thin-layer chromatography (TLC) was conducted on EMD Silica
Gel 60 F254 plates with detection by potassium permanganate (KMnO4), anisaldehyde or UV . For
flash chromatography, 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.
Synthesis of chemical reporters
Known compounds, az-rho, and azido-azo-biotin were synthesized according to literature
procedures.
22,23
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 chloroformate (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 and 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. Purification by silica gel
column chromatography (45:55, ethyl acetate:hexanes) afforded the product (853.2 mg, 52%
yield) as a white solid.
1
H NMR (600 MHz, CDCl3) δ 5.63 (d, J = 8.3 Hz, 1H), 5.11 (t, J = 9.8
Hz, 1H), 5.05 (t, J = 9.6 Hz, 1H), 4.90 (d, J = 8.1 Hz, 1H), 4.59 (s, 1ifH), 4.22 (dd, J = 12.5, 4.4
Hz, 1H), 4.05 (dd, J = 12.4, 1.7 Hz, 1H), 4.00 (t, J = 6.7 Hz, 1H), 3.87 (dd, J = 19.0, 9.4 Hz, 1H),
57
3.75 (dd, J = 9.9, 4.6, 2.2 Hz, 1H), 2.39 (s, 1H), 2.06 (s, 3H), 2.02 (s, 3H), 1.99 (s, 3H), 1.97 (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.
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) 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 (20:80, methanol, methylene chloride). Resulting product 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 temperature. Reaction mixture was concentrated and pyridine
was removed. Extracted with 1 M HCl, saturated sodium bicarbonate, water and brine.
Purification by silica gel column chromatography (45:55, ethyl acetate: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.
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 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
58
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 (15:85, methanol, methylene chloride). 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 pyridine was removed. Extracted with 1 M HCl, saturated sodium
bicarbonate, water and brine. Purification by silica gel column chromatography (50:50, ethyl
acetate:hexanes) afforded the product (123.5 mg, 86% yield) as a white solid.
1
H NMR (500
MHz, CDCl3) δ 6.03 (d, J = 1.6 Hz, 1H), 5.28 – 5.17 (m, 1H), 4.97 (dd, J = 9.8, 3.9 Hz, 1H), 4.69
– 4.60 (m, 1H), 4.28 (dd, J = 8.5, 3.4 Hz, 1H), 4.23 – 4.15 (m, 2H), 4.07 – 3.93 (m, 3H), 2.44 (t,
J = 2.2 Hz, 1H), 2.04 (s, 6H), 1.99 (s, 6H).
13
C NMR (125 MHz, CDCl3) δ 170.66, 170.07, 169.59,
168.09, 155.06, 91.70, 75.10, 73.34, 70.17, 69.00, 65.27, 62.00, 53.03, 51.27, 20.83, 20.71, 20.60.
Cell culture
NIH3T3 cells were cultured in high glucose DMEM media (Cellgro) with 10% fetal calf serum
(FCS, Cellgro) and were maintained in a humidified incubator at 37 ℃ 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 experiments, 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 ℃ for 2 min at 2,000 x
g, followed by washing with PBS (1 mL) two times. Cell pellets were then resuspended and lysed
59
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 centrifugation at 4 ℃ 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)
Soluable cell lysate (200 µg) was diluted with cold 1% NP-40 lysis buffer to a concentration 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 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 proteins 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 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).

60
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.
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
normalized 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
℃ 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 that were pre-washed beads were 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, 2x in PBS (1 ml) and 2x
with 1% SDS in PBS buffer (2,000 x g, 2 min). Beads were then incubated in 25 µL of sodium
dithionite solution (1% SDS, 25 mM sodium 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
61
elution 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% bromophenol blue, 1.4% β-mercaptoethanol) was then added. The samples were boiled for
5 min at 98 ℃, and 60 µg of protein was then loaded per lane for SDS-PAGE separation (Any kD
Criterion Gel, Bio-Rad).
Western Blotting
Proteins were separated by SDS-PAGE before being transferred to PVDF membrane (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 appropriate primary antibody in blocking buffer overnight at 4
℃. 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. 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).
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 permeabilized (0.1% Triton X-100 in PBS for 10
62
min at room temperature). Cell were washed with PBS and blocked for 10 min with 2% FCS in
PBS. Cells were resuspended 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.
63
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66
Chapter 4. An Alkyne-Aspirin Chemical Reporter for the
Detection of Aspirin-Dependent Protein Modification in Living
Cells
*
Introduction
Aspirin, a non-steroidal antiinflammatory drug (NSAID), is one of the most common small-
molecule treatments in the world, and has been utilized in different 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.
3,4

Specifically, the acetylation occurs in a 1:1 stoichiometry 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,
5,6
and more recently, a variety of observational studies and
trials have demonstrated that chronic aspirin use greatly reduces the incidence of cancer and
cancer mortality, with the largest decrease in gastrointestinal cancers.
7-12
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,
8,13-16
aspirin is known to modify other proteins by chemical transfer
of its acetate group to amino acid side-chains.
17
This modification, which we have termed aspirin-
67
*
Balyn W. Zaro (USC) and Stephanie M. Miller (USC) contributed to the work in this chapter.
dependent acetylation, raises the possibility that other protein modification events could
contribute to the pharmacological effects of aspirin. The first experiments 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,
18,19
followed by in vivo demonstration using similar techniques.
20,21

Additional studies demonstrated that the acetylation occurs on the ε-amine side-chains of lysine
residues on fibrinogen.
22
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.
23
Finally, specific antibodies
were used to identify acetylation on lysine 382 of p53 resulting from aspirin treatment of MDA-
MB-231 cells.
24
Aspirin treatment increased p53 nuclear localization and expression of the target
gene p21
CIP1
, although acetylation of 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 aspirin treatment and enabled the enrichment and
identification of 33 of these proteins using mass spectrometry.
25
However, this approach
necessarily suffers from contamination by endogenous lysine-acetylated proteins and no
identification 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)-
68
catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC), have been used in a variety of contexts to
overcome the limitations of traditional biological technologies (e.g. antibodies).
26-28
In fact,
alkyne-bearing probes have been applied to investigate protein glycosylation, acetylation,
methylation, lipidation, other posttranslational modifications, and covalent small molecule
inhibitors.
29-40
Herein, we describe the synthesis and characterization of the CuAAC compatible
aspirin-analog containing an alkyne (AspAlk). AspAlk allowed for the robust fluorescent
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 120 potentially
aspirin-acetylated proteins, representing a range of biological functions.
Results and Discussion
To generate our chemical reporter of aspirin-dependent acetylation, we reasoned that it should
structurally mimic aspirin as closely as possible. The smallest structural perturbation would
incorporate a 3-butynoic ester onto salicylic acid to generate 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
41
and subsequently reacted with salicylic acid to
yield AspAlk (Figure 4-1A). 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),
40
enabled robust visualization of a large variety of proteins (Figure 4-1B). 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
69
protein abundance as judged by Coomassie blue staining (Figure 4-5), suggesting that
modification occurs on specific protein-substrates that are expressed in most cell-lines.
Figure 4-1. AspAlk is a chemical reporter of aspirin-dependent protein modification (A)
Reagents: (a) N,N’-dicyclohexcylcarbo-diimide (DCC), CH2Cl2, 16 hours; (b) salicylic acid,
pyridine, 16 hours, 57% over two steps. (B) The indicated panel of cell-lines were treated with
AspAlk (1 mM) for 6 hours before CuAAC with az-rho, analysis by in-gel fluorescence scanning.
Coomassie blue shows protein loading.
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.
40
In-gel fluorescent scanning showed dose-dependent
70
labeling of a variety of proteins in as little as 50 to 100 µM (Figure 4-2A), which represents a
notable increase in sensitivity over a published immunoblotting method.
24,25
Importantly, a range
of aspirin concentrations used (100 to 300 µM) is achievable in the plasma of patients treated
with a short analgesic dose (600 mg) and others undergoing chronic aspirin-treatment of
rheumatoid arthritis.
42-44
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 phenotypic
cell-based experiments.
45,46
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 concentration of aspirin (250 µM) before CuAAC
with az-rho and analysis by in-gel fluorescence. Co-treatment with aspirin resulted in an
approximately 50% reduction in labeling signal (Figure 4-2B), suggesting that AspAlk modifies
the same proteins as aspirin.
Figure 4-2. Characterization of AspAlk labeling in HCT-15 colorectal cancer cells. (A) HCT-15
cells were labeled with the indicated concentrations of AspAlk for 6 hours before reaction with
az-rho and analysis by in-gel fluorescence 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. Coomassie blue shows protein loading.
71
A significant property of many chemical reporters is the ability to visualize labeling dynamics in
a pulse-labeling experiment. To visualize the rate of AspAlk labeling, 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 4-3A), potentially representing an improvement
compared to previous Western-blotting based reports, where 8 to 12 hour treatments were used.
45,46
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 4-3B). To examine this in more detail we treated HCT-15 cells as above,
followed by harvesting after shorter lengths of time (Figure 4-3C). As in the previous pulse-chase,
more than half of the fluorescent signal was progressively lost within the first 12 hours. This
fraction of signal is consistent with other chemical reporters of enzymatically-reversible protein
acylation pathways
47
; however, the persistence of the remaining signal suggests that at a
percentage of AspAlk modifications are not removed by protein deacetylases.
72
Figure 4-3. Kinetic analysis of AspAlk labeling. (A) HCT-15 cells were treated with AspAlk (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 hours 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.
Finally, to we used AspAlk to identify proteins that are potential substrates of aspirin-dependent
acetylation. HCT-15 cells were treated in triplicate with AspAlk (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 (not necessarily overlapping) in each experimental replicate, (2) a total
of at least three spectral counts in the AspAlk-treated samples, (3) the average spectral counts for
each identified protein in the AspAlk-treated sample must have been at least 3-times greater than
the corresponding average counts int he DMSO negative control and (4) a p-value of at most 0.05
73
(t test). Proteins meeting these conditions were then rank-ordered based on enrichment-ratio and
the average number of spectral counts in the AspAlk-treated sample (Table 4-1). We identified
120 proteins with diverse cellular functions, which importantly contained eight proteins
previously known to be acetylated by aspirin (Figure 4-4A).
18,19,25,48
In this list of identified
proteins were several of the core histone proteins, including H2B, and H3. Given the crucial role
of histone acetylation in transcriptional regulation,
49,50
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 4-4B).
Figure 4-4. Identification AspAlk labelled proteins. (A) HCT-15 cells were treated in triplicate
with AspAlk (1 mM) for 2 hours before reaction with azido-biotin under CuAAC conditions, on-
bead trypsinolysis, and proteomic identification by LC-MS/MS. Representative proteins are
indicated by name. (B) Histones were enriched from HCT-15 cells treated with either AspAlk (1
mM) or DMSO vehicle for 6 hours, followed by CuAAC with az-rho and in-gel fluorescence
scanning. Coomassie-blue staining shows protein loading.
Conclusions
Aspirin-dependent acetylation and subsequent inhibition of cyclooxygenase enzymes (Cox-1 &
-2) is well established as one molecular mechanism contributing to cardioprotection and
74
chemoprevention.
8,13,14,16,51,52
Notably, aspirin is known to acetylate a variety of additional
proteins, giving it the potential to simultaneously affect multiple other cellular pathways that
could also contribute to the effects of chronic aspirin-treatment. However, understanding the
molecular consequences of aspirin-dependent acetylation has been hampered by a lack of tools to
visualize and identify protein targets. To enable the robust visualization and identification of these
modifications, we have developed an aspirin chemical-reporter (AspAlk) that transfers an alkyne
functionality to proteins that can be subsequently 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
proteins 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. AspAlk 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.
36
As noted above,
aspirin concentrations in the plasma of patients receiving an analgesic dose (600 to 650 mg) can
reach 100 to 300 µM.
42-44
However, to our knowledge, the concentrations of aspirin in the
gastrointestinal tract have not been precisely measured, and millimolar concentrations (2.5 to 10
mM) of aspirin have been routinely used in cell-culture experiments in the past.
45,46
Additionally,
other studies have found that repetitive administration of aspirin results in a disproportionately
large increase in serum levels
53
Therefore, although 1 mM AspAlk is somewhat higher than the
observed plasma concentrations, we utilized it to maximize our labeling efficiency 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
75
previously reported studies using anti-acetyl lysine antibodies, which treated cells for 12 hours
with aspirin.
24,25
Crucially, our short labeling-times correspond well to the measured stability of
aspirin in the plasma of human patients.
44
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 protein acylation.
47
However some in-gel
fluorescent signal persisted over 48 hours and could represent protein degradation and/or dilution
through cell division.
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 substrates as possible that could be validated in cell-culture and animal
model experiments in the future. Finally, we validated our proteomics data by visualizing the
aspirin-dependent acetylation of several core histones, suggesting a possible role for aspirin in
transcriptional regulation. 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 proteins 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 aspirin in
cardioprotection and chemoprevention.
76
Experimental Procedures
General Information. All reagents used for chemical synthesis were purchased from Sigma-
Aldrich unless otherwise specified and used without further purification. All anhydrous reactions
were performed under argon atmosphere. Analytical thin-layer chromatography (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 VNMRS-600 at 600 and 125 MHz. Chemical shifts are
recorded in ppm (δ) relative to solvent. Coupling constants (J) are reported in Hz.
Synthesis of 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 hours at room temperature under an argon
atmosphere. The reaction mixture was then diluted with CH2Cl2 (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 stirring for 16 hours 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 NMR (600 MHz, CDCl3)
δ 8.00 (dd, J = 7.8, 1.7 Hz, 1H), 7.63 – 7.56 (m, 1H), 7.34 (td, J = 7.6, 1.1 Hz, 1H), 7.13 (dd, J =
8.1, 1.1 Hz, 1H), 2.82 (dd, J = 8.2, 6.8 Hz, 2H), 2.60 – 2.54 (m, 2H), 2.30 (t, J = 2.7 Hz, 1H).
13
C
NMR (125 MHz, MeOH) δ 171.13, 170.64, 166.46, 150.89, 133.85, 131.56, 126.08, 123.71,
82.12, 69.20, 33.36, 13.85. ESI-MS calculated for C12H10O4 [M+Na]
+
241.05, found 240.00.
77
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% fetal 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.
Preparation of 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 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 protease inhibitor cocktail
(Roche Biosciences) for 15 min and followed by centrifugation 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 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 concentration 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
78
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 hour. Upon completion, 1 mL of ice cold methanol was
added to the reaction, and proteins were precipitated at -20 ℃ for 2 hours. The reactions were
then centrifuged at 4 ℃ for 10 min at 10,000 x g. The supernatant was removed, 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 ℃, 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 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 azido-PEG3-biotin (5 mM, Click Chemistry Tools) for 1 hour, after 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
79
mL ice-cold MeOH, with resuspension of the pellet each time. The pellet was then air-dried for 1
hour. 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, 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 hours. 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 resuspension 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, leaving
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 (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
centrifugation 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/MS Analysis. Extracted 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
80
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 chromatography 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
MASCOT 2.3.02 (Matrixscience, London, UK). Peptides fulfilling a Percolator calculated 1%
false discovery rate threshold were reported. All LC-MS/MS analysis were carried out at the
Proteomics Resource Center at The Rockefeller University, New York, NY , USA. A total of 810
proteins were identified, with 120 fulfilling our criteria as a “hit.” A list of all identified peptides
and proteins will be made available in Excel format upon request.
Acid extraction of histones. HCT-15 cells were treated with DMSO or 1 mM AspAlk for 6
hours. 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 cycles 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 resuspended in 0.4 N H2SO4 and agitated overnight
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 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 resuspended in water.
Concentration was determined by BCA Assay and normalized with 1% NP-40 lysis buffer [1%
81
NP-40, 150 mM NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete Mini protease
inhibitor cocktail] for CuAAC.
Figure 4-5. AspAlk labels proteins in a variety of cell lines. The indicated cell-lines were
incubated with AspAlk (1 mM) for 6 hours before CuAAC with az-rho and analysis by in-gel
fluorescent scanning. Coomassie blue staining shows protein loading.
82
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Appendix
MS Table
1
H and
13
C NMR spectra
109
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
110
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
111
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
112
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
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).
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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).
114
115
O
AcO
AcO
NH
OAc
OAc
O
O
GlcPOC
116
O
AcO
AcO
NH
OAc
OAc
O
O
GlcPOC
117
O
OAc
AcO
NH
OAc
OAc
O
O
GalPOC
118
O
OAc
AcO
NH
OAc
OAc
O
O
GalPOC
119
ManPOC
O
AcO
AcO
HN
AcO
OAc
O
O
120
ManPOC
O
AcO
AcO
HN
AcO
OAc
O
O
121
AspAlk
122
AspAlk

Abstract (if available)

Abstract Post translational modifications (PTMs), including glycosylation and acetylation, have a wide variety of implications in cells. My goal was to explore the molecular consequences of PTMs using chemical approaches to reveal pathways that are important to human disease, including cancer. This manuscript uncovers a variety of chemical probes that have been used to study mucin O‐linked glycosylation, O‐GlcNAc modification, and aspirin‐dependent acetylation.

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Asset Metadata

Creator Bateman, Leslie Anne (author)

Core Title Using chemistry to reveal the consequences of post translational modifications in cancer

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 07/14/2014

Defense Date 06/20/2014

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

Tag acetylation,cancer metabolism,chemical biology,chemical probes,glycosylation,OAI-PMH Harvest,O‐GlcNAc,post translational modifications

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Pratt, Matthew R. (committee chair), Louie, Stan G. (committee member), Zhang, Chao (committee member)

Creator Email labatema@usc.edu,leslie.bateman@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-438440

Unique identifier UC11286360

Identifier etd-BatemanLes-2670.pdf (filename),usctheses-c3-438440 (legacy record id)

Legacy Identifier etd-BatemanLes-2670.pdf

Dmrecord 438440

Document Type Dissertation

Format application/pdf (imt)

Rights Bateman, Leslie Anne

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

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA