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THE SYNTHESIS AND EVALUATION OF DIAzo FUNCTIONALIZED SOLID
SUPPORTS AND THEIR APPLICATION TOWARD THE IDENTIFICATION OF
PHOSPHORYLATED PROTEINS
By

Samantha M. Frawley Cass

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

DOCTOR OF PHILOSOPHY
Chemistry

2009

In)

SI

ABSTRACT

THE SYNTHESIS AND EVALUATION OF DIAZO FUNCTIONALIZED SOLID
SUPPORTS AND THEIR APPLICATION TOWARD THE IDENTIFICATION OF
PHOSPHORYLATED PROTEINS

BY

Samantha M. Frawley Cass

Many cellular processes are regulated by the reversible process of protein
phosphorylation/dcphosphorylation on serine, threonine and tyrosine residues.
Deregulation of the signal transduction cascade upsets this well-balanced system
and is implicated in diseases such as cancer, type II diabetes, cystic fibrosis, and
Alzheimer’s disease. Even though the human genome maps out the structure
and sequence of our genes, it does not provide any insight into post-translational
modifications, such as phosphorylation, which regulate cell function. In order to
understand cellular regulation on a molecular level, post-translational
modifications need to be identified and characterized. Unfortunately, proteomic
techniques relevant to the elucidation of signal transduction pathways have
lagged in development in comparison to genomic technologies. Despite recent
advancements in mass spectrometry techniques, characterization of protein
phosphorylation is often hampered by their low-abundance and signal
suppression. Enrichment of phosphorylated peptides/proteins prior to their
introduction into a mass spectrometer can therefore significantly facilitate
phosphoprotein analysis. Here, we describe a method for the enrichment of

phosphorylated serinelthreonine and tyrosine containing substrates using a

 

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unique solid phase enrichment technique. The method takes advantage of
diazo-functionalized solid phase resins, which react with the phosphate moiety
allowing for non-phosphorylated peptides to be efficiently removed by filtration.
Liberation of the phosphorylated substrates from the resin using aqueous acid
provides the products, which can be readily analyzed and sequenced by tandem
mass spectrometry. The technique described herein has successfully enriched
the phosphorylated substrates from B-casein and ovalbumin digests, in low-
picomolar quantities and is found to be competitive with the current commercially
available enrichment methods. Further application of this phosphopeptide

enrichment technique towards a purified cellular extract will also be discussed.

Copyright by
SAMANTHA M. F RAWLEY CASS
2009

I dedicate this dissertation to my parents. To my mother for being an endless
supply of faith and encouragement and to my father (and fellow chemist); this
work is proof that the apple did not fall far from the tree...

 

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ACKNOWLEDGEMENTS

There are many people that have contributed to the completion of this
dissertation. I would like to take this Opportunity to say thank you to all those
involved with my project and/or development of my graduate career. First and
foremost, I would like to thank my advisor Dr. Jetze J. Tepe. Under his guidance
I believe that I have grown to become an independent researcher. He motivated
and encouraged me to continue to think critically about my own project and I
don’t believe I would have grown as a researcher the way I have without him as
my advisor.

I would also like to thank Dr. Gavin Reid for all of his guidance in the
biological mass spectrometry realm. I have learned a great deal from him these
past few years and I truly appreciate all the time and effort he spent with me to
help improve as a researcher. Gavin is very vocal about exactly what he thinks
and I appreciated all of his “this is how it is” comments because by knowing how
he felt, I could take his advice to improve in my writing, researching, thought
process, etc.

The rest of my guidance committee all deserves my thanks as well. Dr.
Greg Baker, I am not sure if you remember this, but the first semester here I went
to talk to you when l was not sure if I was going to stay because my grades were
not the best. I remember you saying- “Just keep reading and working hard. I

know you can make it." The faith you had in me so early on in my graduate

vi

career, I will never forget. I also would like thank Dr. Babak Borhan for his insight
into many different questions I have had over the years concerning both my
research project and graduate classes. Babak, whenever I have had a question
for you, instead of just answering it yourself, you helped lead me to answering it
for myself; that’s what academics are all about. Dr. Pinnavaia, l have Ieamed a
lot about materials science as a result of our ongoing collaborations and I
appreciate all Of the time that you have taken to help improve both the materials
that l have used and my knowledge of them.

I really want to take the time to thank all of my Iabmates; you guys have
been my support system for the past six years. I feel very privileged that I
worked with the people I did. I appreciate all of the camaraderie and teamwork
with all of my “bio labmates”. Teri, I am so glad that you were there to train me
on all the ins and outs of the bio lab. You are a terrific teacher; you don’t hover,
which I like, but you were always there to answer any and all questions that I
had. Daljinder, thank you for your advice and support (both research and
personal), I appreciate it more than your know. Thu aka ‘the bio-lab organizer’,
you have worked so hard to make it easier on all of the bio-lab members to find
and use whatever it was we were looking for with your organization skills! Thank
you for your years of friendship, conversations and of course egg rolls. I will miss
all of the above. Amanda, we have had many experiences over the last few
years; from ASMS to mini meetings. You have been a great friend as well as a
research associate. We have spent endless hours bouncing ideas off one

another and you are always there to help whenever I need it! Thank you for

vii

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being there. I can’t imagine what my graduate school experience would have
been without you!

Additionally, I would like to thank all past and present Tepe group
members, in particular, Chris and Jason. They have always been there for me
when I needed them and I am privileged to call them my friends. We have all
shared some memorable moments here in graduate school. I won’t go into
listing them-to protect both the innocent and the guilty. Jason (Stinky), you
always knew what to say to make me feel better when things were going bad.
Your ability to always make me laugh helped get me through these past six
years! I have been, and will always be, very grateful for your friendship. Chris
(PhePhe), you are like the brother I never had (and never really wanted). What
will I do without your daily teasing?! In all seriousness, I am really going to miss
talking and laughing with you on a daily basis. You have been one of the best
friends a person could ask for.

I would also like to thank all my friends and family. Particularly my
parents, who have always supported and believed in me. I especially want to
thank my husband, Kyle. You have been so patient with me through this whole
process. From dealing with my moods to fixing an endless number of dinners,
your support has only made me love you more. I am looking forward to spending

the rest of our lives together. Just remember, you have to call me “Doctor” now.

viii

 

 

 

 

CHAD

RA?
31v.

LIST OF TABLES ................................................................................. xii
LIST OF FIGURES ............................................................................... xiii
LIST OF SCHEMES ............................................................................................ xviii
KEY TO SYMBOLS OR ABBREVIATIONS .................................................. xx
CHAPTER 1
PROTEIN PHOSPHORYLATION: PROJECT BACKGROUND ............................. 1
1.1 Introduction to post-translational modifications ................................. 1
1.2 Introduction to the study of protein phosphorylation .......................... 3
1.3 Detection Of phosphorylated proteins ................................................ 9
a. 32F radiolabeling ..................................................................... 9
b. Gel electrophoresis/phosphoprotein staining ........................ 10
c. Phosphospecific antibodies .................................................. 12
1.4 Enrichment of phosphorylated proteins ........................................... 13
a. Immunoprecipitation ............................................................. 15.
b. Affinity chromatography ........................................................ 16
i. Immobilized metal affinity chromatography ..................... 17
ii. Strong ion exchange chromatography ............................ 21
iii. Metal oxide affinity chromatography ................................ 22
c. B-elimination-phosphate modification ................................... 25
i. Chemical tagging ............................................................. 25
ii. Proteolytic phosphopeptide mapping .............................. 30
d. Covalent phosphate addition to a solid support .................... 32
1.5 Quantitative phosphoproteomic analysis ......................................... 35
a. Stable isotope labeling In cell culture (SILAC) ...................... 36
b. Isotope labeling/isotope affinity tags ..................................... 39
c. Phosphospecific staining ...................................................... 43
1.6 Global phosphoproteomic studies ................................................... 45
a. Using IMAC ........................................................................... 46
b. Using TIOz ............................................................................. 49
0. Additional technologies ......................................................... 51
1.7 Current work .................................................................................... 54
1.8 References ...................................................................................... 56
ix

TABLE OF CONTENTS

CHAPTERZ
SYNTHESIS OF DIAZO FUNCTIONALIZED SOLID SUPPORTS AND

QUANTIFICATION OF THE DEGREE OF FUNCTIONALIZATION ..................... 77
2.1 Reactivity of diazo materials with Bronsted acids ........................... 77
2.2 Concept of diazo-phosphate binding ............................................... 80
2.3 Original synthesis of diazo functionalized solid support .................. 83
2.4 Problems associated with original studies ....................................... 86
2.5 Improved synthesis of diazo acetate solid support .......................... 88
2.6 Application of improved synthesis to functionalize additional diazo
supports ........................................................................................... 93
2.7 Synthesis of diazo ketone solid supports ........................................ 96
2.8 Synthesis of phenyl diazo methane solid supports .......................... 97
2.9 Quantification of solid support diazo functionalization ..................... 98
2.10 Experimental procedures ............................................................... 104
2.11 Appendix ........................................................................................ 110
2.12 References .................................................................................... 1 34
CHAPTER 3
APPLICATION OF DIAZO SOLID SUPPORTS IN THE ENRICHMENT OF
PHOSPHOPEPTIDES ........................................................................................ 139
3.1 Introduction to diazo solid support phosphopeptide
enrichment ..................................................................................... 139
3.2 Preliminary phosphopeptide enrichment from a peptide mixture and
a protein digest .............................................................................. 141
3.3 Evaluation of synthetic phosphopeptide enrichment by diazo
supports ......................................................................................... 143
a. radiolabeled quantification .................................................. 143
b. MS-based quantification ..................................................... 147
3.4 Scope of diazo ketone solid support ............................................. 152
3. proteins ............................................................................... 152
b. residues .............................................................................. 158
3.5 Experimental procedures ............................................................... 162
3.6 Appendix ........................................................................................ 1 69
3.7 References .................................................................................... 205
CHAPTER 4

APPLICATION OF PHOSPHOPEPTIDE ENRICHMENT ON A BIOLOGICAL
SAMPLE CONTAINING NF-KB PROTEIN-PRELIMINARY WORK AND FUTURE

PROJECT DIRECTIONS .................................................................................... 208
4.1 Impact Of application towards biological samples ......................... 208
4.2 Introduction to NF-KB ..................................................................... 211
4.3 General signaling pathway of NF-KB ............................................. 214

4.4 Phosphorylation events in NF-KB and their impact in cancer
therapy ........................................................................................... 217
4.5 Determination of phosphorylation events in NF-KB pathway ......... 218
a. previous work ...................................................................... 218

x

4.6
4.7
4.8

4.9

4.10
4.11
4.12

b. proposed work using our approach .................................... 223

Activation and purification of NF-KB .............................................. 228
Enrichment attempts made using diazo ketone solid supports ..... 235
Future project directions ................................................................ 242
a. application on cellular proteins ........................................... 242

b. improvement on enrichment efficiency ............................... 246

i. solution-phase enrichment approach ............................ 246

ii. preparation of new diazo solid support ......................... 251
Conclusions ................................................................................... 254
Experimental procedures ............................................................... 256
Appendix ........................................................................................ 266
References ................................................................................... 278

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LIST OF TABLES

Table 1-1. Summary Of some select global phosphoproteome studies and the

enrichment tools used ...................................................................................... 51-52
Table 2-1. Average percent diazo functionalization results ............................ 103
Table 3-1. Enrichment recovery results using a 32P labeled peptide .............. 146

Table 3-2. Average enrichment recovery for each diazo functionalized solid
support ......................................................................................................... 149-150

Table 4-1. IEF result comparison between unmodified and phosphorylated
peptides from a digested whole cell protein extract ............................................ 221

Table 4-2. IEF result comparison between unmodified and phosphorylated
proteins from a whole cell protein extract ............................................................ 221

Table 4-3. Predicted peptide masses in a p65 tryptic digest containing known
phosphorylation sites .......................................................................................... 245

xii

Figure 1-1.
Figure 1-2.
Figure 1-3.
Figure 1-4.
Figure 1-5.
Figure 1-6.
Figure 1-7.
Figure 2-1.
Figure 2-2.
Figure 2-3.
Figure 2-4.
Figure 2-5.
Figure 2—6.
Figure 2-7.
Figure 2-8.

Figure 2-9.

Figure 2-10.
Figure 2-11.
Figure 2-12.
Figure 2-13.

Figure 2-14.

LIST OF FIGURES

Top five most common post-translational modifications ................. 2
Phosphoprotein enrichment and detection technologies ................ 8
Coordination Of metal cations and IMAC chelating reagents ........ 18

Carboxylic vs. Phosphoric acid Ti02 coordination comparison....23
General concept of SILAC approach for MS quantification .......... 36

General approach for MS quantification from EGF stimulation....38

Global phosphoproteomic analysis approach using IMAC ........... 46
Set-up for diazotization of amino-functionalized resin .................. 87
Prepared diazo-functionalized solid supports ............................... 95
IR of a-diazo acetate Wang resin using HONO gas 1 ................ 110
IR of a-diazo acetate Wang resin using DIC coupling ................ 111
IR of a-diazo acetate Wang resin using EDCI coupling .............. 112
IR of a-diazo acetate Wang resin 2-1 ......................................... 113
IR of a-diazo acetate Wang resin 2-2 ......................................... 114
IR of a-diazo amide tentagel 2-3 ................................................. 115
IR of a-diazo amide megabead 2-4 ............................................ 116
IR Of PEGA a-diazo amide 2-5 ................................................... 117
IR of PEGA HMPA a—diazo amide 2-6 ........................................ 118
IR of PEGA HMPA a-diazo amide dendn’mer 2-7 ....................... 119
IR of 4-butyl a—diazo acetate polystyrene 2-8 ............................. 120
IR of a-diazo acetate polystyrene 2-9 ......................................... 121
xiii

Figure 2-15. IR Of NovaSyn a—diazo acetate polystyrene 2-10 ....................... 122
Figure 2-16. IR of NovaSyn TG HMP a-diazo acetate polystyrene 2-11 ........ 123
Figure 2-17. IR of NovaSyn a—diazo anhydn'de polystyrene 2-12 ................... 124
Figure 2-18. IR of vicinal a-diazo acetate silica 2-13 ...................................... 125
Figure 2-19. IR of NHNMPTS a-diazo amide silica 2-14 ................................. 126
Figure 2-20. IR of a-diazo ketone polystyrene 2-15 ........................................ 127
Figure 2-21. IR of a-diazo ketone polystyrene 2-16 ........................................ 128
Figure 2-22. IR of a-diazo ketone silica 2-17 .................................................. 129
Figure 2-23. IR of benzyloxy phenyl diazo methane polystyrene 2-18 ............ 130
Figure 2-24. IR of phenyl diazo methane polystyrene 2-19 ............................ 131

Figure 2-25. IR of benzyloxy phenyl diazo methane polystyrene 2-20 ............ 132
Figure 2-26. Fmoc—glycine standard curve ...................................................... 133
Figure 3-1. Enrichment recovery quantification procedure using 32P-labeled

peptide ................................................................................................................ 145

Figure 3-2. Enrichment of ovalbumin phosphopeptides ................................ 154
Figure 3—3. Enrichment of B-casein phosphopeptides ................................... 157
Figure 3-4. Enrichment of phosphotyrosine peptide (angiotensin II) ............. 159
Figure 3-5. Enrichment of a synthetic phosphothreonine peptide ................. 161
Figure 3—6. Enrichment of 25 pmol angiotensin II, acidic cleavage ............... 169
Figure 3-7. Enrichment of 25 pmol angiotensin II, basic cleavage ................ 170
Figure 3-8. Enrichment of 100 pmol B-casein non-sequencing grade trypsin

digest ................................................................................................................... 171
Figure 3-9. Enrichment of 100 pmol B-casein sequencing grade trypsin

digest, acidic cleavage ........................................................................................ 172

xiv

Figure 3-10. Enrichment Of 100 pmol B-casein sequencing grade trypsin
digest, basic cleavage ......................................................................................... 173
Figure 3-11. MS of CHKtide standard ............................................................. 174
Figure 3-12. MS of CHKtide phosphorylation assay ....................................... 175
Figure 3-13. MS/MS of phosphorylated CHKtide ............................................ 176
Figure 3-14. H5/D5 calibration curve ............................................................... 177
Figure 3-15. MS of 05 enrichment using Resin 2-1 ....................................... 178
Figure 3-16. MS of D5 enrichment using Resin 2-2 ....................................... 179
Figure 3-17. MS of 05 enrichment using Resin 2-3 ....................................... 180
Figure 3-18. MS of 05 enrichment using Resin 2-4 ....................................... 181
Figure 3-19. MS of D5 enrichment using Resin 2-5 ....................................... 182
Figure 3-20. MS of 05 enrichment using Resin 2-6 ....................................... 183
Figure 3-21. MS of DS enrichment using Resin 2-7 ........................................ 184
Figure 3-22. MS Of D5 enrichment using Resin 2-8 ....................................... 185
Figure 3-23. MS Of 05 enrichment using Resin 2-9 ....................................... 186
Figure 3-24. MS of D5 enrichment using Resin 2-10 ..................................... 187
Figure 3-25. MS Of DS enrichment using Resin 2-11 ..................................... 188
Figure 3-26. MS of 05 enrichment using Resin 2-12 ..................................... 189
Figure 3-27. MS Of 05 enrichment using Resin 2-13 ..................................... 190
Figure 3—28. MS of D5 enrichment using Resin 2-14 ..................................... 191
Figure 3-29. MS of D5 enrichment using Resin 2-15 ..................................... 192
l=‘lgure 3-30. MS of DS enrichment using Resin 2-16 ..................................... 193
l=igure 3-31. MS of D5 enrichment using Resin 2-17 ..................................... 194
Figure 3-32. MS of D5 enrichment using Resin 2-18 ..................................... 195
xv

Figure 3-33. MS of 05 enrichment using Resin 2-19 ..................................... 196
Figure 3-34. MS of D5 enrichment using Resin 2-20 ..................................... 197
Figure 3-35. MS of D5 enrichment using IMAC ZipTips .................................. 198
Figure 3-36. MS Of D5 enrichment using Glygen Ti02 NuTips ....................... 199
Figure 3-37. MS Of D5 enrichment using Glygen ZnOz NuTips ...................... 200
Figure 3-38. MSIMS of ion 2173 from ovalbumin enrichment ......................... 201
Figure 3-39. MSIMS of ion 2553 from ovalbumin enrichment ......................... 202
Figure 3-40. MSIMS of ion 2160 from B-casein enrichment ............................ 203
Figure 3-41. MSIMS Of ion 3234 from B-casein enrichment ............................ 204
Figure 4-1. The classical NF-KB signaling pathway ...................................... 213
Figure 4-2. Known NF-KB phosphorylation sites ........................................... 215
Figure 4-3. General approach for the purification of NF-KB .......................... 225
Figure 4-4. Western blot analysis of nuclear extracts from THP-1 cells
probing for NF-kB p65 subunit + actin ............................................................... 229
Figure 4-5. Western blot analysis of p65 immunoprecipitated nuclear extracts
from CEM and L363 cells .................................................................................... 232
Figure 4-6. Silver stain of p65 immunoprecipitated nuclear extracts from CEM
and L363 cells .................................................................................................... 233
Figure 4-7. Western blot and silver stain comparison ................................... 234
Figure 4-8. MS of the p65 antibody tryptic digest .......................................... 266
Figure 4-9. MS of THP-1 untreated nuclear extract immunoprecipitate tryptic
digest ................................................................................................................... 267
Figure 4-10. MS of THP-1 treated nuclear extract immunoprecipitate tryptic
digest ................................................................................................................... 268
l=igure 4-11. MS of THP-1 treated immunoprecipitate digest enrichment ....... 269

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Figure 4-12. MSIMS Of ion 1738 from the THP—1 treated immunoprecipitate
digest enrichment ................................................................................................ 270

Figure 4-13. MS3 of ion 1738 from the THP-1 treated immunoprecipitate digest
enrichment ........................................................................................................... 271

Figure 4-14. MSIMS of ion 2302 from the THP-1 treated immunoprecipitate
digest enrichment ................................................................................................ 272

Figure 4-15. MS3 of ion 2302 from the THP-1 treated immunoprecipitate digest
enrichment .......................................................................................................... 273

Figure 4-16. MS of THP-1 treated immunoprecipitate digest hydrolyzed
enrichment .......................................................................................................... 274

Figure 4-17. MSIMS Of ion 2231 from the THP-1 treated immunoprecipitate

digest hydrolyzed enrichment ............................................................................. 275

Figure 4—18. MSIMS of ion 1653 from the THP-1 treated immunoprecipitate

digest hydrolyzed enrichment ............................................................................. 276

Figure 4-19. MS of angiotensin II-diazo biotin coupling .................................. 277
xvii

LIST OF SCHEMES

Scheme 1-1. General process of reversible protein de/phosphorylation .............. 4
Scheme 1-2. General B-elimination-phosphate modification approach ............. 26

Scheme 1-3. Enrichment approach using modified phosphopeptide-biotin tag
coupling ................................................................................................................. 28

Scheme 14. Improved modified phosphopeptide-solid support coupling .......... 29
Scheme 1-5. Methodology used in phosphospecific proteolysis ........................ 30
Scheme 1-6. Phosphoamidate modification enrichment scheme ...................... 33

Scheme 1-7. Triphenylphosphine mediated phosphoamidate formation on a

solid support .......................................................................................................... 34
Scheme 1-8. Solid phase phosphopeptide enrichment using diazo-
functionalized polystyrene ..................................................................................... 55
Scheme 2-1. Reactivity of diazo carbonyl moieties towards acids ..................... 79
Scheme 2—2. Theory Of diazo carbonyl-phosphate binding ................................ 81

Scheme 2-3. Scheme for the formation of the covalent phosphoester bond ..... 84
Scheme 2-4. HONO gas mediated diazotization of amino resins ...................... 85

Scheme 2-5. Diazotization reaction using glyoxylic acid p-
toluenesulfonylhydrazone ..................................................................................... 88

Scheme 2-6. Synthesis of glyoxylic acid p-toluenesulfonylhydrazone ............... 89
Scheme 2-7. Synthesis Of a-diazo acetate Wang resin using DIC coupling ...... 90
Scheme 2-8. Optimized synthetic route for a-diazo acetate Wang .................... 92

Scheme 2-9. Application of Optimized diazo synthetic route for both hydroxyl

and amino functionalized solid supports ............................................................... 93

Scheme 2-10. Synthesis of diazo ketone resins .................................................. 96

SCheme 2-11. Synthesis of phenyl diazo methane polystyrene ........................... 98
xviii

Scheme 2-12. Quantification of diazo materials with Fmoc-glycine ................... 100

Scheme 3-1. General mechanism for covalent enrichment of phosphopeptides

from diazo-functionalized solid support ............................................................... 140
Scheme 3-2. Formation Of the 32P labeled synthetic peptide CHKtide ............. 144
Scheme 4-1. Formation of diazo-functionalized biotin derivative ..................... 248
Scheme 4-2. Coupling of angiotensin II to diazo biotin .................................... 249
Scheme 4-3. Synthetic route to prepare diazo ketone biotin derivative ........... 251
Scheme 4—4. Mechanism for the enrichment of phosphopeptides ................... 252

Scheme 4-5. Comparison between diazo ketone and diazo dicarbonyl solid
support ................................................................................................................ 253

xix

 

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SILAC
PFZD
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KEY TO SYMBOLS AND ABREVEATIONS

Post-translational modification

isoelectric point

isoelectric focusing

strong cation exchange

strong anion exchange

high-performance liquid chromatography
liquid chromatography-liquid chromatography
2-dimensional gel electrophoresis

collision induced dissociation

matrix-assisted laser desorption ionization mass
spectrometry

electrospray ionization liquid chromatography
tandem mass spectrometry

electron transfer dissociation

electron capture dissociation

immobilized metal affinity chromatography
iminodiacetic acid

nitrilotriacetic acid

isotopic-coded affinity tag

stable isotOpe labeling by amino acids in cell culture
2 dimensional liquid phase fractionation

hepatocellular carcinoma

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NMR
TFA
TMS
FMOC

epidermal growth factor

epidermal growth factor receptor
sequential immobilized metal affinity chromatography
nuclear factor-k8

I-KB kinases

tumor necrosis factor
dicyclohexylcarbodiimide
ethyldimethylaminopropyl carbodiimide
N,N—dimethyl formamide
tetrahydrofuran

1 ,8-Diazabicyclo[5.4.0]undec-7-ene
dibenzofulvene

nuclear magnetic resonance
trifluoroacetic acid

trimethylsilyl

fluoren-9-ylmethoxycarbonyl

CHAPTER 1

PROTEIN PHOSPHORYLATION: PROJECT BACKGROUND INFORMATION

1.1 Introduction to post-translational modifications

The complexity of the human proteome (the proteins encoded by ~30,000
genes) is a staggeringly two to three orders of magnitude larger than its
corresponding genome, resulting in more than 1,000,000 various forms of
proteins.1 The substantial increase in complexity is the result of both RNA
transcriptional regulation‘?’3 and post-translational modifications (PTMs).1 The
ladder includes ~300 known covalent modifications that occur in proteins.4
These modifications are responsible for protein inlactivation, folding, signaling,
stability, and localization.“5 The importance of these modifications can also be
reflected by the 5% of the genome reserved for encoding the enzymes
responsible for these PTMs.1 The various enzymes involved in the catalysis of
PTMs can be divided into two sub-groups; the covalent addition of some
chemical group (i.e. phosphate, acyl, alkyl groups) and the covalent cleavage of
the peptide backbone. Additionally, many of these transformations require some
sort of co-factor to aid in the covalent addition/cleavage; such as acetyl COA

dependent acetylation, ATP dependent phosphorylation, S-adenosylmethionine

(SAM)-dependent methylation, etc.1

Illustrated in Figure 1-1 are the top five most common types Of covalent

addition; phosphorylation, acylation, alkylation, glycosylation and oxidation.

Phosphorylation Alkylation

 

 

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Figure 1-1. Top five most common PTMs.

All of these modifications change the physical properties of the amino acid
to which they covalently attach. Whether it be from the addition of a negatively
charged phosphate or by changing the hydrogen bonding properties of the N-
terrninus by acetylation of the amine to form the amide. These changes not only
affect the modified amino acid, but the protein it belongs to as a whole.1 The
structural changes that take place in the protein as a result of PTMs, help
illustrate their involvement in signal transduction pathways and how the activity of
the protein changes as a result of PTMs as well. However, the dynamic role that

2

PTMs play in cellular activity/response/grOMh is still largely in question.
Although this area of research is Of great interest, it remains challenging due to
the need to isolate the post-translationally modified protein of interest for
analysis. Isolation and subsequent analysis of the modified protein from an
exceptionally complex proteome is why characterization of these modifications
and the amino acid residues in which they bind is a challenge that has only been

met in a few selective modifications.

1.2 Introduction to the study of protein phosphorylation

In the previous section, it was already addressed that protein
phosphorylation was one of the most ubiquitous PTMs known. The dramatic
change in the physical properties of the amino acid as the result of becoming
phosphorylated is quite substantial. As one might expect, the significant physical
change in both the amino acid as well as the protein itself, proves advantageous
in cellular communication.

Many cellular processes including internal cellular signaling,
differentiation, cell survival, metabolism, regulation and proliferation are regulated
by the reversible process of protein phosphorylation, which is governed by
protein kinases and phosphatases.6‘8 This ATP dependent transformation occurs
by a kinase prompting the addition of phosphate to a protein and a phosphatase

enzyme catalyzing its removal (Scheme 1-1).

Kinase Ox‘P/OH
’ \
Cf” (I) OH
Protein Protein
Phosphatase
ATP ADP

Scheme 1-1. General process of reversible protein de/phosphorylation.

While acyl phosphorylation on glutamic/aspartic acid residues and N-
phosphorylation on histidine and lysine residues can occur in eukaryotes, O-
phosphorylation on serine, threonine and tyrosine residues are the predominant
forms of this modification.9 The human genome encodes for about 500 protein
kinases, about 100 protein phosphatases, and it is estimated that one third of all

10,11

human proteins contain at least one site of phosphorylation. Given the

de/phosphorylation events’ involvement in signal transduction pathways and the

large percentage of proteins that can be phosphorylated, it is understandable that

disruption of this process has been implicated in diseases such as cancer?”

”'15 cystic fibrosis,16 and Alzheimer’s disease?17 Due to the

type II diabetes,
impact phosphorylation has on cell function, phosphoproteins and kinases have
been explored for the last two decades as either possible drug targets or

biomarkers to treat or diagnose a variety of diseases (reviewed elsewhere in 18'

21).

 

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As a result of the importance in studying protein phosphorylation,
conventional biological techniques, such as gel electrophoresis and 32F
radiolabeling of phosphopeptides/proteins have been utilized as the traditional
tools employed in studying/detecting the phosphoproteome. While these
classical methods have well documented protocols available to observe

phosphoproteins (reviewed elsewhere in 2233

), they do require a large amount of
protein starting material. The implementation of mass spectrometry (MS)
technologies to probe the phosphoproteome has added new depth in elucidating
the phosphoproteome. Not only has mass spectrometry been used in
phosphorylation site identification,2“'25 but when coupled with additional
phosphoprotein enrichment methods, MS has become the phosphoproteomic
analytical tool of choice due to both its speed and sensitivity.26 The cooperative
use of phosphoprotein enrichment and mass spectrometry analysis has been
employed in discovering new phosphorylation sites, which could lead to the
discovery Of cancer biomarkers and drug targets.20

Regardless of the development of classical biological assays or mass
spectrometry, proteomic techniques relevant to the elucidation of signal
transduction pathways have still lagged in development when compared to
genomic technologies. The challenge in phosphoproteomic identification is in
part due to the complexity of cellular samples and the low abundance typical of
phosphoproteins relative to their unphosphorylated counterparts. This is why the

development of efficient enrichment technologies is a vital step to probing the

phosphoproteome on a cellular level. Enrichment of specific post-translational

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modified proteins can reduce the complexity of a sample and allow one to more
readily determine qualitative discrepancies between samples. To quantify the
changes in the phosphoproteome between samples, additional methodologies

have also been developed (recently reviewed in 27'28).

Both qualitative and
quantitative data of phosphoproteins is pertinent to identifying potential
biomarkers in particular biological pathways or diseases.

In an effort to understand how phosphorylation-mediated signaling
pathways occur, many different phosphoprotein/peptide separation techniques
have been developed in the last 50 years that utilize physical, structural, or
chemical properties pertaining to the phosphate moiety. The physical effects
resulting from the addition of a phosphate include the potential change in the
isoelectric point (pl) Of the protein, which can be separated from other proteins by
either isoelectric focusing (IEF)29 or strong anion/cation exchange
chromatography (SAX and SCX).30

Additionally, the negatively charged phosphate has affinity for metal
cations, which has been exploited in several types of chromatography techniques
including iron31 or gallium32 and metal oxides such as titanium dioxide33 and
zirconium dioxide.34 The 3-dimensional structural changes that arise as a result
Of phosphorylation allow for the development of phospho—specific antibodies,
which can be used to isolate a specific phosphorylated form of a protein.35 The

specific reactivity of the phosphate group can also be manipulated to further

modify the protein/peptide, which can subsequently undergo enrichment of the

23,36)

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A basic overview illustrating all of the current phosphoprotein detection
and enrichment techniques discussed within this chapter are shown in the
diagram below (Figure 1-2). New tools and modifications on the listed
techniques are constantly emerging in an effort to develop more sensitive,
selective, and quantitative approaches. Currently, there is no one particular
approach that appears to be the ‘holy grail’ in phosphoproteomic analysis. With
respect to global phosphorylation analysis, the application of multiple enrichment
techniques, either in a series or parallel fashion, is generally required to Obtain

the most information possible (reviewed elsewhere in 37).

    
     

 
 

Phosphoprotein]
cell Iysate sample

Phosphoprotein
detection .
32.; or 33p labeling Phosphoprotein
. 41 enrichment
Gel electrophoreSIs
l

I

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42-45

Immunoprecipitation

pSer/pThr-
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Affinity
”’93 chromatography

Strong ion
exchange

 

    
     
 
  
 
  

    
  
 
  
 

Phospho
specific stain
48-50

    

 
 

Phosphate
modification

phospho—amidate
129,131 .132

 
 
 

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61 ,85-87

 
    
   
 

 
   
    
 
 
   
    
  
       

 

   

phosphate elimination
1 16,1 19

 

     
 

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126,127

      
   
  
 

nucleophilic

linker
affinity label
117,118,121

reactive solid
support
1 20,1 22—124

 

 

TIOZ
105,105,109-111

Figure 1-2. Phosphoprotein enrichment and detection technologies flow chart.

8

1.3 Detection of phosphorylated proteins

Prior to routine mass spectrometry analyses, biologists and chemists alike
had to utilize other means of phosphoprotein detection or analysis. The typical
tools employed were either 32F labeling, gel electrophoresis, Edman degradation,
the use of phosphospecific antibodies, or any combination of the aforementioned

38'39). These methods are still valid tools

techniques (reviewed elsewhere in
today and are used routinely in studying phosphoproteomics, but have since
been coupled to mass spectrometry (MS) analysis because of its sensitivity,
versatility and speed (reviewed elsewhere in 28'38'40). While the limitations of the
aforementioned biological tools have been addressed by the employment of MS,
these biological tools are still important when dealing with complex mixtures,
large datasets, and low abundances of protein; all typical scenarios when dealing

with phosphoproteomic analysis. As such, it is important to briefly discuss the

validity of these biological approaches to phosphoproteomic analysis.

1.3.a 32F Radiolabeling

The incorporation of radiolabeled phosphate into a phosphoprotein in vitro
via 32P or 33P—ATP or the in vivo addition of radiolabeled organic phosphate (32F)
and subsequent identification of the radiolabeled protein is considered one of the
classical methods in the detection of phosphorylated proteins. This assay was
first used to confirm that both glucagon and epinephrine stimulate the

phosphorylation of dog liver phosphorylase.41

 

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Since then, this technique has been implemented for many different in
vitro and in vivo phosphorylation studies. Typically, the site of phosphorylation
has been characterized by first purifying and digesting the radiolabeled
phosphoprotein, then isolating the radiolabeled peptides by high-performance
liquid chromatography (HPLC) followed by sequence analysis using Edman
c:legradation.“‘°"14

While this approach has successfully been performed on cell culture to
identify phosphorylation sites,45 there are limitations to this approach in vivo as

32F labeling is inefficient due to high amounts of endogenous ATP present within
cells and some phosphorylated proteins have low phosphate turnover rates, both
of which will result in little incorporation of the radiolabel. Additionally, the
i r'ItrOduction of radioactivity can cause a cellular stress response resulting in
artificial phosphorylation events.38 Edman degradation can also become
problematic by causing phosphate elimination on phosphoserine residues during
analysis and is not capable of sequencing peptides with blocked N-termini. The
employment of radiolabeling on its own is somewhat limited, which is even more
true for complex samples and requires coupling of another technique besides

Edman degradation, such as gel electrophoresis, for further phosphoproteomic

a halysis.46'47

1 - 3.6 Gel electrophoresis/Phosphoprotein staining

In order to separate and detect phosphoproteins within a complex mixture,

SL1 ch as a cellular extract, 1 and 2 dimensional gel electrophoresis or HPLC is

10

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typically the method of choice. In 2-D gel electrophoresis, one dimension is
usually a separation by charge state (ie. the pl), which is referred to as
isoelectric focusing (IEF). The other dimension is separated by molecular weight
of the protein using a polyacrylamide gel. In separating proteins by 1D or 20 gel
electrophoresis, individual phosphoproteins can be selectively visualized using
phosphoprotein specific stains.48 Early in the development of these stains some
issues arose due to a lack of specificity of the stains, which limited the utility of
this technique. However, in recent years a fluorescent-based dye called Pro-Q
Diamond was found to selectively bind directly to the phosphate moiety of
3:)roteins.“9'50 Pro-Q Diamond stain shows great promise in phosphoproteomic
analysis because of its specificity, selectivity and its compatibility with mass
spectrometry analysis. It also is capable of staining either all types of O-
phosphorylated residues, or specifically phosphotyrosine, depending on the
staining protocol. This approach has been used for phosphoprotein analysis on
a variety Of different biological systems/cellular samples.39
The utility of gel electrophoresis and its subsequent staining can also be
fI-J rther characterized by MS analysis. The identification of phosphopeptides by
application Of both techniques has been performed by first separating the protein
Via gel electrophoresis followed by in-gel digestion and MS analysis. The digest
was then subjected to phosphate removal via alkaline phosphatase and
d i1=l"erential analysis is performed, monitoring the loss of 80 within a peptide thus

(:0 hfirming the peptide was phosphorylated.51 Another example of the application

11

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of these two technologies was illustrated by Patton and co-workers who

selectively stained and characterized phosphoproteins from a Jurkat cell Iysate.49

1.3.c Phosphospecific antibodies

In addition to phosphospecific staining on a polyacrylamide gel, another
means to visualize femtomole amounts of a phosphoprotein of interest is western
blotting.52 This technique, however, is limited by the availability and selectivity Of
an antibody corresponding to the protein of interest.” Ideally, the antibody
should recognize either the specific phosphorylation site or a type of
phosphoamino acid (i.e. phosphotyrosine), in order to avoid selectivity issues
associated with the detection of unphosphorylated forms Of the same protein.
There are many phospho-serine, threonine, and tyrosine antibodies that have
been developed and are available with limited cross-reactivity to the
unphosphorylated forms of these residues.“"“‘56 The use of phosphotyrosine
antibodies to study the phosphorylation state of various neural proteins in vitro
and in vivo has proven the technique to be successful.57‘59 Additionally, this
approach can be used to monitor the amounts Of phosphoprotein levels in a
plate-based assay forrnat.60

Foley and co-workers grew 82 cells in a 96-well plate, stimulated the cells,
subsequently fixed the cells onto the plate and probed the plate directly with an
anti-phospho JNK antibody. The primary antibody was then probed with a
fiucrophore linked secondary antibody for visualization. This technique allowed

for the quantification of JNK activation in cells directly.60

12

Chin and co-workers took a different approach employing
immunoprecipitation by first immobilizing 50-100 different antibodies on a PVDF
membrane and then subsequently introduced the membrane to mammalian cell
extracts. The cellular proteins were labeled with a fluorescent dye to visualize
the presence of the protein of interest.61

The application of probing a ZD gel for the different types of
phosphoamino acids using different monoclonal antibodies followed by
subsequent protein mass fingerprinting has been utilized in phosphoprotein
characterization.47 Instead of employing the antibody to visualize the protein of
interest, an alternative application is the use of antibodies for selective

immunoprecipitation of a desired phosphoprotein, which will be discussed further

in the enrichment section.

1.4 Enrichment of phosphorylated proteins

Despite recent advancements in mass spectrometry techniques, including
tandem mass spectrometry (MS/MS), the characterization of phosphopeptides
still remains challenging. Identification of phosphopeptides is Often hampered by
their sub-stoichiometric abundance relative to their unphosphorylated
counterparts, resulting in signal suppression.62 Additional ion suppression can
also result from the formation of phosphopeptide-metal ion complexes.63
Enrichment of phosphorylated peptides/proteins prior to their introduction into a
mass spectrometer can therefore help facilitate phosphoprotein analysis

23,27,28,36,64,65

(reviewed elsewhere in ). Additional problems relating to the lability

13

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of the phosphate group when introduced into the mass spectrometer, includes
dominant neutral losses of 98 and 80 Da in both MS and MSIMS analysis,
creating difficulty in both sequencing the peptide and assigning the site of

7

phosphorylation.66'6 Consequently, an alternative approach uses MS3 to

sequence the phosphopeptides when dominant H3PO4 loss in MS2 occurs.2'°"68'69
However, recent evidence supports the loss of H3PO4 results in a cyclic product
ion whose subsequent MS3 product ions could provide additional insight to
determine the site of phosphorylation within a peptide containing multiple
potential phosphorylation sites.70 Additional difficulty in MS analysis is the
potential for the phosphate to transfer to an unmodified hydroxyl containing
amino acid when analyzed by collision induced dissociation (CID)-MS/MS,
potentially resulting in mis-identification of the site of phosphorylation.71

The problems associated with CID-MSIMS analysis in both the facile
H3PO4 loss and the gas-phase phosphate rearrangement of phosphoproteins has
been addressed via the use of either ETD (electron transfer dissociation)72'73 or
ECD (electron capture dissociation)74 to induce molecular ion fragmentation. The
fragmentation occurs for both ETD and ECD primarily at the N-Ca bonds,
creating c and 2 type ions.75'76 This type of fragmentation allows the phosphate
to remain intact along the peptide backbone, enabling phosphorylation site
identification.77""'2

Identification of the sequence of a peptide and its” respective protein, can

be assisted by comparing the spectra with an MS database, however, the

database only aids in identification if the protein in question is in the database.

14

Additional software utilized to process the excessive amounts Of
phosphoproteomic data acquired from large sample sets, Gygi and Schwartz
developed an algorithm to aid in the identification and classification Of sequence
motifs of phosphorylation sites.83 They applied this algorithm to characterize
phosphorylation sites found from HeLa nuclear protein analysis,84 and found 77%
Of phosphopeptides that were characterized had a proline residue 1+ position (C-
terrninal) and/or a glutamate at the 3+ position. This bioinforrnatics tool can both
be used to analyze large phosphoprotein datasets and as a comparative tool in
the sequence homology between different types of phosphoproteins collected
from global phosphorylation studies described below.

The sub-stoichiometric problem has also been addressed by many groups
using a variety of different techniques, which will be discussed in more detail
herein. The most widely employed enrichment method has utilized the
phosphate moiety’s affinity for metal cations and metal oxides. Alternative
approaches include elimination of the phosphate followed by subsequent
introduction of a more stable modification. Thus, identification assignment of the
phosphorylation site is based on identifying the modified residue (enrichment

procedures reviewed elsewhere in 27""8"’"3'38'40'64).

1.4.a Immunoprecipitation
As illustrated in the previous section, antibodies have been used as a
means of detecting phosphorylated proteins, however, they also can serve as a

phosphoprotein/peptide enrichment tool. Instead of employing the antibody to

15

visualize the protein of interest, an alternative application is the use of antibodies
for selective immunoprecipitation Of a desired phosphoprotein. The antibody of
choice is bound by either affinity (i.e. biotin) or covalently (i.e. agarose) to a solid
support and introduced to a cellular fraction containing the protein Of interest.
The protein is subsequently purified from the rest of the cellular proteins by
collecting the solid support-antibody—protein pellet. The protein is then eluted
from the pellet and enzymatically digested and subsequent MS analysis of the
immunoprecipitated sample can lead to identification of the phosphorylation

sitesaf”86

The application of probing a 2D gel for the different types of
phosphoamino acids using different monoclonal antibodies followed by
subsequent protein mass fingerprinting has been utilized in phosphoprotein
characterization.47

Alternatively, the cellular extract may first be digested, then introduced to

an antiphosphotyrosine antibody, allowing for the global MS identification of

phosphotyrosine peptides.87

1.4.b Affinity chromatography

As mentioned earlier, the addition of the negatively charged phosphate on
the protein can be manipulated to be purified by chromatography. Specifically,
affinity chromatography has been employed utilizing the affinity of the negatively
charged phosphate towards metal cations. This purification approach has been
exploited in several types of chromatography techniques including iron31 or

gallium32 and metal oxides such as titanium dioxide33 and zirconium dioxide.34

16

Additionally, the physical effects resulting from the addition of a phosphate
include the potential change in the isoelectric point (pl) of the protein, enabling
purification by either strong anion/cation exchange chromatography (SAX and

sexy”

1.4.b.i. Immobilized metal ion affinity chromatography (IMAC)

Identifying phosphorylation sites on proteins by isolating phosphopeptides
through exploitation of their affinity for metal cations has been explored for many
years. Originally coined “metal chelating affinity chromatography’, this technique
was introduced in 1975 by Porath and co-workers and used to purify histidine-
tagged proteins.88 When subsequent studies revealed that phosphorylated
amino acids also bind to iron(III)-charged species coordinated to agarose-bound
iminodiacetic acid, immobilized metal ion affinity chromatography (IMAC) was
bom.31

IMAC has been one of the most widely employed and well-known
techniques used in phosphoproteomic studies. This method utilizes metal ions,
such as Ga3+ or Fe‘”, which have been found to have the highest percent
recovery of the negatively charged phosphate.32 The metal ions are first
introduced to chelating agents, such as iminodiacetic acid (IDA) or nitrilotriacetic
acid (NTA), which are immobilized on a resin, bead, glass chip or membrane.
The IDA-metal complex and the NTA complex has three sites and two sites of

available coordination, respectively (Figure 1-3). The coordination sites are only

available to the phosphate at a pH range between 2.0-3.5.32 In an effort to

17

Figure

develop an IMAC material capable of enrichment at physiological pH, an

alkoxide-bridged di-nuclear Zn (II) was developed, referred to as phos—tag (Fig.

1-3).89
0
O
> O

NH 9/15 \ / \

O \ 1’ I \ /N\ ’N /
‘ 2+ ’2+
I \/ .
/ N\/K/N \

(a) 0 (C)

(3"gk

0)

+

E ,0
O -n"
(D ‘\

N2

C)

Figure 1-3. Coordination between metal cations and IMAC chelating reagents.

The phosphopeptide enrichment can be carried out by introducing a
complex biological sample, such as a protein digest or cellular extract, to the
column or surface containing the immobilized metal cations. The phosphate
moiety coordinates to the metal cations allowing for the removal of unbound
peptides. Liberation of the bound phosphopeptides from the affinity material is
accomplished by either washing the column with a basic pH solution, or by
introducing excess free ligand, such as a phosphate buffer. The liberated
peptides are typically analyzed directly by either matrix assisted laser
desorption/ionization mass spectrometry (MALDI-MS) or electrospray ionization
mass spectrometry (ESI-MS) to sequence the peptide and determine the site of
phosphorylation.

There are some problems associated with using IMAC to determine
phosphopeptides/proteins. These challenges include the recovery of multiply
phosphorylated peptides which have a higher binding affinity to the metal cations

18

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and result in poor elution from the support, requiring harsher conditions to
liberate the peptides.66'90 The most problematic issue using IMAC is non-specific
binding due to acidic residues (i.e. aspartic acid and glutamic acid) and electron
donor residues (i.e histidine), which also have affinity to the metal ions. In part,
this issue has been addressed by esterifying the carboxylic acids to methyl
esters using in situ formed HCI in dry methanol.66 It is important to follow the
esterification conditions explicitly to avoid either incomplete conversion or
unwanted side reactions, both of which increase the complexity Of the sample.
Phosphorylation site identification may also become more challenging as a result
of other amino acids such as tyrosine, histidine, and tryptophan binding to the
IMAC material.

There have been many advancements to improve current IMAC
technologies, including fabricating higher loading solid supports such as
mesoporous nanoparticles,91 glass microchips,92 magnetic particles,93'94 metal-
chelating plastic MALDI chip95 and functionalized gold MALDI plates.96'97 Higher
loading has increased the sensitivity of the supports and decreased the amount
of IMAC material required to carry out the enrichment. In an effort to develop a
high-throughput and automated approach to IMAC, HPLC columns have also
been fabricated with metal chelating silica to allow for large-scale analysis or
front-end LC-MS analysis."""98'99

The scope of IMAC technology has been explored by a variety of research
groups, measuring the capacity, selectivity, and enrichment ability of different

materials. The application of IMAC has also been used to probe the

19

 

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phosphorylation sites of a variety of other commercially available

I66 as well as

phosphoproteins and characterizing the phosphoproteome of yeas
various cancer cell lines (discussed later within this review), indicating the
capability Of this technique to handle large datasets.

In addition to the aforementioned limitations of IMAC, an additional
drawback is the inability to fractionate IMAC enriched samples as an additional
purification measure, as IMAC peptides are typically eluted in only one fraction.
This limitation has been addressed by the development of Sequential elution
from IMAC (SIMAC).1°°

In this technique, mesenchymal stem cells were harvested, the proteins
digested and the peptide mixture was introduced to iron-coated PHOS-select
metal chelate beads. The beads were washed with 1% TFA in 20% acetonitrile
to elude the singly phosphorylated peptides and contaminating unphosphorylated
peptides, then multiply phosphorylated peptides were eluted from the IMAC
beads upon introduction of ammonium hydroxide (pH 11.3). All of the IMAC
enriched fractions including the flow-through and washing solutions were then
further enriched using Ti02 chromatography and then analyzed by either LC-MS
or MALDI-MS,100 resulting in the number of monophosphorylated peptides
identified as 179, 232 and 80, respectively. Taking into consideration
phosphopeptide overlaps, a total of 306 unique monophosphorylated peptides
were detected, 55 Of which were identified from the IMAC flow-through, indicating
the importance of a secondary enrichment technique to capture phosphopeptides

that do not bind well to the IMAC column. The resulting multiply phosphorylated

20

peptces

EigIeOI' VII

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be has

Th
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were ml:

peptides that were detected in the flow-through, 1% TFA elution and NH4OH
elution were 8, 11, and 179 respectively.100 The results illustrate the importance
of adding a basic solution to the IMAC column in order to obtain the multiply
phosphorylated peptides, as more than 96% of the those peptides were identified
in the basic elution wash and not in the 1% TFA elution. Further application of
IMAC for global phosphoproteome analysis will be discussed later within the text.

The SIMAC approach was compared to using just Ti02 enrichment and
286 phosphopeptides were identified using TIO2, where 54 Of those peptides
were multiply phosphorylated. The use of the SIMAC technique resulted in the
identification of 492 phosphopeptides where 186 of those were multiply
phosphorylated, illustrating the improvement of enrichment for multiply

phosphorylated peptides when using SIMAC.100

1.4.b.ii Strong ion exchange chromatography (SCX, SAX)

Another technology that employs the negative character of the phosphate
as means of separation is ion exchange chromatography. Enrichment of
phosphopeptides by the use of strong cation exchange (SCX) is achieved by the
negatively charged phosphate’s inability to bind well to the SCX material,
whereas unphosphorylated peptides are more readily retained on the support.
Successful application of this approach has been illustrated on cellular protein
extracts that had been separated by gel electrophoresis and then in-gel

digested.“101 Cooperatively, SCX can also be used as a front-end purification

21

technique prior to TIOz mediated phosphopeptide enrichment as shown by Mann
and co-workers.69

Additionally, strong anion exchange (SAX) can also be used to enrich
phosphorylated peptides.102 As opposed to SCX, in SAX the negatively charged
nature of the phosphate moiety allows it to bind SAX material while their
unphosphorylated counterparts are not retained. Comparatively, both SAX and
SCX have been used to enrich phosphopeptides and it was found that each ion-

3 Dai et al.

exchange method enriched different types of phosphopeptides.10
reported some loss of phosphopeptides from the use of SCX, which was
confirmed by the detection Of phosphopeptides collected from the SCX flow
through fractions.104 Gu and co-workers probed the liver phosphoproteome with
SAX and compared the results obtained with SAX versus using the IMAC
approach. The technique was compared qualitatively using standard protein
digests to IMAC and found to have identified >95% of the singly phosphorylated
peptides, where IMAC identified 66.7%.102 The benefit of SAX over IMAC is the

ability for SAX to fractionate samples, whereas IMAC typically produces only one

fraction, unless SIMAC is performed.

1.4.b.iii Metal oxide affinity chromatography (MOAC)

In an effort to find a material with a higher degree of specificity for the
phosphate group, metal oxides can be explored. In this approach the phosphate
is proposed to bind to the titanium dioxide through a bidentate interaction (Figure

1-4).33 Heck and co-workers described a procedure to trap phosphopeptides in

22

 

a pre-column packed with Ti02 (titansphere) under acidic conditions.
Subsequently, the phosphopeptides can be desorbed from the column under
basic conditions.105 These fractions are trapped on a reversed-phase column for

further separation and subsequently analyzed by LC-ESl-MS/MS.

a) O b) HO
\\ ,OR(H)
/P\ O
O O

l l O. /0
T102 TI02 T102

 

 

Figure 1-4. Hypothesized coordination on Ti02 support comparison between

carboxylic acids and phosphoric acid by Larsen et al.106

While this technique is capable of enriching low femtomole quantities of
phosphopeptides, some acidic, non-phosphorylated peptides also show affinity to
the titanium dioxide column.

This selectivity issue was addressed by Larsen et al., who prepared
microcolumns packed with Ti02 and observed a higher degree of
phosphopeptide specificity when 2,5-dihydroxybenzoic acid (DHB) was present
in the loading solution.106 The increase in selectivity of Ti02 for phosphopeptides
under these conditions was thought to result from competition between DHB and
non-phosphorylated peptides for the remaining Ti02 binding sites.

This hypothesis was confirmed with spectroscopic studies indicating that
substituted aromatic carboxylic acids, such as DHB, coordinate strongly to the
surface of TiOz, while monofunctional carboxylic acids (i.e. benzoic acid or

glutamic acid) coordinate much more weakly with titanium dioxide 107. Larsen

23

also suggested that while there is competition between DHB and other non-
phosphorylated peptides, it is not competing with phosphorylated peptides. This
is due to the difference between DHB and phosphate in their coordination
geometry with TiOz. DHB is a bidentate chelator,107 whereas the phosphate

group is a bridging bidentate,108

which results in both species differing in Optimal
binding sites (Figure 1-4).

Improvement in phosphopeptide selectivity with Ti02 versus IMAC
enrichment was confirmed by Krause and co-workers, who evaluated several
phosphopeptides and their unphosphorylated counterparts for Ti02
enrichment.109 Recently, reproducible protocols have been published using Ti02
packed pipette tips in phosphopeptide enrichment and analysis.“"'111

Additionally, zirconium dioxide packed pipette tips have also been
implemented in the enrichment of phosphopeptides. Comparatively, the two
types of commercially available metal oxide tips were found to be equivalent in
phosphopeptide enrichment, however, the ZI'Oz tips were found to be more
selective for singly phosphorylated peptides while the Ti02 tips were superior for
the enrichment of multiply phosphorylated peptides.34 Both of these materials
have been useful in global phosphoproteome characterization, which will be
explored later on within the text.

Ti02 is the most widely employed metal oxide in phosphoprotein
enrichment; however, its development brought about the exploration Of additional

metal oxides for their enrichment capabilities. Zhang and co-workers have

coated Fe304 magnetic microspheres with either Al203112 or Ga203113 and both

24

 

 

materials were capable of enriching phosphopeptides from commercially
available phosphoproteins and rat liver protein digests. Additionally, Sn02114 and
Nb205115 have also recently been identified as phosphopeptide enrichment

materials.

1.4.c B-Eliminatlon-phosphate modification

Another phosphopeptide enrichment technique that became established
around the same time as phosphO-affinity enrichment, was phosphopeptide
derivatization. Manipulation of the reactivity of the phosphate moiety can be
implemented to purify phosphorylated peptides/proteins from their
unphosphorylated counterparts through the addition of base. Under basic
conditions the phosphate moiety can be eliminated upon the deprotonation of a
carbonyl and the resulting eliminated product can be further modified and
analyzed. The addition of the nucleophile can then be corresponded back to the

original site of phosphorylation.

1.4.c.i Chemical tagging

In the chemical tagging approach, the phosphopeptide is first modified by
elimination of the phosphate group on either phosphoserine or
phosphothreonine, followed by nucleophilic attack of the newly formed Michael

acceptor (Scheme 1 -2).

25

 

O=Fl’-OH l h'l Nuc
O 'OH ~ \ . nuc eop re;
T g ”H; V \ExNjWE’
' §' O H

phosphoserine dehydroalanine

Scheme 1-2. General scheme for B-elimination-phosphate modification

approach for phosphoserine.

The formation of the unphosphorylated derivative eliminates the problem
of difficult sequence analysis from dominant -H3PO4 loss in the MSIMS
spectrum. Prior to routine mass spectrometric analysis, the phosphorylation site
on the peptide derivatives were characterized by Edman degradation.116 Meyer
and coworkers found the nucleophile of choice to be ethanethiol, due to its
addition efficiency and its significant change in elution time allowing for definitive
identification.116 More recently, this route has been used to modify a variety of
phosphopeptides and subsequently analyze the modified product by ESI or
MALDI MS."7'119 The modified proteins undergo proteolytic digestion and their
phosphate site is characterized by the +44 mass unit shift of the modified peptide
observed in MSIMS analysis.119 Validation of the approach was confirmed by the
commercially available protein and additionally, 28 phosphorylation sites were
characterized on the purified human protein.119

While this technique does prove advantageous over the affinity
approaches as it removes the challenges of phosphopeptide sequence analysis
by modifying the phosphate first, there are limitations. The most notable

drawback for the B-elimination approach is its incompatibility to detect

26

phosphotyrosine residues, as they do not undergo B-elimination. Another
limitation is the elimination of unmodified serinelthreonine residues and O-
glycosylated peptides which can result in a “false positive’ modification and
create ambiguous phosphorylation site determination.

Aside from the direct MS analysis of the modified peptide without
additional purification, there are other applications for the phosphopeptide
derivatives that involve phosphopeptide enrichment. The B-elimination product
can be introduced to a nucleophile that is also attached to either an affinity tag or
immobilizing reagent, allowing for selective enrichment of the modified
phosphopeptides. Application of this concept has been employed to enrich
phosphopeptides using the nucleophile ethanedithiol to react with the B-

1" Prior to the introduction of the

elimination species (dehydroalanine).
ethanedithiol, cysteine residues are oxidized to prevent any unwanted
nucleophilic attack by adjacent residues. The terminal sulfur on the modified
(previously phosphorylated) peptide can undergo subsequent nucleophilic attack
to a biotin linked maleimide, forming an affinity labeled (formerly phosphorylated)
peptide. The electrophilic maleimide-biotin incorporated within the modified
peptide can then be purified by avidin beads, which retain the biotinylated

species on the column (Scheme 1-3). The flow-through and washing steps

effectively remove any unmodified (i.e. unphosphorylated) peptides.117

27

 

O=FI’-OH /\/SH
OH HS
0
ifl H3PO4 3‘Njfi"?
H
O
Biotin
ii
017”
B' . O
IOtInw-NH 002”
H BiotinW-N
KI H20 0 H
<—-—
S 3
ml“? it lift
H N
O H

0
Scheme 1-3. Enrichment approach using modified phosphopeptide coupled to a
biotin affinity tag.

While biotin affinity chromatography is an attractive approach because of
its availability and well documented protocols, there are a few limitations using
biotin as an affinity tag. The biotin-avidin interaction is quite strong, making
Blution of the biotin-peptide species sometimes difficult. As a result, other
researchers have utilized this established enrichment strategy but developed a

Solution to the current issues associated with biotin affinity tags. The
incorporation of both the ethanedithiol and the maleimide was performed,
however, the maleimide was bound to an acid cleavable solid support instead of
biotin, thereby eliminating the problems associated with biotin.120 Additional work

maintained the use of biotin as an affinity label, but was first coupled to an acid

28

g

cleavable linker prior to its introduction to the modified peptide and then
subsequently liberated the modified peptide from the affinity column under acidic
conditions.121 The implementation of either the ethanedithiol phosphopeptide
derivative122 or a solid-support bound sulfur123 as the nucleophile has also been
thoroughly explored towards Optimization of this technique.

Further problems arise in the sequence analysis of the biotin-containing
peptides which may be challenging due to fragmentation patterns that are hard to
interpret. This limitation has been addressed in a follow-up publication by Chait
and co—workers, who again incorporated ethanedithiol, but then performed the
enrichment using an activated thiol affinity resin followed by liberation Of the
bound peptide by reduction of the disulfide bond (Scheme 1-4).124 The absence
0f the biotin tag increased the sensitivity of the enrichment, however, the authors

acknowledged still having difficultly addressing the unwanted side reaction, B-

elrrn i nation of serine.

 

H
S S N
e, ”O
H O

Scheme 1-4. Improved modified phosphopeptide coupled directly to a solid

$342734)? Q’Sslgr

H3P04

support.

29

 

 

Cramer and co-workers explored the complimentary use of both IMAC as
an initial enrichment step followed by a subsequent B—elimination of the
phosphate on the IMAC solid support, which eluted to modified phosphopeptides
125. The benefit of coupling the two techniques simultaneously avoids
aforementioned complications inherent of each method, such as improving the
elution Of the phosphorylated peptides by eliminating the phosphate on the IMAC
support. Additionally, the use of IMAC prior to the B-elimination allows

glycosylated and phosphorylated peptides to be distinguished.

1.4-c.ii Proteolytic phosphopeptide mapping

To date there is currently no known protease to selectively hydrolyze
residues at their phosphorylation site, however there has been a way to
accomplish this through phosphopeptide modification. This approach still
implements the documented B-elimination-derivatization sequence, however, the
peptide is modified to form a lysine residue mimic, which upon introduction to
trIv’Psin, will selectively cleave at the formerly phosphorylated amino acids?”127

(Scheme 1-5)

OH H2N NH2 NH2 NH2

e a. a. %.
-OH SH + \: =
$5,311 TwJKgI-Ficujwy a.” = . — snlfl
O O O

O H3PO4

Scheme 1-5. Methodology used in phosphospecific proteolysis.

30

Independently, Shokat and co-workers and Hathaway and co-workers
each developed similar chemistry by derivatizing phosphoserine to form
aminoethyl cysteinem"127 Structurally, aminoethyl cysteine behaves similar to
the natural amino acid lysine, and by introducing the modified peptide to a
protease that cleaves at lysine residues, such as trypsin, it will also selectively
cleave at the modified phosphorylated residue (i.e. aminoethyl cysteine).
Subsequent MSIMS analysis of the digested modified protein can identify the
presence of pseudo-lysine residues, therefore identifying the phosphorylation
sites present within the protein.

The same limitations apply for this method as stated earlier, including the
exclusion of phosphotyrosine identification and unwanted side reactions.
Additionally, characterizing phosphothreonine residues is challenging, as they do

not undergo phospho-ester cleavage readily.126

In a follow-up publication,
Hathaway and co-workers developed a modification of the original procedure to
reduce the problem with unwanted side reactions by discriminating between
Phosphorylated and glycosylated peptides.128 Hathaway observed complete
conversion to the B-elimination product for both phosphorylated and glycosylated
Peptides in the presence of barium hydroxide. However, when ammonium
hydroxide was substituted for the barium hydroxide, 60% of the glycopeptides
remained unconverted. While this reduces the amount of false characterization
Of a glycopeptide as a phosphopeptide, caution still must be issued when making

PhOSphorylation site assignments based solely on this methodology. as the

PhOSphate is no longer intact to confirm its identity. Later studies determined the

31

Optimized conditions to enable the selective elimination of phosphopeptides over
glycopeptides by reducing the amount of barium hydroxide used and decreasing

the amount of reaction time under these conditions.122

1.4.d Covalent phosphate addition to a solid support

Other than techniques relying on B-elimination to derivatize the phosphate
moiety, the phosphate moiety is not inert from other types of nucleophilic
addition. The phosphate itself has access to an arsenal of additional reactivity
utilizing both its nucleophilic and its electrophilic properties. For example,
adaptation of a known condensation between carboxylic acids and carbodiimides
can be applied to the phosphate moiety for phosphopeptide enrichment.129 Prior
to the condensation, this method requires protection Of all the carboxylic acid
residues and C-terminus with ethanolamine to eliminate any unwanted side
reactions, as their reactivity is similar to that of phosphate. Aebersold and co-
workers performed the condensation reaction catalyzed by the carbodiimide
between cysteamine and the phosphate, and upon nucleophilic addition of
ditI'IiOIthreitOI, the disulfide bond was reduced, generating a free thiol. The thiol
0" the modified phosphopeptide was then covalently bound to a solid support by
reacting it with iodoacyl functionalized glass beads followed by cleavage Of the

PhOSphate from the solid support was achieved in the presence of acid (Scheme

1-6),

32

 

 

 

I I
0—P-OH H N/VOH 0=P-N/\/OH TFA 0=P"OH
: ' H ——-> O
EDC °
t-Boc OH t-Boc N t-Boc H
\N \N \/\O \ \/\OH
H O H O
NH2
s—s’ EDC, DTT
H N
o 2 if
?H S 0 9H SH
0 < o
t-Boc III t-Boc N
H O H O
lTFA
OH
O=P-OH O
O + S
H2N V‘OH
0

Scheme 1-6. Phosphoamidate modification-enrichment scheme.

This technique was measured for its enrichment efficiency with a
radiolabeled peptide formed from 32P-ATP, a tyrosine kinase and myelin basic
PTOtein. The labeled protein was digested and its subsequent labeled
PhOSphOpeptide underwent the enrichment protocol and its radioactive counts
were measured after each step, determining an enrichment recovery of ~ 20%.
Furthermore, this approach was introduced to a yeast Iysate, where multiple
Phosphopeptides were enriched and their corresponding phosphoproteins were

identified.130 In a more recent publication, Aebersold and co-workers performed

33

the enrichment by implementing the same EDC-mediated formation of the
phosphoamidate between an amino functionalized dendrimer and the
phosphopeptides.131 This approach first protected the carboxylic acids by
converting them to their corresponding methyl esters as previously described by
Hunt and co-workers.66

Pflum and co-workers developed an oxidation-reduction method for
enrichment utilizing the electrophilic character of the phosphate group, which can
be accomplished by activating the phosphate with phosphine and disulfide, then
coupling the phosphate to a solid-support bound nucIeophiIe.132 The solid
suF>|:Jort-bound phosphoamidate enabled the removal of unphosphorylated
Peptides and subsequent hydrolysis of the phosphate liberated the peptide from

the support (Scheme 1-7).132

NH2

9H 9H <9“ ,VO
0=I';"'OH MeOH 0=I'3-OH 0:5,-”
O HCI O O

ACLRRA AcLRRA Pph3 PYSSPY; A LRRA
Nln/OH OMe DIPEA DMF C \N OMe
H 0

Scheme 1-7. Triphenylphosphine mediated phosphoamidate formation on a

 

SOIId support

Additionally, the current work described within this dissertation illustrates a
method for the enrichment of phosphorylated serinelthreonine and tyrosine
containing substrates using a unique solid phase enrichment technique. The
method employs the use of a functionalized solid support, which reacts

34

 

specifically with the phosphate moiety allowing for non-phosphorylated peptides
to be efficiently removed by filtration. This technique is an alternative approach
to the solid phase enrichment approaches discussed above. Further elaboration
of our approach will be addressed later on within the current work section of this

chapter.

1.5 Quantitative phosphoproteomic analysis

Upon the development Of phosphoproteomic technologies, additional effort
to quantify the phosphopeptides/proteins is important for two different reasons.
First, the importance of the development of quantitative phosphoproteomic
technologies should not be trivialized. A critical facet of phosphoproteomics is
determination of the relative change in abundance of phosphoproteins. The
Change in abundance of phosphoproteins is of particular interest when biological
systems are exposed to stimuli, under duress, or as a result of pathogenic tissue
Signaling. There are a variety of different technologies that have been (and are
currently being) developed. In-gel based quantitative protein staining/labeling
f0I|C>wed by subsequent MS analysis is a common techniques used in protein
quantificationfawfi but it suffers limitations with reproducibility and problems with
efficiently separating very large or very small, >200 kDa and <10 kDa proteins.136
Additionally, phosphospecific stains can be utilized in a microarray format instead
0f the traditional gel electrophoresis.137 The most widely employed quantitative
method, however, involves the implementation of various isotopic labels followed

by sUbsequent MS analysis.69'13°'13& ‘42

35

Secondly, the quantification of the recovered phosphopeptides can be
used to determine the efficiency of these enrichment techniques. This
information can then be used to identify optimal enrichment conditions and
materials. This also can identify biases of different approaches with respect to

the type (and extent) of the phosphopeptides enriched.

1.5-a SILAC route to quantification of cellular proteins

Stable isotope labeling by amino acids in cell culture (SILAC) involves the
metabolic labeling of cellular proteins using stable isotope-coded amino
acicls.13‘"1‘"'142 The isotopically heavy and light labeled cell culture samples can
be treated under different conditions (i.e. exposed to certain drugs or activators in
a particular pathway), then pooled together and the changes in phosphoprotein
levels can be determined quantitatively by MS (Figure 1-5).

Cell Sample #1: ‘ Cell Sample #2:
control-light isotope stressed-heavy isotope

"heavy" label

IIII

"light" label

   

Pooled samples

illIIIs

FIN-Ire 1-5. SILAC approach using mass spectrometry for the quantification of

 

Protein expression levels between normal cells and cells under stress.

36

 

Work by Neubert and coworkers monitored the cellular response to
stimulation by brain derived neurotrophic factor (BDNF), utilizing SILAC to
quantitatively measure changes in the phosphotyrosine proteome in primary

neurons.143

Upon stimulation, the cells were lysed, immunoprecipitated with
phosphotyrosine antibodies, separated by gel electrophoresis and in-gel
digested. The digested samples were analyzed by LC-MS/MS and Neubert
found that after stimulation 18 phosphoproteins had ratios greater than 1.25 or
less than 0.80 and of those phosphoproteins, 13 had ratios greater than 1.5 or
less than 0.67.143

Mann and coworkers illustrated the capability of the SILAC to determine
global phosphorylation changes by employing it to identify and quantify
phosphorylated proteins expression levels in HeLa cells either unstimulated or
stimulated with epidermal growth factor (EGF).69 Different plates of cells were
supplemented with one of the following; unlabeled lysine and arginine, or labeled
lysine “4” and arginine “6”, or lysine “8” and arginine “10”. The cells were
collected after exposure to EGF after 0, 1, 5, 10, and 20 minutes. The collected
cellular proteins were further separated by strong-cation exchange

chromatography and titanium dioxide chromatography for enrichment of

phosphopeptides and then subsequently analyzed by MSIMS (Figure 1-6).

37

 

 

 

 

Lys “0" Lys "8"
Arg ROI Arg I10"
0 min EGF 10 min EGF
1 min EGF 5 min EGF 20 min EGF

 

Pool cell samples

/\

Cytoplasm proteins Nuclear proteins

1 digest l digest

SCX fractionation SCX fractionation

l l

Ti02 enrichment Ti02 enrichment

Figure 1-6. Scheme for SILAC incorporation and enrichment strategy adapted

from 69.

The SILAC global quantification and phosphopeptide enrichment by Mann
and co-workers led to the detection of more than 10,000 phosphopeptides and
the characterization of 6,600 phosphorylation sites (4901 phosphoserine, 670
phosphothreonine, and 103 phosphotyrosine) on 2,244 proteins. In comparison
to the SwissProt database, more than 90% of the identified phosphorylation sites
within the experiment were found to be novel. As a result of the employed SILAC

technique, 1,046 phosphopeptides were found to be more than a 2-fold increase

38

as a result of EGF stimulation, which includes 724 phosphoserine, 106
phosphothreonine, and 53 phosphotyrosine, which all are in some way regulated
byEGF.

Further analysis on identified phosphopeptides containing more than one
phosphorylation site had their corresponding phosphopeptides independently
quantified to determine if different phosphorylation sites on the same protein are
regulated differently. It was found that 77% of those proteins were regulated
differently on different phosphorylation sites. This Observation indicates the utility
of targeting post-translational modifications as biomarkers by identifying specific
forms of a protein as a result of activation. This could also aid in the identification
of drugs targeting inhibition of a specific phosphorylated form of the protein
without inhibiting all of the additional functions the protein is responsible for.
Ultimately, this study determined there are at least 26 transcription factors, 20
transcriptional co-regulators, 21 proteins involved with regulating cytoskeleton
function, 27 phosphopeptides belonging to actin-binding proteins, 12 ubiquitin
Iigases, among others that are involved with EGF signal activation.

Additional quantitative phosphoproteome studies using SILAC has been

145

performed on mouse liver cells,“4 lung cancer cells, and cervical cancer

ceIIs.146

1.5.b The use of stable isotopic labeling or isotope affinity tags to quantify
Many LC-MS/MS approaches have been utilized to determine relative

protein changes?“149 but the method of choice in accurately comparing protein

39

levels within a sample set is stable isotopic labeling of proteins, either
metabolically in cell culture (SILAC) or employing labeling reagents on already
extracted proteins. One type of labeling reagent is incorporated within the
peptide via nucleophilic attack of the labeling reagent by cysteine residues within
the peptide, which is referred to as “isotOpe-coded affinity tag” (ICAT).138'14° The
ICAT reagent consists of three sections; the biotin affinity tag, the linker which
contains either the light or the heavy label and the reactive portion of the
molecule. A newer version of the ICAT reagent is labeled with carbon-13 instead
of deuterium and contains an acid-cleavable linker attached the biotin tag.150

The introduction of the ICAT reagent can label two separate protein
mixtures (light and heavy), the two mixtures combined, and their proteins are
subsequently digested using trypsin. The pooled, labeled digests are first
purified using strong ion exchange chromatography (SXC) to remove excess
ICAT reagent, separated further by HPLC, and subsequently analyzed by MS to
Observe the mass difference in the same protein between the light and heavy
chain labels and determine relative abundance differences between the two
sample sets.150

ICAT has been successfully used in quantifying protein changes in
chromatin,151 mitochondria,152 and microsomes,153 even though there is an issue
regarding incomplete coverage with ICAT technology, since the labeling reagent
only reacts with cysteines, which are present in only 26.6% of the total human
tryptic peptides.154 This labeling approach has also been implemented in the

138,155

analysis of whole cell protein changes, including analysis of prostate cancer

40

 

cells 156 and pancreatic cancer juice.157 However, this technique is limited with
regards to phosphoprotein quantification as this reagent requires the presence of
both a cysteine and phosphorylated residue within the same peptide in order to
quantify the phosphoprotein.

Another approach similar to ICAT but with fewer limitations with respect to
phosphoproteome quantification is called iTRAQ, and is comprised of isobaric
amine-reactive tagging reagents capable of differentially labeling up to eight
samples.158 Pappin and co-workers utilized these reagents to monitor global
protein expression of both wild-type and mutant yeast strains.158 In regards to
quantification of phosphorylated proteins specifically, the iTRAQ approach has
been employed to perform a quantitative comparison between some enrichment
materials such as IMAC and Ti02 surfaces.159

Burlingame and coworkers utilized iTRAQ technology to quantitatively
monitor differences in the phosphorylation sites from proteins extracted from
various brain tissues such as the murine cortex, midbrain, cerebellum, and
hippocampus.160 The various brain tissues were digested, isotopically labeled,
pooled together, subsequently fractionated by SCX and the phosphopeptides
were enriched by TiOz and then analyzed by LC-MS/MS. The results provided
the quantification of 1564 phosphorylation sites on 831 unique proteins.

Additionally, isotopic labels can be incorporated while simultaneously
performing the enrichment. Goshe and co-workers, prepared two yeast protein
samples which underwent cysteine oxidation, B-elimination, and Michael addition

by either isotopically labeled ethanedithiol-D4 or ethanedithiol-H4 and the two

41

separate samples were then pooled together followed by biotin functionalized
affinity tag purification.118 The enriched peptides were subsequently analyzed to
compare differences in abundances of the enriched phosphopeptides. The
isotopic labeling and enrichment developed by Goshe allowed for not only
identification of new phosphorylated proteins, but also changes in abundance of
the same phosphopeptides within two different sample sets.

In another previously discussed enrichment strategy, Aebersold and co-
workers used EDC to condense a phosphopeptide on a dendrimer and quantified
the enrichment efficiency to be >35% by labeling the peptides via methyl

131 In

esterification using methanol (CH30H) or deuterated methanol (CD30D).
the same publication, Aebersold also performed this enrichment-labeling
procedure to determine tyrosine phosphorylation sites in human T cells (Jurkat
cells). The cells were treated with pervanadate for 2 or 10 minutes, then lysed
and immunO-purified using phosphotyrosine antibodies. The immunoprecipitated
samples were digested and the carboxylic acids were converted to their methyl-
do or methyl-da esters. The labeled peptides underwent the dendrimer
enrichment and the products were subsequently analyzed by microcapillary
reverse-phase LC-MS/MS. The analysis led to the identification and
quantification of 97 phosphotyrosine-containing proteins.131 Additional labels,
such as 18O, can be incorporated into peptides by 18O -Iabeled water during

161-163

proteolytic digestion and used to further quantify phosphoprotein

differences between two sample sets.

42

 

1.5.c Quantification of protein phosphorylation using a phosphospecific stain

Protein microarrays have been valid tools in the discovery and analysis of
protein phosphorylation as well as protein quantification. The concept of Pro-Q
Diamond dye, which as previously discussed, selectively binds to the phosphate
moiety Of the peptide and can enable the extent of phosphorylation in a sample
to be visualized in a microarray without the use Of fluorescently labeled
antibodies.137 Initial studies by Patton and co-workers employed this technique
using serial dilutions of four phosphorylated proteins and three unphosphorylated
proteins. The microarray slides were incubated with Pro-Q Diamond stain,
subsequently visualized, and quantified on a microarray analysis scanning
system. The stained phosphoprotein standards were quantified to determine if
Pro-Q Diamond provided linear dynamic range and the technique was found
suitable for the quantification Of proteins at unknown concentrations. The limit Of
detection was also determined for each of the phosphoproteins and found to vary
between 300-600 femtograms, depending on the protein.137

This approach was employed to detect kinase reactions on microarrays by
stamping three specific kinase substrates onto a polyacrylamide slide. The
kinase reactions were completed by incubating the array with the appropriate
kinase and ATP. The success of the phosphorylation, as a result of the kinase
assay, was determined by staining the array with the phospho—specific Pro-Q
Diamond dye. It was found that 156 picograms of phosphorylated peptide were
labeled with the Pro-Q dye, proving the validity of this approach to quantify

phosphoproteins and the changes that take place within the phosphoproteome.

43

This technique was also tested for specificity by arraying a thiophosphorylated
peptide against an in vitro phosphorylated tyrosine kinase peptide. The
sensitivity of detection by Pro-Q was found to be 10.4 picograms for the
phosphotyrosine and 325 femtograms for thiophosphopeptide, which confirmed
the stain’s specificity for the phosphate.137

An additional purification tool is a multidimensional separation system,
PFZD which can be used for separation and quantification of biological

samples‘“‘168

and specifically phosphopeptides when coupled with the Pro-Q
Diamond stain. The PFZD technique involves a peptide-(or protein) based 2-D
liquid phase fractionation, which has shown to be capable of high-throughput
analysis and can be fully automated, enabling excellent reproducibility and great
resolution. Liquid-based fraction collection can be utilized in large-scale
proteomic separation and profiling.169'170

The use of 2-D fractionation has also been employed to specifically
perform differential phosphoprotein mapping. The 2-D separation was carried
out by first separating the proteins by their pl via chromatofocusing (CF) and then
further separating the cell lysate by RP-HPLC.171 Two breast cancer cell
samples were cultured and either treated with a kinase inhibitor (P0173074) or a
DMSO control then harvested and 2-D separated. The separated fractions were
deposited on a microarray surface and stained with Pro-Q Diamond stain to
visualize the presence and relative changes between Iysate samples. Only the

fractions corresponding to the phosphorylated proteins detected in the array data

were subjected to digestion and subsequent MS analysis. Upon analysis it was

44

 

found the +25 phosphoproteins were identified and their difference in expression

levels between the two sets of lysates were characterized.171

1.6 Application of phosphoproteomic enrichment technologies in global
phosphorylation analysis

Aside from the initial application of the developed enrichment technologies
on commercially available proteins, is the importance of their subsequent use on
practical cellular samples. The significance of global phosphoproteomic analysis
is imperative in qualitatively studying the impact of a phosphoprotein and the
extent that its phosphorylation site has on the function Of healthy cells versus
pathogenic cells (i.e. tumorgenic). Global phosphoproteomic analysis allows for
the ‘whole picture’ view of the phosphoproteome, providing a more synergistic
and cooperative system identification. Instead of looking at one particular protein
of interest, global analysis provides information on the complete ramifications of
cellular changes occurring as a result of activation or inhibition of one particular
protein or pathway. This also provides insight on proteins relationship with one
another, either directly or through cross talk. A variety of phosphoproteomic
enrichment technologies have been employed either singularly or cooperatively
to tackle the sample complexity inherent in global analysis. The ultimate goal of
this phosphoproteome enrichment project Is its application towards elucidating
phosphorylation sites within a cellular protein of interest. Further elaboration of
this aspect of the project will be discussed in more depth in chapter four of this

dissertation.

45

 

1.6.a. The use of IMAC to characterize the phosphoproteome

Since IMAC has been the technique that has been the most widely used, it
is the most common approach employed to identify the global phosphoprotein in
a wide variety Of cell lines and tissue samples. In one of the preliminary global
phosphoproteome IMAC enrichment experiments, White and co-workers looked
at a colon adenocarcinoma cell line (HT-29) in an effort to profile the
phosphorylation sites. Characterization of the phosphopeptides within these
cells was accomplished by subjecting the lysates to IMAC capillary
chromatography and subsequently analyzing the fractions by LC-MS.172 The

general scheme for this enrichment is shown in Figure 1-7.

 

 

 

 

o f”;
(96) Cell lysis \ Protein digestion ‘5}
© © 0 l >|§ ”W \"
© © Trizol . - Trypsin I‘Ew’o't
HT—29 cells proteins peptides
IMAC Enrichment
MASCOT LC/MS/MS a“. \
Identified < I Separated _ 9’-
phosphopeptides phosphopeptides
attern phosphopeptides
explorer

Figure 1-7. Global phosphoproteomic analysis via IMAC enrichment (Scheme

adapted from figure in 172).

As a result of this method, 238 phosphorylation sites were found from HT-

29 cells. The experiment was run in duplicate to determine reproducibility, which

46

was found that about 60% of phosphorylated peptides from one analysis were
also identified in a duplicate analysis. The subsequent MASCOT automated
sequence explorer was found to give a ‘false-positive’ identification rate of 11.5
%, as a result of large mass errors. Most of the characterized phosphorylation
sites originated from phosphoproteins involved in basic structural support or
cellular function, such as transcription, translation and mRNA processing. There
were some characterized sites, however, that corresponded to proteins involved
in signaling pathways including Protein kinase C, AMP-activated protein kinase
0, p53, and MAPK 14."?-

In addition to the global study discussed above, quantitative
phosphoproteomic analysis was performed in cells observing the tumor necrosis
factor (TNF) pathway.146 While the impact of this study directly relates to the
pathway we are interested in and will be discussed in more depth in chapter four,
its importance in IMAC-based global phosphoproteome enrichment requires its
mentioning here. Briefly, whole cell extracts of stimulated and unstimulated cells
labeled with either 14N or 15N, digested and separated on a gel-free isoelectric
focusing instrument, which separated the peptides by their pl. IEF is a tool of
choice based on the general idea that upon phosphorylation the isoelectric point
(pl) of a protein or peptide will shift from a higher to lower value based on the
additional negative charge from the phosphate moiety. Further enrichment of the
IEF fractions collected at pH 3 and 4.6 were subjected to IMAC purification
followed by LC-MS. The enriched samples were subsequently pooled together

and analyzed by LC-MS/MS/MS. The results identified a total of 701

47

phosphopeptides between the triplicate analyses, characterizing phosphoserine,
phosphothreonine and phosphotyrosine form both the 14N and 15N isotopically
labeled fractions. From the MSIMS analysis, the comparison between the two
labeled fractions led to the quantification of 223 Of the phosphopeptides. This
study utilized quantification via isotopic labeling of followed by sequential
phosphopeptide purification by both isoelectric focusing and IMAC technologies.
Additional global phosphoproteome analysis using IMAC enrichment has
been explored by other researchers in a variety of cell lysates.68'100'1”"7‘5 The
global phosphoproteomic identification in prostate cancer cells, as reported by
Beranova-Giorgianni and co-workers, was performed by tryptic digestion Of the
whole cell extract and subsequent IMAC purification.175 The IMAC was
performed using gallium spin columns and both the flow-through and elution
fraction were collected and analyzed by LC-MS/MS. Upon analysis of the
fractions collected, 137 phosphorylation sites were identified from 81
phosphoproteins and out of the 137 phosphorylation sites, 124 sites have
previously been identified in various protein databases. For all of the identified
phosphopeptides, their corresponding proteins categorized by cellular function
and 35% of the proteins were involved with RNA processing and transcriptional
regulation, 17% for signal transduction and 14% for translation function. With
respect to biomarker identification, one of the phosphoproteins identified in the
enrichment study was the kinase BRAF, which is known to be mutated in about

7% of all cancers.177 Another identified phosphorylation site within the study

corresponded to heat shock 27-kDa protein (HSP27), which is a typically over-

48

expressed protein in prostate cancer and has been used as biomarker used to

determine poor prognosis in prostate cancer patients.178

1.6.b. The use of Ti02 in probing the phosphoproteome
Veenstra and co-workers employed Ti02 enrichment technique to probe

179 The cells were harvested and

the phosphoproteome of HeLa cells.
subsequently labeled with either 18O-water or 16O-water in the presence of
trypsin, to quantitatively measure phosphoproteome differences and the digests
were enriched for phosphopeptides using a Ti02 column followed by LC-MS
analysis.179 When no enrichment was performed prior to analysis, only 2.1% Of
the unique peptides identified were phosphorylated. This improved to 17.1% with
an enrichment step using 0.1% TFA as the washing solution. Improvement of
the enrichment protocol to 38.2% was achieved upon introduction of ammonium
glutamate as the washing solution. The subsequent MSIMS analysis identified
and characterized a total of 1034 phosphorylation sites from 858 unique
phosphopeptides representing 607 proteins. The characterized phosphorylated
proteins were found to have a variety of different cellular functions including
cytoskeleton maintenance, tumorigenesis, transcription and translational control,
cell proliferation and signal transduction.

The identified phosphopeptides were cross-validated by comparing the
MS2 and MS3 data Obtained to if both sets of data would result in identification of

the same phosphopeptide and phosphorylation site. The data was also analyzed

manually to determine the rate at which ‘false-positives’ are identified by the

49

automated system. From both sets Of analysis, 24% of the phosphopeptides
were identified by the MS2 and MS3 data, while 76% were identified by either
MS2 or MS3 data alone. 74 of the 204 identified phosphorylation sites within the
study had not been previously reported.179 There were many phosphorylation
sites found in this study that were also identified using the same cell line in global
phosphorylation study,69 confirming the validity of this approach in cellular
phosphoproteome analysis.

Recent global phosphoproteomic analysis has been performed by other
research groups, also employing TiOz enrichment, albeit preparing the material in
a different form in an effort to Obtain higher sensitivity and selective results."‘°'182
Ishirama and co-workers developed an on-Iine ZD-LC-MS system where they
manufactured a titania/C18 biphasic column to perform the enrichment of
collected HeLa Iysate trypsinized proteins. The subsequent MS analysis yielded
696 non-redundant phosphopeptides from 512 phosphoproteins.180

In a follow-up publication, Mann and co-workers applied a similar global
phosphoproteome purification study as previously discussed within this
dissertation,69 using SCX and Ti02 enrichment.183 Solid melanoma tumors were
homogenized, the Iysate extracted, digested and subsequently purified by either
SCX fractionation and Ti02 enrichment or multiple incubations of TiOz
enrichment and then analyzed by LC-MS/MS. The SCX-TiOz enrichment
sequence led to the identification of 3999 phosphorylation sites, corresponding to

3621 phosphoserine, 355 phosphothreonine and 23 phosphotyrosine. The

multiple TiOz incubations enrichment identified 2666 phosphorylation sites

50

 

consisting of 2385 phosphoserine, 266 phosphothreonine and 15
phosphotyrosine residues. Collectively, 2250 phosphoproteins were identified

containing a total of 5250 different phosphorylation sites.

1.6.c. Additional technologies used to determine global phosphorylation

While the affinity approaches listed above have proven to be the method
of choice in phosphoproteome analysis, there have been additional methods
employed to enrich cell Iysates that deserve to be recognized as well. In a
previously discussed publication, Aebersold performed the aforementioned
phosphoamidate-dendrimer enrichment and methyl esterification labeling
procedure to determine tyrosine phosphorylation sites in human T cells (Jurkat
cells). The cells were treated with pervanadate for 2 or 10 minutes, then lysed
and immunO-purified using phosphotyrosine antibodies. The immunoprecipitated
samples were digested and the carboxylic acids were converted to their methyl-
do or methyl-d3 esters. The labeled peptides underwent the dendrimer
enrichment and the products were subsequently analyzed by microcapillary
reverse-phase LC-MS/MS. The analysis led to the identification and
quantification of 97 phosphotyrosine-containing proteins.131

Another approach couples the separation ability of 20 gel electrophoresis
and an additional enrichment procedure, such as either phospho-specific
staining,"""185 IMAC,136 or immunoaffinity,"’7'188 followed by MS analysis to
identify phosphoproteins present in cellular extracts. The immunoaffinity

approach towards enrichment is an attractive approach for the identification of

51

 

 

phosphotyrosine residues as they are less abundant than phosphoserine and
phosphothreonine residues. This point is illustrated in the brief summary of some
global phosphoproteome studies on different human cell lines, using various
enrichment techniques all analyzed by MS, shown in Table 1-1.

The use of immunoaffinity for global phosphotyrosine analysis was shown
by Rush et al., who harvested Jurkat cells (leukemia cell line), digested the
proteins and enriched the phosphopeptides with immobilized anti-
phosphotyrosine antibodies.87 The enriched peptides were liberated from the
antibody and then separated and analyzed by LC-MS/MS. This strategy

identified 194 phosphotyrosine sites from 185 phosphopeptides.

 

 

 

 

 

 

 

 

 

Table 1-1.
. Unique
. Cellular Enrichment .
Ref. Cell lIne . . phos.peptides
fraction technique (pS e rszhrszyr)
pTyr Ab.- 9., .
._ pTyr proteins
131 Jurkat T cells Anti pTyr IP covalent 75 pTyr. sites
binding
Human HeLa SCX, then , ,
69 cells Whole cell “02 (4,901.670.103)
Human HeLa SDS-PAGE, phos.peptides
84 cells Nuc'ea' scx 1631219820
Human colon .
172 ad en 0 carcin cm a Whole cell IMAC 238 phos. srtes
174 Humeli(n5?2yeloid Whole cell IMAC 689 phos.peptides
Human prostate 137 phos. sites
175 cancer cells Whole cell IMAC 115 phos.peptides
Human
10° mesenchymal Whole cell SIMAC 306 monophos, 186
muItIply phos.
stem cells
Human HeLa . .
179 cells Whole cell TI02 858 phos.peptides

 

 

 

 

 

 

 

Table 1-1 (cont’d).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

C18-titania
18° Human HeLa Whole cell biphasic 2D— 696 phos.peptides
cells LC
Human
embryonic TiOz chip .
192 kidney 292T Whole cell based HPLC >100 phos.peptides
cells
Human liver Tissue .
102
tissue samples Iysate SAX 305 phos.peptrdes
Human Chang pTyr AD] 2- 16 phos.proteins
184 liver cells Whole cell DE 15 pTyr proteins
Human myeloid Multiple Anti- .
187 K562 Whole cell pTyr Ab. 67 pTyr peptides
Human A431 .
189 cells, xenograft Whole cell IMAC phgg-gpggllg es
tumors ' '
Human
19° mesenchymal Memtbrane TiOz 703 phosphopeptides
stem cells pro eIns
Jurkat T-cells/ . Combined: 209 phos.
19‘ A549 and HCT- Egg; SDmZPéGE' Sites, 185 phos.
116 peptides
P19 embryonal 472 phos. sites on
‘92 carcinoma cells Whole cell MAC 151 phos.proteins
Human HeLa 79 phos.peptides (97
145 cells Whole cell IEF” IMAC sites); TNF-regulated
268 phos.peptides
193 Jurkat T-cells Whole cell IMAC tot al.138 pT y r.
190 phos.peptides
19.. ”#3203232 Whole cell IMAC from 152
p phos.proteins
195 Human heart Tissue IM AC 75 phos. sites
tissue samples Iysate 47 phos.peptides
Table 1-1. Brief summary of some global phosphoproteome studies and the

enrichment tool employed

53

1.7 Current work

Clearly the phosphate moiety can be manipulated in a plethora of different
ways, as illustrated within this chapter. Many research groups employ the 8-
elimination-subsequent modification/enrichment material couplings and a few
additional groups have designed enrichment approaches targeting the
electrophilic character of the phosphate moiety. We have taken the opposite
approach and utilized both the nucleophilicity and acidity of the phosphate group
to selectively bind to a diazo functionalized solid support.196 The carboxylic acids
and C-terrninus are first protected via conversion into methyl esters, leaving the
most acidic proton within the peptide to be on the phosphate. Upon a proton
transfer by the phosphate to the diazo resin, the solid support becomes activated
as a result of the positively charged N2 leaving group. This activation allows for
the negatively charged deprotonated oxygen on the phosphate to attack the solid
support, simultaneously liberating N2 gas in an irreversible reaction. The
phosphopeptide can then be released from the solid support under acidic
conditions by hydrolysis of the phosphoester bond (Scheme 1-8). This approach
has successfully enriched a phosphorylated peptide from a complex protein

digest.

O R O

HzN—ggerj/ankéi/u‘ome

0 CH2 H
O. ,OH
9P
0 \OH
O
"NM—O
O R O
H O R o
HZN-Ei NW/IL J\ A H N- H
\le u EE OMe TFA 2 fix“, #Lngé/ILOMe
0 CH2 _. 0 CH2 H
°:p’°H o O. ,OH
0’ \ 9P
0 O \OH

Scheme 1-8. Solid phase phosphopeptide enrichment by a diazo-functionalized

polystyrene

In the upcoming chapters, I will address this approach to phosphopeptide
enrichment. The development of the synthesis of the various diazo-
functionalized materials and determination of their extent of phosphorylation will
be discussed. Additionally, all of the diazo-functionalized materials were all
evaluated for their phosphopeptide enrichment capabilities. The Optimal diazo
materials were then evaluated for the abilities to enrich phosphorylated peptides
from proteolytic digests of commercially available phosphoproteins. The effort
towards the application of this technology on a cellular system and future

directions will also be addressed.

55

10.

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cells and high confident phosphopeptide identification by cross-validation
of MSIMS and MSIMS/MS spectra. Journal of Proteome Research 2007,
6, (11), 4150-4162.

Imami, K.; Sugiyama, N.; Kyono, Y.; Tomita, M.; Ishirama, Y. Automated
phosphoproteome analysis for cultured cancer cells by two-dimensional
nanOLC-MS using a calcined titania/C18 biphasic column. Analytical
Sciences 2008, 24, (1), 161-166.

Li, Y.; Xu, X. Q.; Qi, D. W.; Deng, C. H.; Yang, P. Y.; Zhang, X. M. Novel
Fe304@Ti02 core-shell microspheres for selective enrichment of

phosphopeptides in phosphoproteome analysis. Joumal of Proteome
Research 2008, 7, (6), 2526-2538.

74

-.-—-.—._,.

182.

183.

184.

185.

186.

187.

188.

189.

190.

Mohammed, S.; Kraiczek, K.; Pinkse, M. W. H.; Lemeer, S.; Benschop, J.
J.; Heck, A. J. R. Chip-based enrichment and nanoLC-MS/MS analysis of
phosphopeptides from whole Iysates. Journal of Proteome Research
2008, 7, (4), 1565-1571.

Zanivan, S.; Gnad, F.; Wickstrom, S.; Geiger, T.; Macek, 8.; Cox, J.;
Fassler, R.; Mann, M. Solid tumor proteome and phosphoproteome
analysis by high resolution mass spectrometry. Journal of Proteome
Research 2008, 7, 5314-5326.

Kim, J. H.; In, Y. J.; Kim, W. K.; Bae, K. H.; Kang, 8.; Lee, S. C.
Differential signatures of protein glycosylation and phosphorylation in
human chang liver cells induced by TCDD treatment. Toxicology Letters
2008, 178, (1), 20-28.

Kristjansdottir, K.; Wolfgeher, D.; Lucius, N.; Angulo, D. S.; Kron, S. J.
Phosphoprotein profiling by page-LC-MS/MS. Journal of Proteome
Research 2008, 7, (7), 2812-2824.

Dubrovska, A.; Souchelnytskyi, S. Efficient enrichment of intact
phosphorylated proteins by modified immobilizes metal-affinity
chromatography. Proteomics 2005, 5, (18), 4678-4683.

Lind, S. B.; Molin, M.; Savitski, M. M.; Emilsson, L.; Astrom, J.; Hedberg,
L., et al. lmmunoaffinity enrichments followed by mass spectrometric
detection for studying global protein tyrosine phosphorylation. Journal of
Proteome Research 2008, 7, (7), 2897-2910.

Zheng, H. Y.; Hu, P.; Quinn, D. F.; Wang, Y. K. Phosphotyrosine
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of immunoprecipitation and immobilized metal affinity chromatography.
Molecular & Cellular Proteomics 2005, 4, (6), 721 -730.

Stover, D. R.; Caldwell, J.; Marto, J. A.; Root, K.; Mestan, J.; Stumm, M.,
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treated human tumor cells and mouse xenografts. Clinical Proteomics
2004, 1, 69-80.

Thingholm, T. E.; Larsen, M. R.; lngrell, C. R.; Kassem, M.; Jensen, 0. N.
TiOz-based phosphoproteomic analysis of the plasma membrane and the
effects of phosphatase inhibitor treatment. Journal of Proteome Research
2008, 7, (8), 3304-3313.

75

191.

192.

193.

194.

195.

196.

Wissing, J.; Jansch, L.; Nimtz, M.; Dieterich, G.; Homberger, R.; Keri, G.;
Wehland, J.; Daub, H. Proteomics analysis of protein kinases by target
class-selective prefractionation and tandem mass spectrometry. Molecular
& Cellular Proteomics 2007, 6, (3), 537-547.

Smith, J. C.; Duchesne, M. A.; Tozzi, P.; Ethier, M.; Figeys, D. A
differential phosphoproteomic analysis of retinoic acid-treated p19 cells.
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Brill, L. M.; Salomon, A. R.; Ficarro, S. B.; Mukherji, M.; Stettler-Gill, M.;
Peters, E. C. Robust phosphoproteomic profiling of tyrosine
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2004, 76, (10), 2763-2772.

Gevaert, K.; Staes, A.; Van Damme, J.; De Groot, S.; Hugelier, K.; Demol,
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Lansdell, T. A.; Tepe, J. J. Isolation of phosphopeptides using solid phase
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76

CHAPTER2

SYNTHESIS OF DIAZO FINCTIONALIZED SOLID SUPPORTS AND

QUANTIFICATION OF THE DEGREE OF FUNCTIONALIZATION
2.1 Reactivity Of diazo materials with Bronsted acids

As discussed briefly in the previous chapter, the goal of this project is to
develop materials capable of selectively and covalently binding to the
phosphate moiety of phosphorylated peptides. There are many moieties that
can, and have, been used to react with phosphates and more specifically,
phosphopeptides by manipulating the electrophilic character of the phosphate.
In particular, there are additional approaches used to obtain covalent binding
between a solid support and a phosphate. Specifically, a phosphate can
become succeptable to nucleophilic attack upon the employment of a
carbodiimide, in a similar fashion as a carboxylic acid. Additionally,
nucleophilic attack may be obtained upon the implementation of
triphenylphosphine to the phosphate. These approaches of employing a
carbodiimide“2 or phosphine3 to activate the phosphate moiety have been
applied to mediate the covalent coupling between the phosphate and a
nucleophile-bound solid support, which then was applied towards
phosphopeptide enrichment.

As an alternative to manipulating the electrophilic character of the
phosphate, deprotonation of the phosphoric acid can result in a nucleophilic
oxygen, leading to further reactivity. A moiety that can both act as a proton

acceptor, while also harboring a site succesptable towards nucleophilic attack

77

led to the exploration of diazo-functionalized materials and their ability to
covalently bind to phosphates.

Specifically, diazo carbonyl groups were explored as the adjacent
carbonyl adds additional stabilization of the diazo moiety. While this stability
of diazo carbonyl compounds enables the ease of preparation of these
molecules, they are still reactive towards a myriad of different moieties.“'5 The
synthetic utility and versatility of diazo carbonyl compounds has been
illustrated in countless publications and elegantly reviewed by Ye and
McKervey.4

Although solution chemistry exploiting the diazo carbonyl moiety has
been widely explored, limited work has been published pursuing the synthetic

6'10 The main diazo solid support

application of this moiety on solid supports.
used for synthetic applications have been a-diazo-B-ketoesters. Upon the
synthesis of these supports, their subsequent application was aimed towards
the preparation of a variety of heterocycles including oxazolones,8 oxazoles,11

2 imidazoles,13 pyrazinones and pyrazines.14 The

indoles,7 imidazolones,1
preparation of these molecules enabled by the reactivity of the diazo carbonyl
resulted in a metal-catalyzed N-H insertion, while liberating N2 from the
support. Additional diazo-functionalized solid supports have been
implimented to prepare a variety of compounds by nucleophilic addition of an
alcohol to the a carbon of the diazo support, again releasing N2 gas.6

The reactivity of the diazo carbonyl moiety towards nucleophilic addition

with Brcnsted acids has also been documented. The addition of hydrogen

78

halides to a diazo carbonyl results in the preparation of a-halogenated
ketones.15 The addition of a Bronsted acid towards an a-diazo carbonyl in
Scheme 2-1 illustrates the potential of these materials to react with
phosphoric acids (i.e. phosphorylated peptides) for enrichment applications,

which will be discussed at more length later on.

Reactive form

 

 

W Riki
H H H—rA‘
0

Scheme 2-1. Reactivity of diazo carbonyl moieties towards acids

The protonation of the negatively charged carbon in the above resonance
form of the diazo carbonyl leads the material to susceptibility towards
nucleophilic attack at the a-carbon. Upon the addition of the conjugate base
nucleophile, N2 gas is simultaneously liberated. This results in the a-substituted
conjugate base carbonyl product, such as the a-halogenated ketone discussed
previously. It can then be hypothesized that additional acidic reagents can be

used in a similar fashion.

79

2.2 Concept of diazo-phosphate binding

The idea behind the covalent binding between the diazo moiety and the
phosphate is identical to the use of the Brensted acid in Scheme 2-1; the
acidic proton on the phosphate (pH 2.8) activates the diazo carbonyl species
followed by the subsequent nucleophilic attack by the corresponding
negatively charged oxygen (i.e. the conjugate base). Theoretically, the
covalent binding of the a carbon by a phosphoric acid or a phosphorylated
peptide should react in the same manner as any other Brcnsted acid, shown
in Scheme 2-2. It is hypothesized that upon introduction of an immobilized
diazo carbonyl material to a phosphoric acid (or phosphorylated peptide), the
subsequent covalent binding (and immobilization) of the phosphate will then
allow for its purification from any unbound materials by filtration. This
hypothesis led to the both concept and development Of the synthesis of diazo-
functionalized materials immobilized on solid supports. The use of these
materials in covalent phosphate binding would provide a means of purifying
phosphopeptides from a complex mixture, such as a proteolytic digest. The
importance and utility of developing a selective phosphopeptide enrichment

approach was illustrated in Chapter 1.

80

Reactive form
R
O O O H
(+3.,N ®2N HZN-gg NW/IL J\ )L
RJKfN R/U\(?,N + \H’ u §§ OMe
H

H

 

 

RJK/Ox /O

/
HO, \9
H CH2 0

MGOTHYNqu’lkEE-th + ~21

O R 0

Scheme 2-2. Theory behind diazo carbonyl-phosphate (phosphopeptide)

binding.

chemistry that were addressed in Chapter 1.16

It can also be reasoned that this covalent enrichment approach to purify

phosphopeptides can reduce the potential specificity problems associated with

affinity chromatography and the limitations inherent to phosphate elimination

81

The current covalent

phosphopeptide enrichment strategies typically include the elimination of the
phosphate moiety from serine or threonine, under alkaline conditions, creating
a Michael acceptor.2'17’19 This allows the peptide to be further modified at the
site of the eliminated phosphate moiety. Disadvantages to this approach
include undesired reactions at the site of glycosylated amino acids within the
peptides, dehydration of unmodified serine or threonine residues, and the
inability to functionalize phosphotyrosine sites within peptides. As mentioned
earlier in this chapter, other covalent enrichment strategies have been
reported that utilize the electrophilic character of the phosphate moiety by
employing a carbodiimide“2 or phosphine3 to mediate the coupling between
the phosphate and a nucleophilic solid support.

It can be envisioned that a diazo-functionalized material can covalent
bind to the phosphate moiety of a phosphopeptides and be applied towards
the enrichment of phosphorylated serine/threonine and tyrosine containing
substrates. The use of a diazo-functionalized solid support to react
specifically with the phosphate moiety would allow for non-phosphorylated
peptides to be efficiently removed by filtration. Liberation of the
phosphorylated substrates from the resin can be accomplished using aqueous
acid to hydrolyze the phosphoester bond, while still keeping the phosphate
intact. The enriched phosphopeptides can be readily analyzed and

characterized by tandem mass spectrometry.

82

 

2.3 Original synthesis of diazo functionalized solid support

The implementation of solid phase enrichment and solid phase
purification techniques have become increasingly common in the chemical
and pharmaceutical industries.’"°'23 One of the advantages of solid support-
mediated purification approaches is the ability to easily isolate the product by
filtration with subsequent removal of any unbound materials by washing.
Further purification can be Obtained by selective/specific cleavage of the
product Off the solid support. Numerous solid phase purification/enrichment
techniques for chemical reactions have been reported in the literature to
illustrate the utility of this approach in synthetic organic chemistry?“30
Applications of this technology in the form of immobilized reagents have also
been applied in total syntheses.“32

The biological equivalent of solid phase enrichment in the field of
proteomics has been illustrated by various covalent and non-covalent affinity
chromatography techniques. One of the most common solid-phase
purification strategies of biological samples is immunoaffinity
chromatography?”4 This approach is used to purify proteins by binding to its
immobilized antibody.35 Additional solid-phase purification on biological
samples has been performed by exploiting the strong biotin-avidin interaction
in which a protein or antibody Of interest is covalently bound to biotin and
subsequently purified on immobilized avidin.36 Additional examples of

purification strategies for biological molecules include synthetically or

metabolically driven modifications of proteins followed by subsequent covalent

83

addition by a reactive affinity probe/solid support?”0 This has proven to be
an effective tool in protein labeling and protein identification.

The use of solid phase enrichment to selectively purify/enrich specific
post-translational protein modifications, however, remains less developed.
The application of a functionalized solid support to specifically purify a post-
translational modified protein from its unmodified counterpart could provide
unprecedented insight into protein mediated cell signaling.

As illustrated above, our approach is based on the reaction between the
diazo functionalized material and the acidic character of the phosphate
moiety, allowing for covalent capture of the phosphate functional group. In
this approach, the covalent enrichment is performed on a solid support.
Protonation of the a carbon of the diazo moiety by the phosphate moiety is
followed by nucleophilic attack by the deprotonated oxygen of the phosphate

on the a carbon resulting in the liberation of N2 (Scheme 2-3).

Scheme 2-3. Scheme for the formation of the covalent phosphoester bond.

Utilizing the benefits of solid-phase organic reactions, this enrichment

approach employs the functionalization of the reactive diazo carbonyl moiety

84

bound to a solid support, allowing for the subsequent purification/removal of the
the peptides that do not contain a phosphate moiety.

The initial synthetic strategy to form a diazo-functionalized solid support
employed the procedure previously used within the Tepe group to prepare a
diazo acetate Wang resin (Scheme 2-4).41 A fluoren-9-ylmethoxycarbonyl
(Fmoc)-glycine protected Wang resin first underwent liberation of the Fmoc
group in the presence Of piperdine to form the resulting free amino-
functionalized Wang resin. The free amine was then oxidized to the
corresponding diazo-functionalized Wang resin upon exposure to the HONO

gas generated from sulfuric acid and sodium nitrite.

“Che .
04L
NHFmoc

1.) 20% piperdine,
DMF

2.) NaNOz, H20,
H2804, CH2CI2

Woes .
O

£N2

Scheme 24. HONO gas mediated diazotization of an amino-functionalized

resin.

Upon the formation of the diazo-functionalized Wang resin, the material

was filtered to remove any reagents or solvent. Confirmation of the formation of

85

the diazo moiety was confirmed by IR, indicating a corresponding diazo stretch at
2110 cm". While this method was capable of preparing the desired diazo-
functionalized material, the diazo IR stretch was never one of the most dominant
stretches even though it is supposed to be a very strong stretch. It is
hypothesized that the reason for the small peak illustrates a poor conversion of

the free amine support to the diazo material.

2.4 Problems associated with original studies

A potential reason for the poor diazotization could be a result of the nature
Of the reaction; incorporation of a gas across a solid support. As described in the
previous section, the preparation of the diazo-functionalized solid support was
Obtained by the addition of H2SO4 to a solution of sodium nitrite. Upon the
addition of acid, HONO gas was generated and bubbled into a vented flask
containing dichloromethane and the amino-functionalized resin (Figure 2-1).
The reaction vessel/s in Figure 2-1 illustrates why set-up was time consuming
and the nature of the reaction required the absence of water (or any other proton
source) in the secondary flask containing the resin, which was accomplished
upon purging the system with nitrogen gas.

Although this procedure did afford the corresponding diazo resin, the
conversion was variable at best and the diazo stretch present in the IR was
modest (see Appendix, Figure 2-2). Additionally, the reproducibility of this
approach was challenging as one day the reaction would afford a modest diazo

stretch, while another day the reaction would yield a significantly smaller diazo

86

stretch. The greatest limitation of this approach was the procedure could only be
applied to primary amino functionalized materials, which limited the scope of the
materials that could be employed and their subsequent further application

towards phosphopeptide enrichment.

 

Amino resin
H2804, HNO3 in DCM

Figure 2-1 Set-up for diazotization of the amino functionalized resin.

As a result of the aforementioned limitations Of this approach, this
prompted the effort to develop an improved route to obtaining diazo-
functionalized solid supports. While developing a new synthetic strategy,
emphasis was placed on developing a route to be applied to a multitude of
materials. Additionally, a more “user-friendly" set-up that could be performed in a

one-pot manner was also desired.

87

2.5 Improved synthesis Of diazo acetate solid support

In the initial effort to move towards a synthetic strategy that could be
applied to a multitude of materials, work by Ouihia and co-workers began the
concept of a new approach. In their work, they performed a coupling of N-
hydroxylsuccimamide to glyoxylic acid p-toluenesulfonyl hydrazone in the
presence of dicyclocarbodiimide.42 Upon the coupling of these two materials, the
hydrazone was subsequently deprotonated, resulting in the liberation of the tosyl
moiety and the formation of the diazo acetate functionalized succinimide
(Scheme 2-5). While they used the prepared diazo materials for other synthetic
applications, this approach however, provided a potential alternative to diazo

moiety preparation.

" DCC
QN-OH + HOJVNNRQ ———> N-O
H O
O 0

Scheme 2-5.Diazotization reaction using glyoxylic acid p—toluenesulfonyl

hydrazone.

This reaction led to pursuing glyoxylic acid p-toluenesulfonyl hydrazone as
a potential source of forming the desired diazo acetate moiety. Upon further
exploration of the use of this material to prepare diazo-functionalized materials,
additional methods were also found. Modifications of those approaches towards
the solid support synthesis of the diazo moiety were also investigated. To

explore this avenue, however, first the glyoxylic acid p-toluenesulfonyl hydrazone

88

must be synthesized. The material was prepared as previously described by
Blankley and co-workers by coupling glyoxylic acid and p-
toluenesulfonylhydrazide in the presence of hydrochloric acid to obtain the
hydrazone product (Scheme 2-6). Compound 2-1 was purified by
recrystallization in 1:2 ethyl acetatezcarbon tetrachloride. This material was

prepared on a large, 20 9 scale, with no problems, in a 70% yield.43

0 O O

H 26MHO 9
H + H2N\ ’8 ; N

O

 

Scheme 245. Synthesis of glyoxylic acid p-toluenesulfonyl hydrazone.

Upon the preparation of compound 2-1, its carboxylic acid moiety was
activated in the presence of diisopropylcarbodiimide and then subsequently
coupled to the Wang resin to form the diazo Wang support in a similar manner to

the reaction used by Oihua and co-workers.

89

N=C=N

 

ll

WOQTOJQ

Resin 2-1
Scheme 2-7. Synthesis of diazo acetate Wang resin using glyoxylic acid p-

toluenesulfonyl hydrazone and and carbodiimide.

The reason for the use of diisopropylcarbodiimide instead of
dicyclohexylcarbodiimide (000) was that the dicyclohexylurea by—product formed
as a result of the carboxylic acid-DCC coupling, crashes out in the reaction.
Typically, this is advantageous in solution-phase reactions. However, as the
product is a resin and purification after the reaction is obtained by filtration, a
different carbodiimide, whose resultant urea by-product is soluble in the solvent,
was desirable.

The initial reaction was run overnight in dioxane, using 6 eq. of the
glyoxylic acid p—toluenesulfonyl hydrazone, which did not afford any of the
desired diazo-functionalized product. Different time points (1, 2, 3 and 4 hr) and
solvents (THF and DMF) were also tried. The conditions that appeared to

indicate the largest diazo stretch were at 2 and 3 hr in DMF (see Appendix,

90

Figure 24). Additionally, a different coupling reagent, EDCI, was used instead
of DIC for different time points (45 min and 4 hrs.) at 0 °C and room temperature,
both with and without lithium hydride. The optimal conditions for the diazotization
were found to be using EDCI, 1.1 eq LiH for 45 minutes at room temperature in
DCM (see Appendix, Figure 2-5). It is believed that the UH was required to
deprotonate the hydrazone to allow for its decomposition in forming the diazo
product.

The IR data still indicated a prominent broad OH stretch at ~3400 cm",
which could be a result of un-reacted starting material. This led to the
exploration of converting the carboxylic acid to a moiety with more reactivity than
with the use of a carbodiimide. The use of 1-chloro-N,N-2-trimethyl-1-
propenylamine has been employed as a chlorinating agent to convert the
carboxylic acid into an acid chloride.”45 As illustrated by Corey and co-workers,
the preparation of the glyoxylic acid chloride p-toluenesulfonyl hydrazone led to
subsequent coupling to an alcohol in the presence of base to obtain the diazo
acetate moiety (Scheme 2-8). Corey then used the prepared diazo acetate to
perform further chemistry.

This approach though, to our knowledge, had not been previously
attempted on a solid support. We believed that it could be applied towards the
synthesis of the desired diazo acetate Wang resin. If this approach was
successful, it could also be applied to additional materials containing either a

nucleophilic oxygen or nitrogen.

91

 

Resins 2-1, 2-2

Scheme 2-8. Optimized route for the formation of diazo Wang resin.

The desired diazo acetate Wang resin was prepared using the glyoxylic
acid chloride p-tosylhydrazone approach outlined above, instead of the
aforementioned previously reported diazotization using HONO gas.41 The free
hydroxyl moiety on a Wang resin (0.89 and 2.2 mmol/g loading) was
converted into the diazo-functionalized solid support in the presence of
glyoxylic acid p-tosylhydrazone,43 1-chloro-N,N—2-trimethyl-1-propenylamine,
N,N-dimethylaniline and triethylamine to afford resins 2-1 and 2-2 (Scheme 2-
8), respectively.“*44

Confirmation in the formation of the diazo-functionality was readily
obtained using IR, in which the diazo stretch has a distinct absorption at 2107

cm'1 (see Appendix, Figures 2-6, 2-7). The yields of the diazo-

functionalization on the solid supports were determined in a modified

92

approach to a previously reported procedure.46 The quantification procedure

and its results will be discussed in a later section.

2.6 Application of improved synthesis to functionalize additional diazo
supports
Once the success of the diazotization using glyoxylic acid chloride p-
tosylhydrazone had been confirmed on the diazo acetate Wang resin by IR,
implementation of this synthetic approach was applied to additional resins
containing hydroxyl moieties. Additionally, it was believed that this route could
also be employed to nucleophilic amino functionalized materials as well, as

illustrated in Scheme 2-9.

9 >::<Cl 0
0—04 JVQTW {N2

R = OH 0' NH? ©TEA Resins 2-3 2- 14

Scheme 2-9. The application of the improved synthesis for both hydroxyl and

amino functionalized solid supports.

The synthetic strategy used to prepare the diazo-functionalized Wang
resin was applied to a range of additional solid supports containing either
hydroxyl or amino moieties (Scheme 2-9, Table 1). A variety of different diazo-

functionalized supports resins 2-3 thru 2-14 were successfully prepared using

93

 

this approach (Figure 2-2) and were also analyzed by IR (diazo stretch at ~2110
cm") to confirm the formation of the diazo moiety (see Appendix, Figures 2-8

thru 2-19 for resin IR data).

94

O

0 “<1. w,
W Q 2 0'54 2

0.89 mmol/g subst. Resin 2-1 Resin 2-3
2.2 mmoI/g subst. Resin 2-2
O O
HN—<‘ HN—<'
Q—©—/ —N2 PEGA —N2
Resin 2-4 Resin 2-5
0 N2

0
NH 0
-/\u/u\/O\©\l o If HN
o \ F)—
N
If 2

HN OJNH

o .
O—_©_j Resun 2-7
_<-_-N2

Resin 2-6

 

 

 

 

 

 

 

 

0 N2 Resin 2-9
0 o
0— PEG r/O—<=N2 0— PEG Jhkko
Resin 2-10 Resin 2-11

 

 

 

 

0— PEG WOWNZ h
0 O 0 N2

Resin 2-12
N
(H3c0)3HSi/\/\o/\K\o/u\¢ 2
O O O
(H3CO)3Sl_/_>Nf O RBSiI‘I 2-13 \E
\ N2

Resin 244 N2

Figure 2-2. Diazo-functionalized solid supports prepared using Scheme 2-9.

95

2.7 Synthesis of diazo ketone solid supports

The reactivity of the diazo-functionality in resins 2-1 thru 2-14 is in large
mediated by the resonance stabilization with the neighboring carbonyl
moiety.5 As a result, diazo ketone supports were also explored for their
potential use in phosphopeptide enrichment. Subsequently, diazo ketone
derivatives 2-15 thru 17 were prepared by the addition of oxalyl chloride to a
carboxylic acid functionalized solid support. Upon formation of the acid
chloride, trimethylsilyl diazo methane was added to achieve the diazo ketone.6
The subsequent work-up for the reaction resulted in liberation of the labile
TMS group to afford resins 2-15 thru 2-17. (Scheme 2-10). Confirmation of
the diazo moiety was confirmed by the IR stretch at ~2100 cm'1 (see
Appendix, Figures 2-20 thru 2-22 resin/silica IR data). Quantification of the
extent of diazotization was performed in the same manner used for the
previously made diazo acetate and amide solid supports. Quantification and

its corresponding results will be discussed in a later section.

0

a C : OH benzene ——Ci>a C :: Cl 1:1 THF/ACN a 3 :NZ

reflux, reflux, 50 hr
16 hr

 

Resins 2-17 — 2-20

Scheme 2-10. The synthesis of diazo ketone resins.

96

2.8 Synthesis of phenyl diazo methane solid supports

Upon the successful formation of the diazo ketone functionalized solid
support, we decided to further explore the range of reactivity of the diazo
moiety, as we replaced the neighboring carbonyl moieties with a phenyl group.
Clearly this substitution would reduce the stability of the diazo moiety,
however, it could yield more reactive towards the phosphate. The phenyl
diazomethane resins were prepared using a modified approach as previously
reported.47 Using three different polymer-bound aldehyde starting materials
(mesh size 50-100, 100-200, and 200-400), each polymer was introduced to
2,4,6-triisopropylbenzenesulfonyl hydrazide to form the corresponding
hydrazone. The resulting hydrazone was heated in anhydrous methanol and
potassium hydroxide, then within minutes the diazo functionalized material
was prepared (Scheme 2-11). It should be noted that the triisopropyl groups
were required on the benzene ring to afford the corresponding product. The
use of benzenesulfonyl hydrazide in the coupling to the aldehyde and
subsequent degradation to the desired diazo product was not obtained. This
coincides with previous work done by Reese and co-worker, who performed
this reaction in solution phase and noticing a drop in yields when a less alkyl

substituted aryl ring was employed.48

97

O
I
N
,2
\
O=CD=O
.4
I
'n
23:
O

+ N _.. , \ll
H H 16hr NI fi
O—cn/Hi 0

KOH in

MeOH

90°C

N2

Resins 2-18 - 2-20

Scheme 2-11. Synthesis of phenyl diazo methane polystyrene.

Confirmation of the diazo moiety was confirmed by the IR stretch at
~2050 cm'1 (see Appendix, Figures 2-23 thru 2-25 resin/silica IR data).
Quantification was performed in the same manner as the aforementioned

materials and its corresponding results will be discussed in the next section.

2.9 Quantification of solid support diazo functionalization

Upon the successful preparation of the twenty different diazo-
functionalized solid supports 2-1 thru 2-20, an effort went in to develop an
approach to determine the degree of functionalization for each material. This
became less straight-forward than initially anticipated, as quantification of
solid supports can be potentially more challenging than for solution materials.
As such, unique techniques or methods can be employed to obtain
reproducible results.”50 While quantification by IR was attempted, it proved
to be inconsistent, which required the development of an alternative method to

98

obtain accurate representative yields of conversions.46
The implementation of the incorporation of fluoren-9-
ylmethoxycarbonyl (Fmoc) as a means of quantification has been shown in

“5'51 Its use has also been cited to characterize the

previous publications.
degree of functionalization in commercially available polymers. An
established protocol that quantifies Fmoc-derivatized solid supports by
measuring the amount of liberated dibenzofulvene (DBF) was modified to
quantify the amount of diazo functionalization for each solid support.

To determine the overall conversion of the starting alcohol, amine or
carboxylic acid resin to its corresponding diazo functionalized solid support,
an excess of Fmoc-glycine in DMF was added to obtain the Fmoc coupled
resin product in a similar covalent binding manner to that of phosphopeptides.
Initially, the Fmoc-glycine solution coupling was extended to two days,
however, the resin was analyzed by IR and found a substantial amount of
diazo still present (IR stretch at ~2000-2130 cm"). It is believed that this
could be due to the difference in acidity of the carboxylic acid (pKa ~5) versus
a phosphoric acid (pKa at 2.8).

As a result of the hypothesis, the coupling was attempted again in the
presence of an equivalent of p-toluene sulfonic acid. Upon reaction
monitoring by IR, there was no diazo stretch present after 4 hours. These
conditions were then introduced into two separately synthesized batches of

each support. The reaction afforded the coupling of the Fmoc-group to the

diazo material. The Fmoc-glycine addition to the diazo-functionalized

99

materials was confirmed using IR, resulting in the disappearance of the diazo
stretch at 2100 cm'1 and the addition of a new ester stretch at 1720 cm”. The
coupled product subsequently underwent liberation of the DBF group in the
presence of DBU, which typically proceeds in a quantitative conversion
(Scheme 2-12). The use of an Fmoc-glycine standard curve was employed
for both batches of each solid support to determine the amount of released

DBF (see Appendix, Figure 2-27).

0
O Hoi/HYO .0 O
O—DJVNZ 0 s O—CI/‘H

TsOH (cat)

 

5% DBU in
DMF

DBF

C02 + O—D/EL’OVNHZ + 000

l I

Measure concentration
at 304 nm

Scheme 2-12. Quantification of diazo functionalized solid support conversion

using Fmoc-glycine coupling.

100

The amount of released DBF was converted back to the total Fmoc-
glycine bound to the solid support, which corresponded to the amount of
Fmoc-glycine that reacted with the diazo moiety. The amount of liberated
DBF was then compared to the manufacturers loading value for the starting
material to determine the overall percent diazo conversion. Quantification of
each support was performed in two separate trials and then reported as an
average (Table 2-1). While these values did indicate high reproducibility, it
should be noted that these % diazo conversion values are a “minimum level of
conversion” rather than % yield of diazo-formation, as they are a result of
liberated DBF, not diazo formation.

Out of all the resins (2-1 thru 2-12) prepared as shown in Scheme 2-9,
the material found to have the largest percent diazo conversion was the
dendrimer (2-7). It is hypothesized the reason for this is due to the length of
the linker thereby increasing the accessibility of reagents in both the diazo
functionalization and the Fmoc derivatization. In general, it appears that the
mesh size is involved in the variability of functionalization for resins 2:1-12 as
most of the materials around 100-200 mesh size appear to be the most
functionalized (resins 1, 7, 9, 11). It is surprising that resins 1 and 2, however,
are not closer in % conversion, as the only difference is in their level of
substitution. This could be a result of a loss in accessibility of the hydroxyl
moiety in the more functionalized support. Resins 4 and 8 have mesh sizes
outside of the 100-200 range, which could explain their relatively low

conversion as a result of poor interaction with the reagents as a result of their

101

mesh size. The poor conversion of resin 12 is likely due to the relative
instability of the diazo anhydride product formed. The extent of
functionalization of the diazo ketone supports 15-16 appear to correlate
directly with their degree of substitution, which illustrates the likelihood that
reagent accessibility is relative to each materials’ level of substitution. The
diazo ketone silica 17 appears to be more accessible as its’ percent diazo
conversion is the highest out of the three diazo ketones. The mesh size
hypothesis also holds up when comparing the degree of functionalization for
the diazomethylbenzene resins 18-20 as well, as resin 19 was found to have

the highest degree of functionalization.

102

Table 2-1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Resin Solid support % diazo conversion

1 Diazo wanflesin (0.89 mmoI/g) 72 1 8
2 Diazo wang resin (2.2 mmoI/g) 46 13
3 Diazo amide tentagel 52 1 2
4 Diazo amide megabead 27.8 1 0.1
5 Diazo amide PEGA resin 76 1 2
6 Diazo amide HMPA PEGA resin 53 1 6
7 Diazo amide dendrimer 80 1 3
8 4-diazo acetate polystyrene 25.9 1 0.5
9 Diazo acetate polystyrene 68 1 2
10 Nova Syn TG diazo acetate 40 1 3
11 Nova Syn TG HMP diazo acetate 72 1 8
12 Nova Syn diazo anhydride 23 1 10
13 Diazo acetate silica N/A*
14 Diazo amide silica N/A*
15 Diazo ketone resin (1.0 mmomq) 22.3 1 0.1
16 Diazo ketone resin (2.0 mmol/gL 36 1 7
17 Diazo ketone silica 41 1 3
18 Diazomethylbenzene (50-100 mesh) 39 1 3
19 Diazometfylbenzene goo-200 mesh) 81 1 12
20 Diazomethylbenzene (200-400 mesh) 55 1 2

 

 

Table 2-1. Average percent diazo functionalization results from two separate
batches of each diazo resin using Fmoc-coupling quantification.
*The following materials were generously donated and starting material

loading values were not available.

103

2.10 Experimental procedures

Materials. All organic chemical reagents were obtained from Sigma Aldrich
Chemical Co. All polystyrene resins were purchased from either Novabiochem,
Sigma Chemical Co., or Advanced Chemtech. Carboxylic acid silica was
purchased from Silicycle. The hydroxyl and amino functionalized silica was

generously provided by the Pinniavaia lab.

Initial diazo Wang resin synthesis.

. O OQJOJLNZ

Commercially available Fmoc-Glycine Wang resin 100-200 mesh (0.5 g) was
added to 20% diperidine in N,N-dimethylformamide (10 mL). The resulting
mixture was stirred at 25° C for 2 hr. The product was filtered off with N,N-
dimethylformamide (3x 30 mL) and dichloromethane (3x 30 mL), collected, and
placed in dichloromethane (10 mL). An adjacent flask containing NaNOz (4.6 g)
in H20 (10 mL) was stirred at 25° C when 10% HZSO4/H20 (5 mL) was added.
The resulting HONO gas produced, was bubbled into the dichloromethane/resin
mixture. After 17 minutes at 25° C, the resin was filtered off, washed with
dichloromethane (4x 30 mL), collected, and stored at 0° C. The resin was
characterized by IR.

IR: 2105 cm" (diazo stretch) KBr pellet

104

Diazo Wang resin synthesis using diisopropylcarbodiimide.

w O OGJOJL—“Z

Commercially available Wang resin (0.1 9, ~02 mmol) was added to a solution of
glyoxylic acid tosylhydrazone (0.24 g, 10 equiv.) in N,N-dimethylformamide (10
mL) and the solution was stirred at 0 °C for 10 minutes. To the reaction flask a
solution of diisopropylcarbodiimide (0.93 mmol) in N,N-dimethylformamide (1.5
mL) was added dropwise and allowed to stir at room temperature for 2 hours.
The resin was filtered off, washed with dichloromethane (4x 30 mL), collected,
and stored at 0° C. The resin was characterized by IR.

IR: 2111 cm'1 (diazo stretch) KBr pellet

Diazo Wang resin synthesis using EDCI.

H_/ O O‘Q4OflN2

Commercially available Wang resin (0.1 g, ~0.1-0.2 mmol depending on
substitution) was added to a solution of glyoxylic acid tosylhydrazone (10 equiv.)
in dichloromethane (10 mL). To the reaction flask, EDCI (10 equiv.) and an
excess of lithium hydride (1.1 eq) was added and the reaction was allowed to stir

at room temperature for 45 minutes. The resin was filtered off, washed with

105

dichloromethane (4x 30 mL), collected, and stored at 0° C. The resin was
characterized by IR.

IR: 2111 cm'1 (diazo stretch) KBr pellet

Diazo acetate/amide resin synthesis.

o—ofJR
Resins 2-1 - 2-14

Commercially available hydroxyl or amino resin (0.1 g, ~0.03 to 0.3 mmol
depending on substitution) was added to a solution of glyoxylic acid
tosylhydrazone (10 equiv.) and 1-chloro-N,N-2-trimethyl-1-propenylamine (10
equiv.) in dichloromethane (10 mL). N,N-dimethylaniline (10 equiv.) was added
drop-wise to the resulting mixture, then stirred at room temperature for 5 min.
Triethylamine (10 equiv.) was added to the mixture and stirred at 0°C for five
minutes and allowed to warm to room temperature and stirred for 10 minutes.
The resulting resins were washed thoroughly with dichloromethane (3x30 mL)
and collected by filtration.

IR: ~211o cm'1 (diazo stretch) KBr pellet.

106

Diazo ketone resin synthesis.

5.1,

O
Resins 2-15 - 2-17

In a 25 mL round bottom flask, commercially available carboxypolystyrene resin
(0.5 g) was added to a solution of oxalyl chloride (10 equiv.) in benzene (15 mL)
and refluxed at 85° C for 16 hr. The acid chloride product was filtered and
washed three times in tetrahydrofuran (30 mL) and three times in
dichloromethane (30 mL). The acid chloride (0.25 g) was added to a solution of
1:1 acetonitrile and tetrahydrofuran (15 mL) in a 25 mL round bottom flask. To
the flask a solution of trimethylsilyl diazo methane (10 equiv.) was added and
stirred at room temperature for 50 hr. The resulting resins were washed
thoroughly with tetrahydrofuran (3x30 mL) and then dichloromethane (3x30 mL)
and collected by filtration.

IR: ~21oo cm“ (diazo stretch) KBr pellet.

Phenyl diazo methane resin synthesis.

/NZ
W
Resins 2-18 - 2-20
In a 25 mL round bottom flask, commercially available aldehyde functionalized
polystyrene resin (0.5 g) was added to a solution of 2,4,6-

triisopropylbenzenesuIfonyl hydrazide (2 equiv.) in tetrahydrofuran (10 mL) and

stirred under nitrogen at room temperature for 16 hr. The hydrazone product

107

was filtered and washed three times in tetrahydrofuran (30 mL) and three times
in dichloromethane (30 mL). The hydrazone (0.1 g) was added to
tetrahydrofuran (10 mL) in a 25 mL round bottom flask. The flask was heated to
90 °C in an oil bath and a solution of potassium hydroxide (0.1 g) in anhydrous
methanol (1 mL) was added dropwise. The reaction stirred until the resin turned
red (about two minutes). The resin was washed three times in tetrahydrofuran
(30 mL) and collected by filtration.

l R: ~2050 cm‘1 (diazo stretch) KBr pellet.

Determination of diazo functionalization.
In a 10 mL vial, the diazo functionalized resin (0.050 g) was added to a solution
of Fmoc-glycine (10 equiv.) in 1:2 DMF and tetrahydrofuran (1.5 mL), dry p-
toluenesulfonic acid (1 equiv.) was added and stirred overnight. The resin was
collected, washed three times in tetrahydrofuran (30 mL), and transferred to a 25
mL round bottom flask containing a solution of DBU (0.04 mL) in N,N-
dimethylforrnamide (1.96 mL) and stirred at room temperature for 60 minutes.
The reaction was diluted in acetonitrile (8 mL) and an aliquot of solution
Containing the liberated Fmoc (2 mL) was removed. The 2 mL aliquot was
diluted further in acetonitrile (23 mL) and the absorbance of the solution was
measured at 304 nm. The amount of Fmoc present in each sample was then

quantified using the prepared F moc standard curve.

108

Preparation of glyoxylic acid tosylhydrazone

To a 100 mL water in a 250 mL round bottom flask, add glyoxylic acid (9.2 g, 0.1
mole) and warm in a hot water bath to 60 °C. Meanwhile, in a separate 250 mL
round bottom flask add p-toluenesuIfonylhydrazide (18.6 g, 0.1 mole) to a 50 mL
aqueous solution of hydrochloric acid (2.5 M) and heat to 60 °C in a hot water
bath. Add the p-toluenesulfonylhydrazide solution to the glyoxylic acid flask,
while maintaining the 60 °C temperature and then the reaction continued to stir at
60 °C for 10 minutes. The flask is allowed to cool to room temperature and then
it was placed at 4 °C overnight to aid in crystalization. The crude product was
collected by suction filtration, washed with cold water (4 x 200 mL), filtered off
and dried in vacuo. The crude product is dissolved in boiling ethyl acetate (100
mL), filtered to remove any insoluable material and then diluted with carbon
tetrachloride (200 mL), cooled to room temperature and placed 4 °C overnight to
aid in recrystalization. The recrystallized product was washed with cold 1:2 ethyl
acetatezcarbon tentrachloride (300 mL), collected by suction filtration and dried in
vacuo. IR (NaCl plate): 3205, 1710, 1350, 1172 cm". 1HNMR (500 MHz,
DMSO-d5): 6 = 2.37 (s, 3H), 3.41 (br s, 1H), 7.18 (d, J = 2.5 Hz, 1H), 7.42 (d, J =
6.5 Hz, 2H), 7.70 (d, J = 6.0 Hz, 2H), 12.27 (s, 1H). 13cullllR (500 MHz, DMSO-
ds): 6 = 21.02, 127.11, 129.91, 135.74, 137.47, 144.06, 163.61. MS (calc. for M“

= 242.2); found 169.8 (M-CzHgNOZ), 155.0 (M-C2H3N202).

109

 

 

 

Appendix
Figure2-3. o
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I 3440.36 III. :
96.5I I III
I 3022-97IL291498

 

4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers

2-3. IR spectrum of a-diazo ester wang resin (0.89 mmol/g loading) made from
HONO gas.

110

Figure 2-4.
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Resin 2-1

1000 ”II II

 

 

 

 

 

 

 

 

 

 

 

 

 

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3404.20 I I 1502.12
92 III
I--____--- - - I'M-2020.40 - -__ - -_____ -

Wavenumbers

2-4. IR spectrum of o-diazo ester wang resin (0.89 mmol/g loading) made from
diisopropylcarbodiimide coupling.

111

Figure 2-5.

0 Q HQJM

 

1:4IIIIII
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1598.81 V1168.68
4000 3500 3000 2500 2000 1500 1000 500

Wavenumbers

2-5. IR spectrum of a-diazo ester wang resin (0.89 mmol/g loading) made from
EDCI with excess LiH, 45 minutes.

112

Figurl

95

85

8O

75'

70

2+3.

0 Q W34“

Resin 2-1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

80‘ I
1511.64

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7°IL.__--_.._.__- -__-. -- .1 - - , , _
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers

I I
I
/ \
1451.27
1681.8 /192,oo

 

 

 

 

 

2-6. IR spectrum of a-diazo ester wang resin (0.89 mmol/g loading) made from
route shown in Scheme 2-9.

113

 

Fig

III

9!

90

85

80

75

2-7_

 

Figure 2-7.

0 Q W48

Resin 2-2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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4000— 3500 3000 2500 2000 1500 Tooo 500
Wavenumbers

2-7. IR spectrum of u-diazo ester wang resin (2.2 mmol/9 loading) made from

route shown in Scheme 2-9.

114

Fig

II

2‘8.

 

 

Figure 2-8.
0

HN—/<=N
0'5 2

Resin 2-3

1°8I“"’”’ ’“ I“ * . * III
WW I “KI/NFW/IIIIILI III ‘

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4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers

I
I
I
I
I
I
I
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I
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2-8. IR spectrum of diazo amide tentagel made from route shown in Scheme 2-
9.

115

,I,I||i (I‘I‘I‘

 

 

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IIIIIII IHI II I III1\1.
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1500 1000 500

2000

a

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Resin 2-4

Wavenumbers

2500

116

Figure 2-9.

 

(”Vb xww
2920.54

 

3000

3500

 

2-9.

4000

2-9. IR spectrum of diazo amide megabead made from route shown in Scheme

Figure 2-10.

“NJL
—~2

Resin 2-5

100~r ‘“‘ _‘ *7 7 v , , 7,, —w——-_4Wr,ir ,,

A If ”V I

2109.76 II
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2871.09 I
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80‘ 1643.06 I
75* 1108.12
MEMHSEOE , ' 3000* “2500‘ 2000.- E00 w 1000 500'
Wavenumbers

2-10. IR spectrum of PEGA diazo amide resin made from route shown in
Scheme 2-9.

117

 

Fig

10!

95

85

75-

70

t /

Figure 2-11

 

 

 

 

Resin 2-6
100177777777“ iiiiii Ave 4 ............
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4000 $500 3000 2500 2000 1500 1000 500
Wavenumbers

2-11. IR spectrum of PEGA HMPA diazo acetate resin made from route shown
in Scheme 2-9.

118

 

Fig:

Figure 2-12.

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II I

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6000 2500 2000 1500 _1000" __ 500
Wavenumbers

4000 35:00

2-12. IR spectrum of diazo amide dendrimer made from route shown in Scheme
2-9.

119

 

 

Fig

70

66

62

Figure 2-13.

 

 

 

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4000 3500 3000 2500 2000 1500 1000 50

Wavenumbers

2-13. IR spectrum of 4-butyl diazo acetate polystyrene made from route shown
in Scheme 2-9.

120

 

Figu

100

95

85

80

75

70

Figure 2-14.

 

 

 

 

 

 

 

 

Resin 2-9

100I,-,r_2 + 2 + + + + - - fl~ #— 2 + ~~—+ ,J ~~~—-~II—- I

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4000” _ ”9600““ flap—00* * {500 2000 [1600— 1000 .504

Wavenumbers

2-14. IR spectrum of diazo acetate polystyrene made from route shown in
Scheme 2-9.

121

Figure .

100

9"."

95

85-

4900 f

245. IF

Figure 2-15.

 

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2500 2000 1500 1000 500
Wavenumbers

 

 

 

._T_._._—._-_—_——_. __ _...—.— __ T' __.7 m

4000 3600 3000

 

2-15. IR spectrum of NovaSyn diazo acetate resin made from route shown in
Scheme 2-9.

122

 

Fig

100

95

85

80*

Figure 2-16.

 

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Wavenumbers

2-16. IR spectrum of NovaSyn diazo acetate TG HMP resin made from route
shown in Scheme 2-9.

123

Figure

100'

95‘

85

2~17_

Figure 2-17.

 

 

 

0— PEG WOfiI/WIZ

 

 

 

o o
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1001 I
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3° 1109.87\ ‘
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4000 3500 3000 23:0 b2:00 1500 1000 500
avenum rs

2-17. IR spectrum of NovaSyn diazo anhydride resin made from route shown in
Scheme 2-9.

124

 

Figur

100‘

70'

 

Figure 2-18.
0
(H3ICOI3,HSI/\/\o/\I/\QJJ\¢N2
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Resin 2-13
10m~— _, —- has i * ~~ ,, a - , M, e e a ”-6 a 41
Mb“ II
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4000 3500 3000 2500 2000 1500 1000 5'00

Wavenumbers

2-18. IR spectrum of vicinal diazo acetate silica made from route shown in

Scheme 2-9.

125

 

 

Figure

30

20

10

2-19.

 

 

Figure 2-19.

0
(H3CO)3Si—/—>N—/— o

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4000 3500 3000 2500 2000 1500 1000 5017

Wavenumbers

2-19. IR spectrum of NHNMPTS diazo amide silica made from route shown in

Scheme 2-9.

126

 

_.—.—- u:-

Figu I

100‘ 7

95

85'

401

2-20.

Figure 2-20.

Resin 2-15

 

1000

 

 

 

 

2000

 

 

 

2600

 

95I
90“
85‘

 

1600

Wavenumbers

 

2-20. IR spectrum of the diazo ketone polystyrene (1.0 mmol/9 loading) made

from route shown in Scheme 2-9.

127

Figure 2-2'

2-21.

 

 

 

 

Figure 2-21.

Resin 2-16

100 4 ' ' W 7 II " T

”III/1» III I I II I III «I IIIII I
.f I

961 W" I
I

2

 

 

 

 

 

I I . .
I I II
I II I I 1491.65
_ I /
92 II I 1622.22
I II 1599.64
III II II
”A II I Ii II
I I I 2098.05
III
aaifi___,_,_._+ __ F _.2 __ _ . . ,.1___ 22% _ f_I
4000 3500 3000 2500 2000 1500 1000 500

Wavenumbers

2-21. IR spectrum of the diazo ketone polystyrene (2.0 mmol/9 loading) made
from route shown in Scheme 2-9.

128

Figure 2

100

95

2~22.

 

 

 

 

Figure 2-22.

 

 

 

 

0
WW
Resin 2-17
100_-,,,,,2-, *’*’”“#*“* ******“” I
I
I
I
WMAVJXWPJWMfi/\\VAWH I I
2112.01 I
95' 1749.1 _ I
1651.86‘ I
i
I
I
°°i I
851 I
I
4300“"3500‘ ’ £00 _ 2500 é 531]: “"7530 7600 500

Wavenumbers

2-22. IR spectrum of diazo ketone silica (1.6 mmol/9 loading) made from route
shown in Scheme 2-9.

129

 

Figur

75‘

70-

55*

2-23.

 

N2

Resin 2-18

 

‘hulH

 

 

InflIIIIII I II III IIIIIII
I- III I IIIII. IIII'
‘IIII‘III
I IIHI I IIIHIIHUIV

 

III
II..I I III .1 III I. I [III III
AIIIuIII

irIIIIIIII I l
IIIIIIIIIHIIglII 5

[J

 

Figure 2-23.

100~

 

 

951
901
051
soI

diazo methane polystyrene

2000
(StratoSpheres, 50-100 mesh) made from route shown in Scheme 2-9.

Wavenumbers
1 30

2500

IR spectrum of benzyloxy phenyl

75 ‘
70*
2-23.

Figure

100 __

95‘

80

75*.

 

 

Figure 2-24.

O< >//N2

 

 

 

Resin 2-19

100 ' I
/N I I

/ /" A III II II II I III
I N W" I . I I’ IIIIIII' II , I
955 I In I III I III II” III III / IWII
. 2054.87 III I I I I
W I 6/ II I I II I
90« ”J I I I
V I I III I
I ‘ I
1672.91 I I
851 I II I I
III I
II I
/I " I
1478.12 I
754 145325 1373.31 I
4000 3500 3000 2500 2000 1500 1000 500 I

Wavenumbers

2—24. IR spectrum phenyl diazo methane polystyrene (100-200 mesh) made
from route shown in Scheme 2-9.

131

Figure.

100*“

70-

 

 

 

 

Figure 2-25.

0 Q 0'04

 

 

 

 

Resin 2-20
100- M\ I
/// \“x/ /fl’II/I III/III ”III II IW
9°‘ / I IV“ I I II II I
I / 2051.67 I I I II I
/ ‘ I I I
°°‘ I J I I I I
I I I
I I I I I I
70I \/ II I I
I I I
60" I II I I
I II I
III I
III II I
1480.19I: I I
40» 1453.79 1369.89 I
I
4000 3500 3000 2500 2000 1000 1000 500

Wavenumbers

2-25. IR spectrum of benzyloxy phenyl diazo methane polystyrene (ZOO-400
mesh) made from route shown in Scheme 2-9.

132

 

Figure 2-1

Ce
rban
Abso

° Std

2.31,

 

 

 

Figure 2-26.

lfl‘ifl’ii ’ i "‘ A'IIKfJ ‘ ' ‘ _ ‘ ””"T””” R'” ' ' SD

1.81

 

1_...I ..... 4 , /
1_51_I....- .. . ........ ,,,..//n",.... I

1.411.... . , ._ //

I
131.1. . / .
I . '

I /(
111—L {/7 f I f I I f I . 7 . r T

0 0.5 1 1 .5 2
Concentration (pmol/mL)

Linear Fit: y = A + Bx: A _B_ Q
' Std (Standards@Exp01: Conc vs MeanVaIue) 1.074 0.444 0.994

Absorbance

 

2.31. Fmoc-glycine standard curve

Six concentrations of Fmoc glycine were subjected to Fmoc liberation in
the presence of base (DBU). The amount of liberated Fmoc was measured in
absorbance at a wavelength of 304 nm. Subsequent analysis to quantify the
amount of liberated DBF present was determined by the above standard curve

and its corresponding linear equation.

133

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Corey, E. J.; Myers, A. G. Efficient synthesis and intramolecular
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138

139

AF

31

ofsoI
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enncr
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Wfihn]
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DUC1e1

CHAPTER 3
APPLICATION OF DIAZO SOLID SUPPORTS IN THE ENRICHMENT OF
PHOSPHOPEPTIDES
3 .1 Introduction to diazo solid support phosphopeptide enrichment

As briefly mentioned in the previous chapter, the biological equivalent
of solid phase enrichment in the field of proteomics has been illustrated by
various covalent and non—covalent affinity chromatography techniques. As
discussed earlier, some of the most commonly used solid-phase purification
strategies include immunoaffinity chromatography“2 or synthetic modifications
of proteins followed by subsequent covalent addition by a reactive affinity
probe/solid support?"6 This approach has proven to be an effective tool in
both protein labeling and protein identification.

Covalent solid support purification is therefore an attractive approach
for phosphopeptide enrichment. Briefly described in Chapter One was an
enrichment method for phosphorylated serine/threonine and tyrosine
containing substrates using a diazo-functionalized solid support. This
approach first had the carboxylic acid residues and C-terminus protected via
their conversion into methyl esters using methanolic hydrochloride as typically
seen in IMAC based approaches.7 Once the carboxylic acid residues have
been converted to their corresponding methyl esters, the most acidic proton
within the peptide is on the phosphate. Upon protonation of the diazo moiety
by the phosphate, the resin would become activated and susceptible towards

nucleophile attack at the a carbon by the negatively charged oxygen on the

139

 

phl
sol

BIT]

Sch

Dho:

phosphate. This material utilizes both the nucleophilicity and acidity of the
phosphate group to selectively bind the phosphate to the diazo functionalized
solid support, which is unique to any other published phosphopeptide

enrichment strategy (Scheme 3-2).

9:
I

HO
\ 40
HO’P\(')
HC 0
H 2
M O N
Writ Wit-W2

O H H
O O—
HO’P‘o 2 Ho’P‘o
H C O H C O
H 2 H 2
M O N M O N
° WY Ykn %%—~H2 ° WY fiukii-NHz
O O O R 0
Scheme 3-1. General mechanism for the covalent enrichment of

phosphopeptides using a diazo-functionalized solid support.

Following trapping of the phosphorylated substrate, non-phosphorylated

peptides can be removed by filtration. The phosphorylated substrates are

140

 

sub

bon

3.2

prot

subsequently liberated from the resin upon hydrolysis of the phospho-ester
bond using aqueous acid. The collected eluent containing the phosphorylated
peptides can then be analyzed and sequenced by tandem mass spectrometry.
The development of the optimal enrichment conditions/solid support and its
subsequent application to successfully enrich phosphorylated substrates from
B-casein and ovalbumin digests, in low-picomolar quantities will be discussed

herein.

3.2 Preliminary phosphopeptide enrichment from a peptide mixture and a
protein digest

Initial phosphopeptide enrichment studies used the diazo acetate Wang
resin that had been prepared as previously described.8 The diazo-
functionalized support was introduced to both a four peptide mix, which
contained angiotensin II (a phosphotyrosine peptide), and a B-casein tryptic
digest. The amounts of peptide mixture used for the enrichment was 5 nmol,
however, only 1/200th of the sample was analyzed (25 pmol). The enrichment
did prove successful at this level using both acidic and basic cleavage
conditions (see Appendix, Figures 3-6, 3-7). Cleavage under basic conditions
yielded the fully hydrolyzed angiotensin II (1127 mlz), while cleavage under
acidic conditions kept the methyl ester protecting groups intact on angiotensin
II (1155 m/z). Additionally, the B-casein enrichment also did prove successful,
using 10 nmol of digest (50 pmol was analyzed). Initially, the digestion was

performed on trypsin purchased from Sigma Aldrich. Although the enrichment

141

 

fill

091
nee
he
run.

U89

and

DMF
”DIS:
9003

@YCC

IWWII
CLam

‘denu

did work, there were many ions that corresponded to the same
phosphopeptide due to incomplete digestion, which further complicated the
spectrum (see Appendix, Figure 3-8). This problem was circumnavigated by
the use of sequencing-grade trypsin purchased from Promega. This change
resulted in also successful enrichment, albeit, less complicated spectra (see
Appendix, Figures 3-9, 3-10).

While this approach was successful; in order to be competitive with
other current enrichment however, the amount of phosphopeptide used
needed to be reduced. This led to the optimization of the enrichment, both in
the type of resin used as well as the conditions in which the enrichment was
run. A variety of time-points for the enrichment were performed as well as the
use of a variety of different solvent systems and cleavage conditions.

All of the enriched samples were analyzed by a MALDI-TOF instrument
and some samples illustrated successful enrichment (overnight with the use of
DMF solvent) by the dominant phosphopeptide ions and the low baseline
noise. However, other samples spectra looked poor, containing low
phosphopeptide ion abundances and high signal to noise baseline (ethylene
glycol enrichment).

All of the various enrichment conditions and analysis, however, only
provided qualitative data. It was then determined that the implementation of a
quantitative enrichment approach would provide more conclusive data to

identify the best enrichment conditions.

142

33

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Synthe
phOSp

3.3 Evaluation of synthetic phosphopeptide enrichment by diazo supports
There were two methods used to determine the optimal enrichment
material. The first approach employed the synthesis of a 32P radiolabeled
phosphopeptide followed by subsequent enrichment and determination of the
amount of recovered 32P radiolabel post enrichment. The other approach
enlisted the use of a deuterium-labeled phosphopeptide, which could be used
to quantify the amount of unlabeled phosphopeptide in a similar manner as

the labeling approach discussed in Chapter 1.9'10

3.3.a Evaluation of diazo supports for enrichment recovery using a
radiolabeled phosphopeptide

Both quantification approaches have their own advantages and
disadvantages. The advantage in the employment of a radiolabeled
phosphopeptide for enrichment quantification is its ability to monitor each step
of the enrichment process. With this approach, we were able to identify
optimal enrichment and cleavage conditions, while MS-based quantification
approaches can only measure the “final enriched product”.

To determine the phosphopeptide recovery for each of the enrichment
conditions used, a 32P radiolabeled phosphopeptide was prepared. A
synthetic peptide, CHKtide (KKKVSRSGLYRSpSMPENLNRPR) was
phosphorylated with either unlabeled or radiolabeled ATP and checkpoint

kinase 2 (CHK-2), using the standard assay protocol (Scheme 3-2).11

143

KKKVSI

Schem

using

confirr
to the
thru 2
both h
Mush;
Confin
Chlorr

the SL

paper

radiol

32
KKKVSRSGLYRSPSMPENLNRPR 73%- KKKVSRSGLYRSPp3ZSMPENLNRPR

Scheme 3-2. Formation of 32P labeled synthetic peptide.

The phosphorylated peptide was separated from the remaining ATP
using ion exchange chromatography and the phosphorylation status was
confirmed by MALDI-ion trap MS analysis of the unlabeled peptide both prior
to the phosphorylation assay as well as after (see Appendix, Figures 3-11
thru 3-13). The resulting spectra indicated the presence of the phosphate
both in an 80 Da mass shift from 2701 to 2781 as well as MSIMS data which
illustrated the -98 loss of H3PO4. A negative control was also performed to
confirm the complete removal of the radiolabel during the ion exchange
chromatography step, using 32P labeled ATP and CHK-2 enzyme but without
the substrate.

No radiation was detected by the Geiger counter on the ion exchange
paper after the chromatography washes, indicating there was no remaining
radiolabeled ATP. After the 32p labeled CHKtide was isolated from the ATP,
the carboxylic acid residues and C-terrninus were converted to their
corresponding methyl esters with methanolic hydrochloric acid. The peptide
was then subjected to enrichment using the diazo acetate resin under a
variety of different solvent conditions, DMF and THF were found to correspond
to the highest phosphopeptide recovered.

Additionally, the conditions in which the phosphopeptides were cleaved

off of the solid support were also explored. The use of both NH4OH in water

144

and T
The ft
Afier

most

water
solut'll
hypot
the h
soluth
condl

matel

thed

IMAC

FiQUrl

peptic

and TFA in water were attempted both at 4, 6, and 8 hrs as well as overnight.
The resin and the cleavage solution were then surveyed for their radioactivity.
After exposure of either of these two solutions to the resin, it revealed that
most of the radiolabel was still left on the resin when exposed to NH4OH in
water. It was also found that more radiolabel was recovered in the TFA
solution, however, the solid support still contained radioactivity. It was then
hypothesized that this could be due to a lack of the resin swelling, preventing
the hydrolysis. As a result, dichloromethane was added to the cleavage
solution, which was found to improve recovery of the radiolabel. These
conditions were then subsequently used to compare different enrichment
materials.

The radiolabeled phosphopeptide was subjected to enrichment using
the diazo acetate Wang resin (resin 2-2), diazo ketone resin (resin 2-17),

IMAC, T102 or ZrOz pre-packed tips, using their reported protocols (Figure 3-

 

 

1).
i2;- € I : . ' I. - ~
; : _g remove ~ g cleave II; . I I
_- .1 . I -" ~ I BE 2": ._ 'd
3 i .3 3 add 3 ,3 unbound .- peptl es ITI
I. ‘I - Tl»: -’l‘=.
3 I resin ‘2 peptides '3 . .- from reSIn I: "3
,, : . .- - . “
'.It , ' 3‘ i ' 1‘ ,;' ‘ I' ‘
i“: 3‘3- 2: a72‘ * L3: If a!
1"“ ."-"“"' ““‘"‘-‘¢ 331.; _-_--.;’:’ 11.2323. '- -.-T?§ E‘HNTT‘V’VS

Figure 3-1. Enrichment recovery quantification procedure using 32P-labeled

pepfide

145

 

per

 

Tat

ehr

9an

 

Once each of the phosphopeptide enrichment techniques were
performed, the amount of recovered 32F was determined by scintillation
counting and then compared to that of the radiolabeled sample prior to
enrichment. Triplicate analyses revealed that the enrichment efficiency was
15% for the diazo ketone resin, while the diazo acetate resin was poorer with
only 5% enrichment efficiency. In comparison, the enrichment efficiencies
obtained from the IMAC, Ti02 and Zl'02 pre-packed tips were 1%, 14%, and

15%, respectively (Table 3-1).

 

 

 

 

 

 

Table 3-1.
Enrichment resin % Radiolabeled peptide recovered
Diazo ketone resin 2-16 15 (i5)%
Diazo acetate wang resin 2-2 3.9 (iO.2)%
IMAC Zip-tips 1.0 (i0.1)%
Glygen TiOz tips 14 (i4)%
Glygen ZI'Oz tips 15 (i0.5)%

 

 

 

 

Table 3-1. Enrichment recovery results using a 32F labeled peptide.

This data clearly suggests that the diazo ketone resin is competitive in
enrichment capabilities to the commercially available affinity methods. While
the use of a radiolabel enables quantification during each step of the

enrichment process, there is a disadvantage of this approach. As a result of

146

 

imple
5am;
ehhc

ahah

radic
mass
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phOE

othEI

implementing a radiolabel, no MS analysis is possible on these enrichment
samples. This is an issue as the application of this phosphopeptide
enrichment technique would be on samples which require MS and MSIMS
analysis instead of scintillation counting.

Since these samples are only measured for their amount of recovered
radiolabel and not whether or not the phosphopeptide ion is observable in a
mass spectrometer or purified from other unphosphorylated peptides present
within the sample, it is important to obtain quantification of the enrichment

procedure as it would take place upon the application of “real” samples.

3.3.b Evaluation of diazo supports for enrichment recovery using an H5 and
D5-labeled synthetic phosphopeptide

While the radiolabeled enrichment recovery experiment revealed the
optimal enrichment conditions, the development of a MS-based quantification
approach would be ideal as actual samples undergo the same protocol as
those analyzed during the quantification. As discussed previously in Chapter
One, various labeling approaches have been implemented in phosphopeptide
enrichment both for quantification in enrichment as well as identifying

phosphorylation changes in cells as a result of stimulation.9"°'12'18

3 we used

In a similar quantification approach discussed previously,1
two synthetic phosphopeptides, one was an unlabeled peptide (H5) and the
other was labeled with deuterium (D5). For our quantification study, one of

the two peptides will undergo the enrichment and prior to MS analysis, a

147

km

9an

ass
the
phc
COL
rec!
phc
sell
est
enr
ml):
phc
on

soII

Coll

dLF

Ont

enr

San

known amount of the other peptide can be added to determine the amount of
enriched peptide.

Each of the twenty synthesized diazo functionalized resins was
assessed for their phosphopeptide enrichment ability in an effort to determine
the most efficient enrichment solid support. This study used both a synthetic
phosphopeptide, CH3CH2COLFTGHPEpSLEK (H5) and its isotopically labeled
counterpart, CDachcOLFTGHPEpSLEK (D5) to determine enrichment
recovery. The D5 labeled peptide (10 pmol) was added to a three non-
phosphorylated peptide mixture (20 pmol) to additionally confirm phosphate
selectivity. Following the conversion of the carboxylic acid moieties to methyl
esters,7 the peptide mixture was subsequently exposed to each of the different
enrichment materials (~20 mg) overnight in tetrahydrofuran. The enrichment
mixture was filtered off to remove unbound peptides and the resin-
phosphopeptide sample was subsequently exposed to 50% trifluoroacetic acid
on 40% dichloromethane overnight to liberate the phosphopeptide from the
solid support. The liberated phosphopeptide samples were subsequently
collected and concentrated under nitrogen.

The enrichment procedure for each material was carried out in
duplicate, desalted and an aliquot (1/10th of the total peptide) was spotted
onto a MALDI plate. Prior to ion trap MS analysis, an amount of the H5
methyl ester peptide corresponding to the theoretical 100% recovery of the
enriched peptide (1 pmol) was added to the sample plate with the enrichment

sample. A comparison of the ion abundances in the resulting mass spectra

148

 

beh~e
pephc
avera
depu:

ldaUI

when
SUppl
differ.
Whol

enfic!

S:
a)
U

—r
CD

9:

m

 

//T7 / Ff7a/

between the H5 and enriched 05 peptide determined the amount of D5
peptide that was recovered and the two recoveries for each material were
averaged and reported in Table 3-2. The enrichment recovery values
depicted at < 10% is a result of the amount of noise present in the spectra
relative to the enriched ion (see Appendix, Figures 3-14 thru 3-37).
Theoretically, the ionization ability of both peptides should be identical
when using the same amount of material. However, to take signal
suppression Into consideration, a standard curve was performed using five
different amounts of 05 while maintaining the same amount of H5 peptide (1
pmol) (see Appendix, Figure 3-38). From the ratio of H5lD5 found in the
enrichment spectra, the actual amount of D5 present was determined using

the prepared standard curve .

 

 

 

 

 

 

 

 

Table 3-2.

Resin Solid support Recovery
1 Diazo wang resin (0.89 mmol/g) ~ 10 %
2 Diazo wang resin (2.2 mmol/9) < 10 %
3 Diazo amide tentagel 12 %
4 Diazo amide megabead < 10 %
5 Diazo amide PEGA resin < 10 %
6 Diazo amide HMPA PEGA resin 17 %
7 Diazo amide dendrimer 20 %

 

 

 

 

 

149

 

Ta

 

 

 

Table 3-2 (cont’d).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Resin Solid support Recovery
8 4-diazo acetate polystyrene < 10 %
9 Diazo acetate polystyrene < 10 %
10 Nova Syn TG diazo acetate < 10 %
11 Nova Syn TG HMP diazo acetate < 10 %
12 Nova Syn diazo anhydride < 10 %
13 Diazo acetate silica 15 %
14 Diazo amide silica a 10 %
15 Diazo ketone resin (1.0 mmol/9) 17 %
16 Diazo ketone resin (2.0 mmol/9) 24 %
17 Diazo ketone silica < 10 %
18 Diazomethylbenzene (50-100 mesh) < 10 %
19 Diazomethylbenzene (100-200 mesh) < 10 %
20 Diazomethylbenzene (200-400 mesh) < 10 %
- Millipore ZiptipMc (IMAC) < 10 %
- Glygen NuTip Ti02 z 10 %
- Glygen NuTip ZrOz ~ 10 %

 

 

Table 3-2. The average enrichment recovery for using each diazo-

functionalized solid support.

In addition to the quantification of the twenty different diazo-

functionalized solid supports, three commercially available affinity enrichment

150

Ies
en

iOn

Cor
aCe
Coil
aVa

MS

materials; Millipore Ziptips, and Glygen Ti02 or Zl'Oz packed tips were also
used for phosphopeptide enrichment following either the manufacturer’s
protocol or an established procedure.19 The commercially available materials
were found to have enrichment capabilities somewhat less than the most
successful diazo resin used. The optimal material based on its
phosphopeptide enrichment recovery was found to be the diazo ketone resin
16.

Comparatively, the enrichment recovery of 16 and 15 correlates to their
level of diazotization illustrated in Table 3-2. This trend was also consistent
when comparing materials 1 and 2. The diazo dendrimer support, which was
found to have the highest degree of diazo-functionalization, also exhibited a
promising enrichment recovery. Unfortunately, the diazo dendrimer
enrichment sample also contained additional polymer degradation ions present
in the mass spectra as a result of the cleavage conditions making this material
less desirable. The diazo amide PEGA HMPA also exhibited promising
enrichment recovery as well, but this material also had polymer degradation
ions present in the spectra.

Additionally, it is interesting to note that the MS quantification data
corresponds to the relative radiolabeled data when comparing the diazo
acetate Wang resin and the diazo ketone solid support. Both sets of data also
coincided with the relative extent of enrichment for each of the commercially
available materials. The overall decrease in the enrichment efficiency for the

MS quantification could be the result of either the affinity purification and/or the

151

vahabflhj

OI
enrichml
likely du
specificil
commen
they bul

which ag

E‘Jaluatel

ova'buml

variability of affinity enrichment depending on the composition of the peptide.
Overall, the diazomethylbenzene supports were relatively inefficient in
enrichment regardless of their degree of functionalization. This was most
likely due to their higher degree of reactivity, which could result in a loss in
specificity relative to their diazo carbonyl counterparts. Additionally, it is worth
commenting that when these materials were exposed to the peptide mixture,
they bubbled substantially, illustrating significant liberation of nitrogen gas,

which again could illustrate their reactivity.

3.4 Scope of diazo ketone solid support

The ultimate goal of this project was to implement a successful
enrichment procedure on a complex peptide mixture, such as a proteolytic
digest. By developing the optimal enrichment conditions, as well the most
efficient enrichment diazo-functionalized material, the use of this data can be

applied to the phosphopeptide enrichment of complex mixtures.

3.4.a Application of diazo ketone enrichment on a proteolytic digest

The data in Table 3-2 identified the diazo ketone polystyrene resin 2-16
as one of the optimal enrichment solid support. This material was therefore
evaluated for phosphopeptide enrichment from a proteolytic digest of
ovalbumin. Ovalbumin protein was digested with trypsin and subjected to
methanolic hydrochloride to form its corresponding methyl esters. An aliquot

of the digest (40 pmol) was subjected to the enrichment procedure using the

152

diazo ketone resin overnight in tetrahydrofuran. The enrichment sample was
subsequently filtered, cleaved off the solid support, desalted and an aliquot of
the sample (4 pmol) was analyzed by MALDI-ion trap MS and compared with
the original digest to highlight the phosphopeptide purification using the diazo
resin (Figure 3-2).

One of the two phosphopeptides of ovalbumin, containing residues 341-
360 (EWGpSAEAGVDAASVSEEFR), was the predominant peptide enriched
by the diazo ketone resin, confirming the ability of the enrichment to be
performed on a more complex mixture. There is also the loss of -98 (H3PO4)
corresponding to ion 2075.6, indicating the loss of a phosphate moiety, most
likely a result of post-source decay. The second phosphopeptide
corresponding to residues 63-85 (LPGFGDpSlEAQCGTSVNVHSSLR) was
also enriched. The only ion present in the enrichment sample that was not
due to a phosphopeptide was ion at m/z 1816, which corresponds to residues

324-340 (lSQAVHAAHAEINEAGR), an abundant ion present in the digest.

153

Fig

1 o 41(Fuf —\lullf- (I airmic—flqg

1 O aria-C—\a-lr- «I a!.u.-C~n«&

Fig

f0 rn

Figure 3-2.

fl

0

O
l

phosphopepfide
A I

*

 

 

   

 

I phosphopeptide

*

  

 

Relative Abundance ° 0

1200 1600

2000 2400 2800 3200 3600 4000
2173.7
100- *

o B

O

o

2

E-

E 2075.6

:3 'H3P04

.9

4 1816.7 *

0' 2553.9
E

‘U

a:

 

 

 

1200 1600 2000 2400 2800 3200 3600 4000
m-‘z

Figure 3-2. Enrichment of ovalbumin.

Depicted in Figure 3-2 is A) MALDI mass spectrum of 1 picomole of the

formed methyl ester tryptic digest of ovalbumin. (B) MALDI mass spectrum of

154

4 pl
corre
the it
(reslc
and -
tbnm
was

mefil
hope
HIPC

3.39,

2-16
8000
pmol

MALl

4 picomole enriched ovalbumin tryptic digest. The ion at mlz 2173.7
corresponds to methyl ester form of phosphopeptide (residues 341-360) and
the ion at mlz 2553.9 corresponds to the methyl ester form of phosphopeptide
(residues 63-85). The ions at mlz 2093.7 and 2075.6 corresponds to the -80
and -98 loss of HPO3 and H3PO4 from the 2173.7 ion, respectively. To further
confirm the identification of the two phosphorylated peptides, MSIMS analysis
was performed for each ion. The MSIMS of 2173 ion showed a loss of
methanol (2140.5 ion) and the loss of 98, corresponding to H3PO4 (see
Appendix, Figure 3-39). The MS/MS of the ion at 2553.9 also indicated a
H3PO4 loss of 98 (2455.6 ion) which corresponds to (see Appendix, Figures
3-39, 3-40).

Additionally, phosphopeptide enrichment using the diazo ketone resin
2-16 was performed on a B-casein tryptic digest (40 pmol). The sample
enrichment protocol was performed as described above and an aliquot (4
pmol) of the desalted enrichment sample was subsequently analyzed by
MALDI-ion trap MS (Figure 3-3).

The enrichment results primarily consisted of the monophosphorylated
peptide containing residues 48-63 (FQpSEEQQQTEDELQDK) and the
tetraphosphorylated peptide containg residues 16-40
(RELEELNVPGElVEpSLpSpSpSEESlTR). The ion corresponding to the
multiply phosphorylated peptide is smaller, however, and this could be due to
either ion suppression or a sub-stoichiometric amount of tetraphosphorylated

peptide in the digest.20 The additional ion at m/z at 2244.7, is salt adduct of a

155

Sf‘

me
4 l
the
32:
(re:
of t
HIF
illus
Gm

302.

peptide containing residues 199-217 (DMPIQAFLLYQEPVLGPVR), which
was the only ion that did not correspond to a phosphopeptide. The remaining
small ions present all corresponded to either de-methylation or salt adducts of
the phosphorylated peptide ions.

Illustrated in Figure 3-3 is A) MALDI mass spectrum of 1 picomole of
methyl ester converted tryptic digest of B-casein. (B) MALDI mass spectrum of
4 picomole enriched B-casein digest. The ion at mlz 2160.6 corresponds to
the methyl ester form of phosphopeptide (residues 48-63) and the ion at mlz
3234.8 corresponds to the methyl ester form of tetraphosphorylated peptide
(residues 16-40). Each of these phosphorylated peptides were also analyzed
by MALDI-ion trap MSIMS (see Appendix, Figures 340, 3-41). The MSIMS
of the 2160.6 ion contained a dominant -98 Da loss, again corresponding to
H3PO4. The MS/MS of the multiply phosphorylated peptide ion 3234
illustrates a dominant ion at 3122, corresponding to both the loss of a methyl
group and H3PO4. There is also another loss of H3PO4 from the 3122 ion at

3024, confirming that it is a multiply phosphorylated peptide.

156

Flgl

Figure 3-3.

100‘ A

phosphopepfides

’1‘
K
i

1200 1600 2000 2400 2800 3200 36m 4000

     

 

 

 

Relative Abundance ° 0

2150.5
100 -

 

3234.8

 

Relative Abundance °o

1200 1600 2000 2400 2800 3200 3500 4000
Ill-"Z

Figure 3-3. Enrichment of B-casein phosphopeptides.

157

Dfl
{F I
Dr:

30

It is important to make note of the different type of phosphorylated
peptides enriched in the two different phosphoprotein digests. The two
phosphopeptides enriched in the ovalbumin sample contained more non-polar
residues than the two phosphopeptides enriched from the B-casein digest.
Additionally, this enrichment approach successfully enriched both
monophosphorylated peptides and multiply phosphorylated peptides as well.
While all of the phosphopeptides present in each of the digests were
phosphoserines, it was hypothesized that this enrichment approach would be

compatible with each of the different types of phosphorylated residues.

3.4.b Application of diazo ketone enrichment on various phosphorylated
residues

To confirm that the diazo enrichment technique was compatible with all
the different phosphoresidues, two synthetic phosphopeptides, one containing
a phosphotyrosine (DRVleHPF) and the other a phosphothreonine residue
(LFTGHPEpTLEK), were each independently spiked into an unphosphorylated
protein digest of bovine serum albumin, esterase and phosphorylase b
(Figures 3-4, 3-5). The mixtures were then subjected to the enrichment
procedure using diazo ketone resin 2-16. Each phosphopeptide was
successfully isolated from the mixture, illustrating the ability of the diazo
ketone resin to indiscriminately enrich each of the different phosphorylated

residues.

158

Figure 3-4.

DRVpYIHPF
(Angioleusinm

I

1154.3

8

Relativealbuudame'o ...

 

1000 1500 2000 2500 3000 3500 4000
111-12

1154.2

Relativealblmdmce'o 8

 

 

‘ ‘.A_-_J I
llllllilillllll‘lllI‘Ilf1llTlfil

1000 1500 2000 2500 3000 3500 4000
11152

Figure 3-4. Enrichment of phosphtyrosine peptide angiotensin II.

159

The spectra shown in Figure 3-4 illustrate (A) Spectra of angiotensin II (1
picomole) spiked into a three non-phosphorylated protein digest (0.1 pg of each).
8) Spectra of an aliquot (25 picomole) of the enrichment of angioteinsin lI
(DRVpYIHPF). 100 picomole of angiotensin II was spiked into a digest
containing 10 pg each of three unphosphorylated proteins and exposed to the
diazo ketone 2-16 solid support for enrichment. This indicates the success of the

solid-phase technique for phosphotyrosine peptides.

160

Figure 3-5.

 

 

 

LFTGHPEIDTIFK
100-
° ‘
O
.3.
5 1393.9
2 *
=3.
3’
.2:
‘5
'3
at
1200 1600 2000 2400 2800 3200 3600 4&30
m-‘z
B 1393.9
100 *
O
0
§
0:
E
.5
"I
1”
.fi
‘5
'8
m

 

1200 1600 2000 2400 2800 3200 3600 4000
Ill-‘2

Figure 3-5. Enrichment of a synthetic phosphothreonine peptide.

The spectral data shown in Figure 3-5. illustrates: A) Spectra of
LFTGHPEpTLEK. (1 picomole) spiked into a three non-phosphorylated protein

digest (0.1 pg of each). B) Spectra of and aliquot (15 picomole) of the

161

enrichment of synthetic peptide, LFTGHPEpTLEK. 75 picomole of the synthetic
peptide was spiked into a digest containing 7.5 pg each of three
unphosphorylated proteins and exposed to the diazo ketone 16 solid support for
enrichment. This indicates the success of the solid-phase technique for

phosphothreonine peptides.

3.5 Experimental procedures

Materials. Sequencing grade trypsin was purchased from Promega. 2,5
dihydroxybenzoic acid was provided from Laser Biolabs. C18 desalting ZipTips
were provided by Millipore. The NuTip Ti02 and Zl‘Oz were purchased from
Glygen Co. Bovine B-casein, ovalbumin, and all organic chemical reagents were
obtained from Sigma Chemical Co. Polystyrene resins were purchased from
either Novabiochem, Sigma Chemical Co., or Advanced Chemtech. Carboxylic
acid silica was purchased from Silicycle. The H5 and D5 synthetic peptides were
generously provided by the Reid lab. Two functionalized silica materials were

generously provided by Prof. T. Pinnavaia (Michigan State University).

Preparation of ovalbumin digests. Ovalbumin (100 pg) was dissolved in 6 M
urea-50 mM Tris-HCI (20 pL). A 10 mM solution of dithiothreitol (5 pL) was
added to the protein solution and heated at 65°C for 1 hr. The solution was
cooled to room temperature and 50 mM ammonium bicarbonate (160 pL) and
100 mM iodoacetamide (10 pL) was added then incubated at room temperature

in the absence of light for 1 hr. 20 pg of trypsin was dissolved in 50 mM acetic

162

acid (40 pL) and an aliquot of the trypsin solution (10 pL) was added to the
protein solution then incubated for 16 hr. at 37°C. The reaction was quenched
with glacial acetic acid (11 pL). The digest was dried down under nitrogen and

subsequently stored at -80°C until their use.

Preparation of unphosphorylated protein digests. Bovine serum albumin
(100 pg), esterase (100 pg), and phosphorylase b (100 pg), were dissolved in 6
M urea-50 mM Tris-HCI (60 pL). A 10 mM solution of dithiothreitol (15 pL) was
added to the protein solution and heated at 65°C for 1 hr. The solution was
cooled to room temperature and 50 mM ammonium bicarbonate (480 pL) and
100 mM iodoacetamide (30 pL) was added then incubated at room temperature
in the absence of light for 1 hr. 20 pg of trypsin was dissolved in 50 mM acetic
acid (40 pL) and an aliquot of the trypsin solution (30 pL) was added to the
protein solution then incubated for 16 hr. at 37°C. The reaction was quenched
with glacial acetic acid (33 pL). The digest was dried down under nitrogen and

subsequently stored at -80°C until their use.

Preparation of B-casein digests. B-casein (10 nmol) was dissolved in 1 M
ammonium bicarbonate (10 pL) and water (14 pL) and incubated for 5 minutes at
37°C. The solution was sonicated for 1 minute. 20 pg of trypsin was dissolved
in 50 mM acetic acid (40 pL) and an aliquot of the trypsin solution (10 pL) was

added to the protein solution then incubated for 16 hr. at 37°C. The digests were

163

divided into 10 pg aliquots, lyophilized under a steady stream of nitrogen gas and

stored at -80°C until their use.

Preparation of methyl ester converted digests. Methanolic hydrochloric acid
was prepared by the addition of acetyl chloride (320 pL) to anhydrous methanol
(2 mL). An aliquot (200 pL) of this solution was then added to 10 pg dried
digests and incubated at room temperature for 2 hr. The methyl ester converted
digest was then dried down under nitrogen, dissolved in methanol and stored in
100 pmol aliquots at -20°C. For the unphosphorylated protein digest, the 100 pg
digest was incubated in an aliquot of methanolic hydrochloric acid (500 pL) for 3

hr. and then dried down under nitrogen and stored at -20°C.

Enrichment of phosphorylated proteins using diazo-functionalized resins.
Briefly, the diazo functionalized material (20 mg) and tetrahydrofuran (1 mL) were
added to an aliquot of either a methyl ester converted protein digest (40 pmol) or
a methyl ester converted purified phosphopeptide (70-100 pmol) spiked into a
unphosphorylated protein digest (7.5-10 pg of each protein). The mixture was
rocked gently overnight at room temperature. The resin was recovered and
washed with tetrahydrofuran (5x 500 pL), acetonitrile (4x 500 pL) and then
methanol (1x 500 pL). Cleavage of the captured species from the resin was
completed by incubation in a solution 50% trifluoroacetic acid, 40% DCM, 10%
water (500 pL). The mixture was rocked gently overnight at room temperature,

allowing for full interaction between the resin and the cleavage solution. The

164

resin was filtered and the elution fraction was collected. The resin was washed
once with methanol (500 pL) and added to the original fraction. The combined
eluents were concentrated under nitrogen. Prior to analysis, the samples were
diluted in water (20 pL) and desalted using Millipore C18 Zip Tips following the
manufacturer’s protocol. Briefly, the zip tip was moistened with acetonitrile (3x10
pL), then equilibrated with 0.1% trifluoroacetic acid (3x10 pL) and introduced to
the enrichment sample (18x10 pL). The bound peptides were liberated from the
zip tip with 50:50 0.1% trifluoroacetic acidzacetonitrile (5 pL). All desalted

samples were subsequently stored at -20°C.

Quantification of enrichment for diazo-functionalized resins. The
enrichment procedure was performed as described above in duplicate, with each
diazo solid support using the D5 labeled synthetic peptide (10 pmol) and a non-
phosphorylated peptide mixture (20 pmol of 3 peptides). The samples were
dissolved in 0.1% trifluoroacetic acid (20 pL) and subsequently desalted using
Millipore C18 ZipTips. Briefly, the zip tip was moistened with acetonitrile (3x10
pL), then equilibrated with 0.1% trifluoroacetic acid (3x10 pL) and introduced to
the enrichment sample (18x10 pL). The bound peptides were liberated from the
zip tip with 50:50 0.1% trifluoroacetic acidzacetonitrile (5 pL). An aliquot of the
desalted enrichment product was spotted on the MALDI plate (0.5 pL) and the H5

peptide (1 pmol) was spotted directly on top of the enrichment sample to

determine enrichment recovery.

165

Phosphorylation assay of CHKtide with P32 ATP. A 2mM aqueous solution of
CHKtide (2.5pL) was added to a 50mM MOPS, pH 7.0, 2.5 mM EDTA solution
(5pL). To the CHKtide solution, an aliquot of 25 nglpL solution of CHK-2 in
20mM MOPS, pH 7.5, 1 mL EDTA, 0.03% Tritton-X-100, 5% glycerol, 0.1 % B-
mercaptoethanol, 1 mg/mL bovine serum albumin (2.5pL) and sterile water (5pL)
was added. A solution (90pL) of 37.5mM magnesium chloride and 250pM ATP
in 20mM MOPS, pH 7.2, 25mM B-glycerol phosphate, 5mM EGTA, 1mM sodium
orthovanadate, 1mM dithiothreitol was added to 10pCi/pL solution of
SOOOCi/mmol [y-32P]ATP (mph. The 9:1 ATP-[y-32P1ATP (10pL) was added to
the assay and incubated at 30°C for 10 minutes. 20pL of the assay was spotted

on a 2 cm circle of P81 ion exchange chromatography paper, allowed to dry and
washed three times for five minutes in 0.75% phosphoric acid (40mL) and then
once in acetone (40mL) for five minutes. The peptide was removed from the
paper by adding 10% ammonium hydroxide (1.5mL). The filtered peptide was
dried down under nitrogen. Methanolic hydrochloric acid was prepared by
adding acetyl chloride (320pL) to anhydrous methanol (2mL) and subsequently
added (100pL) to the phosphorylated CHKtide and incubated at room
temperature for 2hr. The methyl esterified, phosphorylated peptide stock was

made into aliquots and dried under nitrogen.

Enrichment of radiolabeled CHKtide using diazo ketone functionalized
material. To a dried methyl esterified radiolabeled CHKtide aliquot (10pL), N,N-

dimethylfomtamide or tetrahydrofuran, (1mL) was added along with the diazo

166

ketone-functionalized solid-phase material (~10 mg). The mixture was shaken
overnight at 25°C. The material was washed with N,N-dimethylformamide
(2.5mL), acetonitrile (2.5mL) and methanol (2.5mL) and each washing was

removed. 90% trifluoroacetic acid (500pL) or 10% trifluoroacetic acid in

dichloromethane (500pL) was added and the resin and shaken for 6.0 hr. The
resin was filtered off and the eluent was collected and dried under nitrogen.
Dried enrichment sample was diluted in water (50pL) and an aliquot (10pL) was
added to a solution of HiSafe 3 scintillation cocktail (800pL) and water (190pL).

Radioactivity was measured by a Wallac 1414 Spectral Scintillation Counter.

Quantification of enrichment for diazo-functionalized resins. The
enrichment procedure was performed as described above using the D5 labeled
synthetic peptide (10 pmol). The enrichment samples were pulled up in 0.1%
trifluoroacetic acid (20 pL) and subsequently desalted using Millipore C18
ZipTips. Briefly, the zip tip was moistened with acetonitrile (3x10 pL), then

equilibrated with 0.1% trifluoroacetic acid (3x10 pL) and introduced to the

enrichment sample (18x10 pL). The bound peptides were liberated from the zip

tip with 50:50 0.1% trifluoroacetic acidzacetonitrile (5 pL). An aliquot of the

desalted enrichment product was spotted on the MALDI plate (0.5 pL) and the H5

peptide (1 pmol) was spotted directly on top of the enrichment sample to

determine enrichment recovery.

167

 

w gppfifl
lineal quad“

Laser 0‘3:

156%

 

Mass Spectrometry. MS and CID-MSIMS analyses were performed using a
linear quadrupole ion trap mass spectrometer equipped with a Matrix Assisted
Laser Desorption Ionization (MALDI) source (model vMALDl-LTQ, Thermo, San
Jose, CA). Desalted enrichment samples were spotted on the MALDI plate (0.5

pL) and then an aliquot of 50 mglmL DHB matrix in a solution of 50:50

acetonitrile:0.1 % trifluoroacetic acid (0.5 pL).

168

 

Figure 34

 

Appendix

Figure 3-6.

1155.2
100—

8
l

Relaivelntenslty

l

’9. ii;
; f 'I
. . Wt. 2'.
7' ' Aw‘ 4‘“ L“ 4 ALL... __ - A A ‘_

0‘1" ’il * 'I‘ ‘l l l
500.0 1400.2 2303.4 3200.8 4100 8 51010

 

 

Figure 3-6. MALDI-TOF MS of the enrichment of 25 pmol of angiotensin II,
using 90% TFA for the cleavage conditions. (methyl ester

converted angiotensin II peptide mlz = 1155).

169

Figure 3-7.

 

 

1127.1
100—
a
U)
E I
g 50 _ i
.2 I
E f
0 f
a: 1
I
"1,111 .13
“a”: " 2......
0‘l l l ’ l l ' ‘ I
500.0 1400.2 2300.4 3200.3 41005 51010

mlz

Figure 3-7. MALDI-TOF MS of the enrichment of 25 pmol of angiotensin II,
using 30% NH4OH for the cleavage conditions. Under basic
conditions, the methyl esters are hydrolyzed, resulting in the
recovery of the unmodified peptide (mlz 1127).

170

Figure 3-8.

100- 2551.3

4257.3

I
I
I
l
!

Rolalveinlensiy
8
l

l

l

 

   

l l l |
490.0 1300.4 2209.8 3200.2 41 (X16 5101.0

Figure 3-8. MALDI-TOF MS of the enrichment of 100 pmol of methyl ester
converted B-casein non-sequencing grade trypsin digest.

171

Figure 39.

2145.4
100—

 

Relatlve in ten slty
8
|

 

..._ {J
«D 5"
nun-n..—

 

 

It 1 l I
III. . It
If} ‘ fiT, .
M. ’ 1‘1 1 An”
, KM 3,13 _ . 3 l‘ L
499.0 1399.4 22%.8 3200.2 4100 6 5101.0

Figure 3-9. MALDI-TOF MS of the enrichment of 100 pmol of methyl ester
converted B-casein non-sequencing grade trypsin digest, under

90% TFA cleavage conditions.

172

 

Figure 3-10.

 

 

 

2058.1
100— 3
1980.3 1
\"
_a»
U
c .
2
'3 5° --;
.2 '
33
O
m
1
3140.7

 

 

 

 

41m.0 5101.0

Figure 3-10. MALDI-TOF MS of the enrichment of 250 pmol of methyl ester
converted B-casein non-sequencing grade trypsin digest, under
30% NH4OH cleavage conditions.

173

Figure 3-11.

Relativeintensity

 

 

 

 

_I-
o 8 8
g 1111111121
0
A
O
C.)
O
A
‘o”
O
to
D
8
3
M
01
8 100
NI
SEE
3'18-
w o:
o o
C) _n
0 9°
2
o:
‘9’
9,, 'de
a, NJ
0
h
o
Q
0

Figure 3-11. MALDI-ion trap MS of CHKtide standard

174

Figure 3-12.

Relative intensity

009
09
*-00l

WI»

 

 

 

8‘

0

§

0

3
38‘ g?
"I
E g 8:3
3.

 

0099
#917999

 

Figure 3-12. MALDI-ion trap MS of the CHKtide phosphorylation assay

175

Figure 3-13.
Relative inten sity

9
DOI-

 

008

l
-, r r a,
PQ'POSL

 

0003

9118M 91.1.261-

  

 

M
45
8 88
:30)
23.0-
o-3 -
oo 31
'2. $23
3 .. o
.l-o
No
(I)
s»
M
~d

Figure 3-13. The MALDI-ion trap MSIMS spectra of the CHKtide phosphorylated
ion at mlz 2781.

176

 

Figure 3-14.

 

1-05 05le Calibration Curve
0.95

0.85
0.75
0.55
0.55
0.45
0.35
0.25
0.15
0.05

"0‘05 0 0.2 0.4 0.5 0.8 1

Amount of D5 peptide (pmol)

  
    
 

y 1' 1.062x - 0.027
R2 = 0.987

I... . . r... . .... . .-... _ . T" .. ...-.. I

Relative intensity ratio of 05 to H5
pepflde

 

 

Figure 3-14. H5lD5 MS calibration curve.

Various amounts of the synthetic labeled D5 peptide (50, 100, 250, 500,
750, 1000 fmol) were spotted with 1000 fmol of H5 peptide and the calculated
ratio between the two peptides were plotted against the actual relative
abundance in the spectra. The standard curve was then used to determine the
amount of D5 present within the enrichment sample by comparing the relative

abundance of H5lD5 in the sample.

177

Figure 3-15.

 

 

 

 

 

 

 

 

 

 

H5
100_ 14335.5
.3 '-
m —l
g _
s 50...
5 -
a '_' 05
o: _ 1440.5
0 I l l l I
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100— n
'5 - l
2 .—
s 50—
g " U 05
or: - 1440.5
0—
1425 1430 1435 1440 1445 1450
mlz

Figure 3-15. MALDI-ion trap MS of D5 enrichment using diazo acetate Wang
resin (Resin 2-1).

178

 

Figure 3-16.

 

 

 

 

 

 

 

 

 

 

 

 

H5
1435.5
100— n
g _
E 50-‘
3 -
§ _
g - 05
.. 1440.5
0_ I 1 r1
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
.g' __ n
g _
g -

a " U 05
‘r 1 1440.5
0_"1'9'¢F7*l| f1? IT! II
1425 1430 1435 1440 1445 1450
ml?

Figure 3-16. MALDI-ion trap MS of D5 enrichment using diazo acetate Wang
resin (Resin 2-2).

179

Figure 3-17.

 

 

 

 

H5
1435.5
.4? ‘
m I:
g _
E 50—
'5 : 05
c: _ 1440.5
O—d 5.. A A - A. ‘AMAM
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5

 

 

Relative intensity

 

 

 

 

1425 1430 1435 1440 1445 1450

Figure 3-17. MALDI-ion trap MS of D5 enrichment using diazo amide tentagel
resin (Resin 2-3)

180

 

Figure 3-18.

 

 

 

 

 

 

H5
1435.5
100-— “
.3‘ -
a _
‘5 -
.5 50—
% -
'6 : 05
n: g 1 1440.5
0"_T‘T‘T_I ll 7 I II I I I
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100-m
4 1

 

Relative inten sity
0|
0
L .

 

 

 

 

 

_1 D5
4 1440.5
o“'fi‘§‘*‘7_hII I I I] I I I
1425 1430 1435 1440 1445 1450

mlz

Figure 3-18. MALDI-ion trap MS of D5 enrichment using diazo amide megabead
resin (Resin 2-4).

181

Figure 3-19.

 

 

 

 

 

H5
1435.5
2' ..
.5 .1
§ -
.s 50—
¢
.> ‘4
8 _
g .. 05
.. 1440.5
0 II I I I
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100-—

 

 

Relative inten sity
OI
O
I

 

 

 

1425 1430 1435 1440 1445 1450
mi:

Figure 3-19. MALDI-ion trap MS of D5 enrichment using diazo amide PEGA
resin (Resin 2-5)

182

 

Figure 3-20.

H5
1435.5

4
8
IILI
:3-

 

 

Relative intensity
01
O
I

 

 

 

 

 

 

 

   

 

Z 05 i
_ 1440.5 2
0 _..AAA . _.. - -, _ , - - ‘L
1425 1430 1435 1440 1445 1450
mlz i
H5
100‘ 14F55
z~ _
.5 _
g ..
g 50..
15 ..
0: 1
0 l l
1425 1430 1435 1440 1445 1450

mlz

Figure 3-20. MALDI-ion trap MS of D5 enrichment using diazo acetate PEGA
HMPA resin (Resin 2-6).

183

Figure 3-21.

 

 

 

 

 

 

 

 

 

 

H5
1435.5
.2‘ ‘
a; ..
C
2
s 50..
O -I
§ _ D5
3 1440.5
m -l
0 I I I r I T
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100% n
5 :
§ 4
5 50—
° -
5 _ 05
g _ 1440.5
1
0 AAA - - _ L -‘A A .. _ A
1425 1430 1435 1440 1445 1450

mlz

Figure 3-21. MALDI-ion trap MS of DS enrichment using diazo amide dendrimer
resin (Resin 2-7)

184

Figure 3-22.

 

 

 

 

 

 

 

 

 

 

 

H5
1435.5
.3' -
a - 1
g ..
.E 50—
.9 H
5 - U
° 5 05
[I _ 1440.5
0%.? II? I'T" I FTfi
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100—
2? -.
'6 d
g ..
E 50—
% 4
g d D5
.. 1440.5
0—
1425 1430 1435 1440 1445 1450
mlz

Figure 3-22. MALDI-ion trap MS of D5 enrichment using 4-diazo acetate
polystyrene (Resin 2-8)

185

Figure 3-23.

 

 

 

 

 

 

 

 

 

H5
1435.5
100-1
.. 1
.2- ‘
65 ..
g _,
.3 50-1
e> -
g _
.. 05
a: ‘ 1440.5
0— II I I I FI
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100— "
2‘ ..
'55 ..
c
s _-
.E 50...
g " 05
0: ‘ 1440.5
0....
1425 1430 1435 1440 1445 1450
mlz

Figure 3-23. MALDI-ion trap MS of D5 enrichment using diazo acetate
polystyrene (Resin 2-9).

186

Figure 3-24.

 

 

 

 

 

 

 

 

 

 

 

 

H5
1435.5
100—
. 1
.03.; g n
g .4
s 50—
° -
‘5. - u 3
§ - 05 3+
_ 1440.5 _
o
1425 1430 1435 1440 1445 1450 3
mlz
H5
1435.5
100— 1
.g' _ H
g ..
'5 - U
6? H 05
_ 1440.5
0 I‘I‘I I [Fl I
1425 1430 1435 1440 1445 1450
mlz

Figure 3-24. MALDI-ion trap MS of D5 enrichment using NovaSyn diazo acetate
resin (Resin 2-10).

187

Figure 3-25.

 

 

 

  

 

 

 
 

H5
1435.5
100!
4 1
.33 _
3 '1
s -
E 50—
3 -
E ..
o
m -1
0 .1 {+1 I I I I? I I I I
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100—
‘ 1

 

 

Relative intensity
at
O
I

 

 

 

 

.1 D5
1 1440.5
0—‘f r T I 1 Fl 1 'f‘ I I I I

1425 1430 1435 1440 1445 1450
mlz

Figure 3-25. MALDI-ion trap MS of D5 enrichment using NovaSyn TG HMP
diazo acetate resin (Resin 2-11).

188

 

Figure 3-26.

100%

l

50—

0—
1425

 

é
[III

I

1

Relative intensity
0|
0
I

O
1L1

 

1425

H5
1435.5

 

 

 

 

 

 

 

 

 

 

 

 

U 1
D5
1440.5
I l I I
1430 1435 1440 1445 1450
mlz
H5
1435.5
U D5
1440.5
II I I I
1430 1435 1440 1445 1450

Figure 3-26. MALDI-ion trap MS of D5 enrichment using NovaSyn diazo
anhydride resin (Resin 2-12).

189

Figure 3-27.

 

 

 

 

 

 

 

 

H5
1435.5
100— a
.g' _
é _
g . 05
C! ‘ 1449-5-
0 -.‘AAA - -- -, A _A,A _ -
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100— n
2' _
.5 ..
g _
.5 50...
'5 4
_ - D5
5'? 1 1440.5
0...
1425 1430 1435 1440 1445 1450
mlz

Figure 3-27. MALDI-ion trap MS of D5 enrichment using vicinal diazo acetate
silica (Resin 2-13).

190

1' .{I- .1993 q

 

 

Figure 3-28.

 

 

 

 

   

 

 

 

 

 

 

H5
1435.5
1007 11
g ..
E ‘50—; h
g - s:
E! "' ; '
0
1425 1430 1435 1440 1445 1450 11
mlz 9
H5
1435.5
100- n
g I
.E 50—
% -
a " 05
0‘ " 1440.5
fi
0 A‘A-. ,- .. _A‘AA“-
1425 1430 1435 1440 1445 1450
mlz

Figure 3-28. MALDI-ion trap MS of DS enrichment using diazo amide silica
(Resin 2-14).

191

Figure 3-29.

 

 

 

 

 

 

 

 

 

H5
1435.5
100— 1
.3' "
£3 ..
é _.
"33 so:
'5 -
a) _ 05
a: _ 1440.5
0— [I I I I [f
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100— n
.E' '
a, —
g _
5 50—
3
g _ D5
Ct: _ . 1440.5
0; AA -. . .- __A.A - -
1425 1430 1435 1440 1445 1450
mlz

Figure 3-29. MALDI-ion trap MS of D5 enrichment using diazo ketone resin
(Resin 2-15).

192

Figure 3-30.

 

 

 

 

 

 

 

 

 

 

 

 

H5
1435.5
100— 1
.5" '
a; ..
§ _
.s 50_
3 " r
g 05 i
0: 1440.5
0—
1425 1430 1435 1440 1445 1450 4:
mlz ;
H5
100- 147155
g _ 1
C
2 .—
.s 50..
o _ D5
5 _ U 1440.5
6
m —
0 I] l T l l T
1425 1430 1435 1440 1445 1450
mlz

Figure 3-30. MALDI-ion trap MS of D5 enrichment using diazo ketone resin
(Resin 2-16).

193

Figure 3-31.

 

 

 

 

 

 

 

 

 

 

 

 

H5
1435.5
100— 1
‘3‘
a
g _
E 50—
2:. 5
05
0: 1440.5 3
o l I I F F
1425 1430 1435 1440 1445 1450
"V2 1
H5
1435.5
100—- n
{-3 I n
g _
c
'3
,>
5
0
a:
1425 1430 1435 1440 1445 1450
mlz

Figure 3-31. MALDI-ion trap MS of D5 enrichment using diazo ketone silica
(Resin 2- 17).

194

Figure 3-32.

 

 

 

 

 

 

 

 

 

 

H5
1435.5
100— h
.2."
as
5
s 50-
.> -
§ -
g - D5
- 1440.5
0—
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100— n
4%“ I n
g _
.s
O
,>
5
O
a:
1425 1430 1435 1440 1445 1450
mlz

Figure 3-32. MALDI-ion trap MS of DS enrichment using StratoSpheres phenyl
diazo methane (Resin 2-18).

195

 

Figure 3-33.

 

 

 

 

 

 

 

 

 

H5
1435.5
100— n
g ..
s 50—
.g -
3 : 05
_ 1440.5
0— T l I I
1425 1430 1435 1440 1445 1450
ml!
H5
1435.5
100— n
.E‘ '-
0) .—
g ..
.5 50—
.> "'
5 _
g - D5
.. 1440.5
°‘7
1425 1430 1435 1440 1445 1450

mlz

Figure 3-33. MALDI-ion trap MS of D5 enrichment using phenyl diazo methane
polystyrene (Resin 2-19).

196

 

Figure 3-34.

H5
1435.5

100— n

l

l

 

 

 

Relative inten sity
0'!
C)
l

 

 

°1
1425 1430 1435 1440 1445 1450
miz
H5
100__ 1415.5

 

 

 

 

Relative intensity
0!
O
l

 

 

1425 1430

 

Figure 3-34. MALDI-ion trap MS of DS enrichment using phenyl diazo methane
polystyrene (Resin 2-20).

197

 

Figure 3-35.

 

 

 

 

 

 

 

 

 

 

 

 

 

H5
1435.5
‘3‘
a; _
c
2 —
E 50..
o
.> -
8 _
g - D5
_ J 1440.5
oTfilrlIII711Irl
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100—- n
é" I n
c:
2 —
S 50..—
-§ - l
E - D5
.. 1440.5
o—IIIIII113'ITIvafii—‘F—rfi
1425 1430 1435 1440 1445 1450
mlz

Figure 3-35. MALDI-ion trap MS of D5 enrichment using Millipore IMAC Ziptips.

198

 

Figure 3-36.

 

 

 

 

 

 

 

 

 

 

 

H5
100— 14:65.5
2‘
m —
§ _
s 50..
g -
‘6 ' . 05
0‘ : 1440.5
0——,%¢; [111—r leI l
1425 1430 1435 1440 1445 1450
mlz
H5
1435.5
100— R
g _ ”
g _
.5 50—
'> -
g - l 05
0: " J 1440.5
°—-flir°: [Ill—7 IIrTl Ill
1425 1430 1435 1440 1445 1450

mi:

Figure 3-36. MALDI-ion trap MS of DS enrichment using Glygen TlOz NuTips.

199

Figure 3-37.

 

 

 

 

 

 

 

 

 

 

H5
1435.50
100— n

E
a) .-
C
s -
.5 50—
.>
5 ‘ 05
O
a: : 1440.5

0"" I I I I 'I I

1425 1430 1435 1440 1445 1450
mfz
H5
1435.50
100—' n

.23 -
m -
c
8 -
.s so—
'3 -
Tn : 05
0‘ _ 1440.5

0— I I I I II I

1425 1430 1435 1440 1445 1450

mlz

Figure 3-37. MALDI-ion trap MS of 05 enrichment using Glygen ZrOz NuTips.

200

Figure 3-38.

 

 

 

 

 

Relativeinlensitv
_II
OI O
O O O
on
O 11L L Li 1 l [J
O
---I
A
A-
O P
o—
—l'
A—
g__
O_"_as
M
LC!)
“‘0!
”'4
“’Q
P
" a:
A b
8- w
0}
M
O
O
O
a; M
o 2
0 NM.
fic’I
Q
9‘0 ~-
89 3%
a 53°
C) 011
O

 

Figure 3-38. MALDI-ion trap CID MSIMS spectra of ion at m/z 2173 from the
ovalbumin enrichment.

201

 

Figure 3-39.
Relative tnten slty

O
as
—ml

DOLL

 

WI

nu
mu

m
\ Him
2995?! 659862
372793

 

’oa‘- l-
essesz
C‘H—

3

Figure 3-39. MALDI-ion trap CID MSIMS spectra of ion at mlz 2553 from the
ovalbumin enrichment.

202

Figure 340.

Relativeintensity

 

 

 

0 d
o o 8
8 I I I I I I I I ’
o
_L
D
0*
o
“—I
33_
o
o‘—
’—h
_“Nl
P33
_. ,‘cn
8—-'°"
A
0‘”?
113m
—01§
_ ' I
*— 3 3...:
..._' NI 3'0
'- 31 3%.?
_——-~
M NO
34 an
o 0‘

Figure 3-40. MALDI-ion trap CID MS/MS spectra of ion at mlz 2160 from the B-
casein enrichment.

203

 

-ZoOI
.159
80 II 33.8

-2601
.150“

.. -quo.
I 88.8

I www.— .mw was: .mn mocha

 

Relative inten sity
01
O

 

- ‘iIII )I'Illln . ..',iIP It-" Pl . I I'I.l.l|l‘lltlll’lri'»l ,I II. 'E'IIIII.III|'I’IIU . . li'ln II. III?

08 Ammo m 30 ~30 ago 580
:5

Figure 3-41.

Figure 3-41. MALDI-ion trap CID MSIMS spectra of ion at m/z 3234 from the B-

casein enrichment.

204

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207

CHAPTER4
APPLICATION OF PHOSPHOPEPTIDE ENRICHMENT ON A BIOLOGICAL
SAMPLE CONTAINING NF-KB PROTEIN-PRELIMINARY WORK AND FUTURE
PROJECT DIRECTIONS
4.1 Introduction

In the previous chapter, application of the enrichment approach was
illustrated using commercially available proteins. Upon the successful
enrichment of commercially available proteins, the next logical step is probing for
phosphopeptides from biologically important samples. The application of the
enrichment approach on a more complex matrix is important for a few reasons;
(1) successful enrichment illustrates the capabilities of this approach and (2)
enables the identification of potentially new phosphorylation sites. As mentioned
in chapter one, phosphoproteome analysis of a dynamic system (i.e. cellular
extracts) could identify relevant phosphorylation events linked to the pathology of
a specific disease through protein (mis-)regulation.

Our objective was to implement our enrichment strategy in an effort to
probe the cancer phosphoproteome. Identification of these phosphorylation
events could provide insight towards the identification of possible cancer
biomarkers. The importance and impact that biomarker identification has on
cancer treatment can be considered a three-faceted approach involving
prognostic, predictive and diagnostic markers.1 Prognostic biomarkers indicate
the severity of the type of tumor and the logical outcome for the patient. To date,
prognostic-type biomarkers are probed through DNA or gene-expression

signatures in the form of microarrays, rather than proteomic studies, so our

208

 

enrichment strategy is not applicable to the identification of prognostic
biomarkers. The other two types of biomarkers, however, include proteomic
analysis. As a result, both are avenues to explore the phosphoproteome and to
identify biomarkers using our enrichment strategy.

The first of the two is predictive biomarkers, which can indicate how a
patient will respond to treatment or which type of treatment they should receive.
Utility of these biomarkers includes the observation that breast cancer patients
who have an up—regulated ERBBZ gene benefit better from the drug
trastuzumab, where other patients with over-expressed estrogen receptor are
given tamoxifen to treat the disease.1 These identified genomic over-
expressions do have proteomic ramifications, which can lead to further insights
into the potential of proteomic detection and treatment strategies. For example, it
have been found that the lung tumor drug, gefitinib, can more effectively treat
cancer when there are mutations in DNA for the kinase domain of the epidermal
growth factor receptor (EGFR).2

Based on the cancer’s resulting phenotype caused by the genetic
mutation, this study clearly has opportunities to identify more specific biomarkers
by probing the phosphoproteome. Furthermore, characterization of the tumor
phosphoproteome cannot just include the presence (or absence) of a protein but
also requires analysis of changes in protein levels, which can also become a
significant biomarker as well.

Diagnostic biomarkers could lead to much earlier detection of specific

types of cancer, which would result in earlier treatment and greater rate of

209

 

survival. Statistically, cancer patients who are diagnosed at an advanced stage
of the disease have a five-year survival rate of less than 20%, depending on the
type of cancer. This number changes to almost 90% for patients who are
diagnosed with cancer at the early stage. In early detection patients, the ten year
survival rate is 80%.3 Clearly, this illustrates both the importance in early
detection and determining the vehicle to allow for early detection, namely disease
markers.

The concept of biomarkers originated from early studies which involved
probing for tumor-suppressor genes and other genomic molecules to study the
proteomic impact of the corresponding genomic profile. The limitation of
genomics in the realm of diagnosis and prognosis has been shown in some
cancer cell lines by the lack of correlation between mRNA abundance and
protein levels.4 Studying specific oncogenic pathways and the regulation of
those pathways through signal transduction, however, could provide valuable
insight on the disease's specific molecular signature. Understanding the
molecular impact of the disease and how it varies from healthy tissue would allow
looking, not just at how to diagnose the patient at an early stage, but also at
potential drug targets that would be specific for only the tumorgenic and not the
healthy tissue.

There are many pathways that have been researched for their role in the
pathogenesis of cancer, but discussion of all relevant pathways exceeds the
specified objective of this project, as I will discuss the relevant pathway l pursued

for phosphopeptide enrichment. As mentioned before, the phosphoproteome in

210

 

 

cancer cells is of interest because of the impact that phosphorylation has on mis-
regulation of signal transduction pathways within tumorgenic cells. Using our
enrichment strategy will allow for the monitoring of how and where these
pathways become activated (i.e. de/phosphorylated) or unactivated within the
tumor provide valuable information to identify both potential biomarkers and drug

targets.

4.2 Introduction to NF-KB

The transcription factor protein, nuclear factor-KB (NF-KB), has been
thoroughly studied since it was first discovered in 1986.5 It is now known to
consist of a group of similar proteins RelA (p65), RelB, c-Rel, p50 and p52, all
containing a Rel homology domain, which is responsible for both DNA-binding
and homo/hetero dimerization. The NF-KB proteins are responsible for
transcribing a variety of different genes involved in metastasis, cell proliferation,
inflammation, cell survival and suppression of apoptosis (reviewed extensively in
6'7). This protein has been widely researched in an attempt to understand how
this one transcription factor is involved with so many intracellular signals and
such a wide variety of stimuli.2 It is believed that the ability of NF-KB to take part
in the regulation of so many different processes is due to post-translational
modifications, such as phosphorylation.3 Although there have been a few of the
phosphorylation sites on the p65 subunit which have been determined, to date, it

is hypothesized that all the protein’s phosphorylations have not fully been

characterized.4 These modifications are of much interest to help understand how

211

 

this protein is involved in so many different pathways and in response to so many
different stimuli.

As previously mentioned, NF-KB is composed of homo— and heterodimers
of Rel proteins, the most common being the p50/p65 heterodimer.5 Typically NF-
KB exists in its inactive form in the cytoplasm bound to the inhibitory protein, IKB,
until activated by outside stimuli. Activation of the NF-KB pathway can occur in a
variety of different ways including the introduction of a chemotherapeutic agent
such as camptothecin or a cytokine like TNF-q, etc. It is these activators that
initiate the classical NF-KB pathway and are believed to be involved in both
cancer pathology and inflammatory diseases. Activation of the classical NF-KB
pathway is initiated within the cell, it mediates a series of phosphorylations on I-
KB by IKK kinases. The phosphorylations target l-KB for ubiquitinylation where l-
KB is subsequently degraded by the 26S proteasome. Upon the degradation of I-

KB, NF-KB (p50, p65 subunits) is translocated into the nucleus (Figure 4-1).1'7

212

 

Cytokines (TNF-a)

Extracellular

   
   

Cytoplasm

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
    
 
    
 
 

- Senne
Chemotherapeutlcs T kinases
IKK
Kinases
_ -  ®®
‘P657 ,A» ‘ P65_ j.--
* "“3 P50 , ' ' "“3. P50,
tr ubiquitinylation
_ .@ (,9
P65 " _
proteasome
. ' I I-KB
' .P65- degradation
~P50 ,.
Nucleus
NF-KB
translocation

 

Inhibition of
apoptosis

.ifp65' Gene
5‘ p50, transcription

Figure 4-1. The classical NF-kB signaling pathway.

 

 

 

213

Apoptosis regulates the number of cells as well as limiting the number of
mutated cells,8 however, this programmed cell death system is down-regulated in
cancer cells, resulting in tumor cell growth and chemotherapeutic resistance.9
The role that the NF-KB pathway plays in apoptosis is of particular interest to

provide some insight into regulating this process as a means of adjuvant therapy.

4.3 General signaling pathway of NF-KB

The inactive form of NF-xB is sequestered in the cytoplasm by a complex
of inhibitory-KB proteins (l-KB). Upon activation of the “classical” pathway, l-KB
becomes phosphorylated by l-KB kinases (IKKs) and the resulting
phosphorylations on l-KB target it for ubiquitinylation, followed by degradation by

8‘10). Upon

the 26S proteasome (comprehensive NF-KB pathway reviews
degradation of I-KB, the nuclear localization sequence of NF-KB is accessible and
subsequently translocated into the nucleus, where it initiates the transcription of
multiple genes. Depending on the origin of the activation, the pathway does vary
on how and in what form NF-KB is translocated into the nucleus, which would be
expected from the variety of genes NF-KB is responsible for transcribing. The
known phosphorylation sites of each of the subunits of NF-KB are illustrated in
Figure 4-2. The number of different phosphorylation sites and the corresponding
kinases responsible for these events illustrate the dynamic response to stimuli by
NF-KB. The exposure of different stimuli results in phosphorylation events at

different sites of NF-KB and the variance of phosphorylation events invoke the

transcriptions of different genes. An overview of the known phosphorylation sites

214

 

of each of the NF-KB subunits shown in Figure 4-2, and their corresponding

kinase, if known, is also listed.

[24] [25] [26]
PKAc GSKBB lKKb
I / \ / \
$337 $903S907 89278932

Cl?

p105 (p50) [Rel Homology Domain Ankyrin repeats Death Domain

 

 

 

 

 

 

 

phosphorylation
sites
[27- -28] [27-28]
IK
/ I/KK8\\ / ‘(a\
899 810881158123 8866 8870 8872 !

 

®<€®® @9999]

p100 (p52) Eel Homology Domain Ankyrin repeats Death Domain

 

 

 

 

 

 

 

 

 

 

phosphorylation
sites
[35-37] [41-44]
[31-32] IKKb, IKKb,
[29] [30] PKAc [29] [33] [34] IKKe, [38-39] [40] IKKa,
? ’l? M8K1/2 ’l? PKCE, I? GSIK3b Cl|1k1 Cll<2 RSKI
8%)5 $4 8§6 86318311 T435 8468 8505 S529 S536
phospizerylatioJ Rel Homology Domain Transactivation Domain (TAD)
sites
[45] [46] [45]
r r i
T84 8368 8552

(l9 C? 9'9

phospfirzlrglatioJ Leucine zipper Rel Homology Domain Transactivation Domain (T AD)]

 

 

 

 

 

 

 

 

sites
[50-51]
[47481 I491 PKCx,
PFA ? ‘|? NIIK
8267 $4 8460 S471
phosghsrijlation[ Rel Homology Domain Transactivation Domain (TAD) ]
sites

Figure 4-2. Some of the known phosphorylation sites of the NF-KB subunits

(adapted from a figure in 9 and phosphorylation sites identified in “'38 ).

215

The different kinases responsible for the phosphorylation events shown in
Figure 4-2 illustrate how different stimuli can evoke an NF-KB response leading
to the transcription of a variety of genes.39

Specific types of activation of NF-KB, such as the chemotherapeutic drug
camptothecin, can also result in the subsequent transcription of pro-survival
signals and is an important contributor to the tumorgenic properties involved in
many kinds of cancer including pancreatic, breast, gastric, and prostate.“°“13
Understanding the signal transduction pathways that NF-KB is involved in would
provide valuable information in determining its impact and role in the pathology of
the disease. Additionally, it allows for the potential to identify biomarkers
associated with the specific types of cancer to help in early diagnosis, prognosis
and subsequent treatment.

Many chemotherapeutic drugs also induce NF-KB activation,“ which in
turn can result in resistance to chemotherapy by NF-KB mediated anti-apoptotic

4547 Since the NF-KB pathway is activated by

gene transcription.
chemotherapeutic drugs and up-regulated in cancer cells, this provides
therapeutic utility to develop additional drugs to inhibit NF-KB, which would

45'48'51 Many research programs have

sensitize cancer cells to chemotherapy.
developed small molecule inhibitors of NF-KB as adjuvant drugs in an effort to
treat cancer more effectively (reviewed in 52). Additionally, the development of
these drugs and determination of their mode of inhibition provides a higher

understanding on how the regulatory process of NF-KB works, specifically

identifying how and where NF-KB is becoming modified and by which proteins.

216

 

4.4 Phosphorylation events in NF-KB pathway and their impact in cancer
therapy

The importance and impact that biomarker identification has on cancer
treatment has already been addressed earlier in this chapter. Many post-
translational modifications of NF-kB have been characterized and been
extensively reviewed 3'9 and it is at these sites of modification where many drug
targets and biomarkers lie. It has already been illustrated that different
modification sites along the activation pathway of NF-KB have been utilized to
prevent the subsequent transcription of anti-apoptotic genes. Nonspecific
inhibitors of IKK, such as curcumin and quercetin, have been developed to
prevent activation of NF-KB by preventing the phosphorylation of I-KB, which
indicate that this adjuvant treatment with chemotherapeutics could effectively
sensitize tumors.”55

Additional drugs have been developed to inhibit other proteins involved in
the NF-KB pathway, preventing the transcription of NF-KB regulated anti-
apoptotic genes,56 such as bortezomib, which is clinically used in the treatment of
multiple myeloma.57 Other NF-KB pathway inhibitors have been shown to exhibit
chemotherapeutic efficacy such as ALLN,58'59 MG-132,6°'61 lactacystin,62 and
salinosporamide.63

Clearly, this pathway and its respective proteins play a role in drug

discovery and can also play a role as cancer biomarkers as well. The

phosphorylation events which lead to activation and subsequent phosphorylation

217

 

of NF-kB, are critical for the cancer cells oncogenicity, which has been illustrated
by a study on NF-KB activated mouse epidermal cells 54 and on tumor growth of

mouse colitis-associated cancer.65

4.5.a Previous work to determine NF-KB phosphorylation sites

As illustrated in the previous section, the impact in identifying
phosphorylation events can prove to be important in the development of
chemotherapeutics. As such, the relevance of probing the phosphoproteome in
both the diagnosis and treatment of cancer is clear.

Extensive research towards the identification of phosphorylation sites of

NF-KB has been performed (reviewed in 7'9'10'6%9

) and is continuing to be an
avenue of study. One such study performed phosphorylation analysis of the NF-
KB pathway using tissue from patients with either stage I or stage II non-small
cell lung cancer (NSCLC).70 In this study, 88 patients with NSCLC had either a
biopsy or surgical removal of the tumorgenic tissue, which was used in the data
analysis. Along with the tumor tissue, normal lung tissue was also collected as a
control. The collected tissues were lysed and the subsequent nuclear extracts
were collected. Through the use of both staining methods and electromobility
shift assays (EMSA), as would be expected, there was an increase of NF-KB p65
subunit in the tumor cells when compared to that of the normal cells.

It was also found that the expression of phosphorylated l-kBa was

increased in the tumor tissues compared to the normal lung tissues, even though

the total amount of l-KB within the cytoplasm was consistent for both normal and

218

 

tumor lung tissues. It was also found that 46.6% of patients had NF-KB over-
expressed in their tissue, which was also shown in correlation with the severity of
the cancer. Additionally, poor survival ratings were linked to both NF-KB and pl-
kBq, which coincides with the results obtained in previous studies."72 The use
of NF-kB as a biomarker in prognosis was found in other studies to correlate with
poor prognosis of other types of cancer as well, including pancreatic, gastric, and
breast. “4373
Using a different approach, another study performed quantitative
phosphoproteomic analysis in cells observing the tumor necrosis factor (TNF)
pathway.74 This pathway has long been identified to work synergistically with the
NF-kB pathway. Since NF-KB proteins are responsible for transcribing a variety
of different genes involved in metastasis, cell proliferation, inflammation, cell
survival and suppression of apoptosis, NF-ch has been implicated in cancer
(reviewed extensively in 6'7). The impact TNF-a has on cancer cell survival
cooperatively with NF-KB has been a topic of increasing interest. Costas and co-
workers found that TNF-q could, in part, be controlling tumor growth by the
activation and complex formation between NF-KB and estrogen receptor (ER).75
In this quantitative study, Yates and co-workers grew HeLa cells in either
14N or 15N amino acid mix to isotopically label the proteins, allowing for
quantification.74 The whole cell extracts were obtained for both TNF-a treated
and untreated cellular environments. The collected whole cell proteins were
proteolytically digested with trypsin and subsequently separated on a gel-free

isoelectric focusing instrument, which separated the peptides by their pl.

219

 

Additionally, another fraction containing undigested whole cell proteins, was
subjected to IEF prior to digestion. IEF is a tool of choice based on the general
idea that upon phosphorylation the isoelectric point (pl) of a protein or peptide will
shift from a higher to lower value based on the additional negative charge from
the phosphate moiety. An aliquot of each of the IEF fractions were subjected to
LC/LC-MS/MS analysis referred to as multidimensional protein identification
technology (MudPlT).76 The corresponding results obtained from the IEF
fractions for both the intact proteins and the peptides are provided in Tables 4-1
and 4-2.

As one would expect, the data from the digested then IEF sample
suggests that some enrichment is occurring as the number of phosphorylated
peptides goes down with the increasing pH. The sample that first undenrvent IEF

and then was digested resulted in no real enrichment of the phosphopeptides.

220

 

Table 4-1.

Table 4-1.

unmodified (unmod.) and phosphorylated (phos.) peptides identified in MudPlT

 

 

 

 

 

 

 

 

IEF unmod. phos. phos./
fractions peptides peptides unmod.
pH 3 555 12 1.00
pH 4.6 592 13 1.02
pH 5.4 1867 13 0.32
pH 6.2 1445 0.00
pH 7 1350 0.17

Total non-
redundant 5242 41

 

 

 

 

IEF of digested whole cell protein extract, a comparison between

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4-2.
IEF unmod. phos.
fractions proteins peptides peptides
pH 3 388 801 3
pH 4.6 327 811 1
pH 5.4 403 1241 7
pH 6.2 566 1723 5
pH 7 516 1085 8
Total non-
redundant 1714 5435 23
Table 4-2. IEF of whole cell protein extract, comparing the unmodified and

phosphorylated peptides and their corresponding proteins by MudPlT.

221

To further enrich the complex mixture of peptides, the IEF fractions from
pH 3 and 4.6 were subjected to IMAC purification followed by LC-MS, since they
indicated the highest percentage of phosphopeptides. The enriched samples
were subsequently pooled together and analyzed by LC-MS/MS/MS. The results
identified a total of 701 phosphopeptides between the triplicate analyses,
characterizing phosphoserine, phosphothreonine and phosphotyrosine form both
the 14N and 15N isotopically labeled fractions. From the MSIMS analysis, the
comparison between the two labeled fractions led to the quantification of 223 of
the phosphopeptides. Even more interesting was the identification of 33
phosphopeptides (containing 56 phosphorylation sites) which were up-regulated
by at least two-fold and 15 phosphopeptides (containing 23 sites) were down-
regulated by two-fold or more as a result of TNF-q activation.74

To eliminate the possibility that some of the results were due to biological
isotope effect, the reciprocal experiment was also performed (i.e. the 14N cells
were activated with TNF-d while the 15N labeled cells were left untreated). While
the reciprocal experiment did enrich many of the same phosphopeptides, none of
the up or down regulated phosphopeptides were quantified. The authors
hypothesize a few rationales for this observation; including reduced ability to
identify the 15N peptides, variability in sample handling, or experimental variation.
Even though a large number of the phosphopeptides could not be quantitatively
determined due to low signal to noise ratio of the precursor ion peak, they still

could be identified and could be compared to the other data sets qualitatively.

222

 

This led to the identification of 79 unique phosphopeptides that were
present in the treated samples but not in the untreated, therefore deeming those
as TNF-a regulated.74 Among those phosphopeptides characterized from protein
kinase alpha (PKA) which is known to phosphorylate and activate NF-r<B.77 A
number of the remaining characterized phosphopeptides belonged to proteins,
which have been implicated in apoptosis.

The Yates study encompasses the use of both isotopic labeling of cells to
be used in quantification of phosphoproteins, sequential phosphopeptide
purification by both isoelectric focusing and IMAC technologies. The subsequent
MSIMS analysis yielded the identification of many phosphopeptides and their
corresponding phosphoproteins involved with the activation of the TNF pathway.
The overlap in the identification of proteins that become phosphorylated as a
result of TNF-q activation in cancer cells, indicates the utility of the combination
of technologies to probe the phosphoproteome for cancer biomarkers. This
report also illustrates the validity of our approach to probe for phosphorylation

sites upon the activation of NF- KB.

4.5.b Proposed work using our enrichment approach

The exposure to cells using the different stimuli suggested above, followed
by isolation, phosphopeptide enrichment, and characterization of the
phosphorylation sites on NF-KB, noting the differences in these sites as the
stimuli varies is of particular interest. Since NF-KB is responsible for transcribing

many different gene products, phosphorylation events occurring on NF- KB are

223

 

known to be involved with NF-kB’s ability to evoke this specific gene
transcription.1°'39

Our approach to elucidating the phosphorylation sites of NF- KB within
cells employs the activation of the classical pathway by stimulating cancer cells
with TNF-q followed by subsequent extraction of the nuclear proteins.
Theoretically, the activated sample will provide a build-up of NF-KB, containing
known phosphorylation sites and potentially unknown phosphorylation sites as
well within the nucleus. While the control sample (no stimulation with TNF-a) will
not provide any substantial nuclear NF-KB. Upon nuclear extraction, the success
of the activation of the NF-KB pathway can be confirmed by Western blotting and
NF-KB can be subsequently purified by immunoprecipitation using a p65 subunit
anfibody.

The immunoprecipitation is a vital step to the overall success of the
phosphopeptide enrichment and if the IP is not performed prior to enrichment,
two scenarios are possible. Either (1) the most abundant phosphoproteins, such
as actin, will be enriched and detected, or more likely, (2) there will be some
enrichment of many, many different phosphopeptides but none of which in a
large enough quantity to be able to sequence any of them. Additionally, if some
phosphopeptides from a nuclear extract were enriched and detected,
identification of the protein a phosphopeptide belongs to is more challenging as
the number of proteins present is significantly larger. As a result, the
immunoprecipitation technique has been utilized as an efficient way to isolate

and concentrate the protein of interest, NF-KB.19’78‘79

224

 

Once the nuclear extracts are purified by a p65 antibody, the success of
the IP can be confirmed by running a portion of the sample on a 10%
polyacrylamide peptide gel, followed by either silver staining or western blotting
analysis. Concurrently, a larger amount of both unstimulated and stimulated
purified (by IP) protein samples will then be subjected to proteolytic digestion

(Figure 4-3).

I..."

+1“.- -
G’E&

 

         

 

agarose ag arose

nuclear extract / \

Confirm purification dlgesflon and
enrichment of IP

Figure 4-3. General scheme for the purification of NF- KB.

The prepared lP digests can be introduced to our optimal solid support,
the diazo ketone polystyrene, allowing for phosphopeptide enrichment, followed
by subsequent analysis by MALDI-ion trap mass spectrometry. The
phosphopeptide analysis of the enrichment samples will provide the isolated
phosphopeptides in unactivated and activated samples. Upon MSIMS analysis

of all of the containing phosphopeptides, characterization of the identity of each

225

 

of the phosphorylation sites and the corresponding differences can then correlate
the phosphorylation events that are a result of TNF-a stimulation.

The phosphoproteome information illustrates the downfield signaling
response to TNF-q. The phosphorylation sites can then be correlated with the
known genomic repercussions (genes transcribed). Ultimately the correlation
between phosphorylation events and the genomic consequence of the event is
vital to further understanding the dynamic relationship between NF-KB activation-
post-translational modifications and the resulting genes it is responsible for
transcribing.

Upon the successful identification of the phosphorylation sites that
resulted from the activation by TNF-a, this enrichment approach could be used
after exposing cells to different types of stimuli to activate the NF-KB pathway as
well. It has been previously reported that NF-KB can be activated by various
signals such as lipopolysaccharide, viruses, ultraviolet light, double-stranded
RNA, as well as chemotherapeutic drugs and ionizing radiation.”81 The
identification of each of the phosphorylation site/s of NF-KB with regards to the
stimuli used represents the impact that each stimuli has on the transcription
outcome.

In addition to identifying the differences in the phosphoproteome relative
to the exposure of the different stimuli, we will gain new insight into the
modifications NF-KB undergoes with respect to cancer is of particular interest as
well. Determination of what genes are up-regulated because of these

modifications, specific inhibitors can be designed at the site of the

226

 

phosphorylation to produce a more efficient drug in the treatment of cancer. This
method for correlating cell signaling to genomic profiles first entails the
determination of the exact phosphorylation sites of the different activated forms
of NF-KB.

As part of the long-term project forecast is the implementation of a gene
chip experiment on all of the genes NF-KB is known to transcribe or activate.
Correlations between activated/deactivated NF-KB genes and other NF-KB
regulated genes could then be determined while simultaneously identifying the
role phosphorylation has on the activation of each of those genes.

The identified phosphorylation sites from the various stimuli compared to
the cDNA profiles for NF-KB regulated genes, indicate a correlation between the
activation (cellular signaling) of the protein and the genomic ramifications from
the activation. This information on the link between protein signaling and its
genomic consequences is especially relevant in the understanding on a cellular
level of how cancer develops. Comprehending how cancer advances from a
synergistic protein—genomic standpoint, more specific drugs can be designed to
target various steps in the anti-apoptotic pathway in which NF-KB is involved.
This insight will both indicate how to improve the current therapies which are not
effective, as well as facilitate a resource for the development of new treatments

for cancer.

227

mn-

 

 

 

4.6 Activation and purification of NF- KB

Various cell lines have been treated with TNF-a to activate the NF-KB
signaling pathway, these cell lines include THP-1 and CEM cells (both leukemia
cell lines), RPMI 8226 and L363 cells (both are multiple myeloma cells, which
contain constitutively active levels of NF-KB). Upon activation of the various cell
lines using TNF-a, the cells were harvested and the nuclear proteins were
isolated. The nuclear extracts of both the un-stimulated cells and the stimulated
cells, which contain increased amounts of NF-KB, were then subjected to a
Bradford assay to determine the protein concentration of each of the nuclear
extracts. Activation of the NF-KB pathway was confirmed by probing for the p65
subunit in a Western blot on the obtained nuclear extracts and comparing the
intensity of the bands detected (Figure 4-4).

Depicted in Figure 4-4 is the analysis of a set of eight THP-1 nuclear
extracts; four of which were obtained without any stimulation (untreated #1-4)
while the other four samples were stimulated with TNF-q for 30 minutes prior to
the extraction of their nuclear proteins (treated #1-4). The protein bands shown
on the left-hand side (Lane 1) are used as molecular weight standards and the
size of the circled band is 66,200 Da, which corresponds to about where the p65
subunit should be at. The samples in each lane are as follows: (1) Pre-stained
molecular weight standards, (2) THP-1 N.E. Untreated #1, (3) THP-1 N. E
Untreated #2, (4) THP-1 N.E. Untreated #3, (5) THP-1 N.E. Untreated #4, (6)
THP-1 N.E. Treated #1, (7) THP-1 N.E. Treated #2, (8) THP-1 N.E. Treated #3,

(9) THP-1 N.E. Treated #4.

228

 

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Figure 44. Western blot analysis of p65 activation in THP-1 cells after TNF-a

activation, probing for both p65 and actin.

Clearly, this Western blot confirms that the activation of NF-KB was
successful upon the stimulation with TNF-q. However, to confirm that the
activation is in fact a result of the stimulation and not due to an inconsistency in
protein loading, another Western blot is performed on the same membrane, but
this time probing for actin.

Actin is a very abundant protein present in all cells, whether the cells are
stimulated or not. By probing for actin, protein loading consistency can be
determined if there is equal amounts of actin detected for each sample. This
second blot illustrated in Figure 4-4 was performed and which shows similar
amounts of actin present in all of the samples, so it can be reasoned that the
change in the amount of p65 is a result from the activation and not due to loading
errors.

The extent of activation for THP-1 cells were compared by using both

large cell culture plates (1 x 40 mL plate) and small cell culture plates (4 x 10 mL

229

 

plate) to see if it the plate size affected the extent of activation. The figures
shown above are from large plate nuclear extracts. While activation was also
confirmed for the small plate nuclear extracts, it showed more variability in
activation, as some would show thick, dark bands corresponding to p65 in the
Western blot (very activated) while others would be very faint. This led to the
conclusion that only large cell culture plates should be used for future
experiments.

Once activation was confirmed by Western blot analysis, a way to
circumnavigate the problem of insufficient amounts of NF-KB protein relative to
large number of other proteins present in the nuclear extracts was needed. A
way to selectively isolate NF-KB is using an antibody with an epitope specific for
the p65 subunit. The use of an antibody with specificity towards p65, like the one
used for Western blot analysis, could be employed to purify the protein instead of
how it is used in western blot analysis as a means of detection.

The nuclear extract is first pre-cleared to remove any proteins that non-
specifically bind to lgG by introducing lgG to the extract. The lgG and the
proteins that bind to it are removed by the use of protein A/G-agarose.
Antibodies (lgGs) have affinity to protein A and protein G and by immobilizing
these proteins on a solid support such as agarose, centrifugation will separate
the pellet from the rest of the unbound nuclear proteins. This allows for removal
of proteins which non-specifically bind to antibodies by adding both lgG and

immobilized protein A and G.

230

 

Similarly, the antibody of interest can be conjugated to agarose prior to
introducing it to the nuclear extract and once the nuclear extract has been pre-
cleared, the antibody will bind with the protein of interest and upon centrifugation,
the pellet will contain the antibody and its corresponding protein. Upon the
completion of this immunoprecipitation (IP), confirmation of the success of the
NF-KB purification can be performed once again by Western blot analysis and
silver staining.

Western blot analysis proved to be a little more challenging as a different
antibody from a different host animal must be used to probe as the primary
antibody. The best option was the mouse antibody containing the N-terrninal
specific epitope; the antibody used in the immunoprecipitation is specific for the
C-terminal end of the p65 protein.

This particular antibody was not proficient at the 1:1000 dilution of the
antibody, which is the dilution used for the C-terrninal antibody. Exposure times
extended upwards of four hours for the N-terrninal antibody when used at the
1:1000 dilution. Illustrated in Figure 4-5 is a western blot probing for p65 in
immunoprecipitated nuclear extracts from two different cells lines.

The description of the contents of each lane is as follows: (1) Pre-stained
molecular weight standards, (2) p65 antibody (sc-372) control, (3) lgG-Protein
NG control, (4) lgG-p65 antibody control, (5) CEM-IP Untreated #2, (6) CEM-IP
Treated #3, (7) CEM-lP Treated #3, (8) L363-IP Untreated #2, (9) L363-IF

Untreated #4, (10) L363-IF Treated #2, (11) L363-lP Treated #4.

231

 

Lane#: 1 2 3 4 5 6 7 8 9 10 11

_w _ _ _ J' _ JJ . .1 N-.. p65

Q.

Figure 4-5. Western blot analysis of immunoprecipitated nuclear extracts from

CEM and L363 cell lines

This Western blot illustrates that NF-KB is present in the
immunoprecipitated samples and as expected for the CEM extracts, the
untreated IP contains less p65 (lane 5) than the two treated lP samples (lanes 6-
7). The L363 lP samples are different, though, as this particular cell line is from
multiple myeloma cells and has constitutively active NF-KB present in the nucleus
already. This is illustrated in the IP samples as well, as it appears that both the
untreated and the treated samples both contain relatively similar amounts of NF-
KB.

Aside for performing Western blot analysis on the immunoprecipitation, a
second 10% polyacryamide gel was also run and subsequently silver stained to
visualize the purification success of the IP since Western blot analysis will
provide the information in whether or not a protein is present, but it will not elude

to the number of proteins within a sample.

232

 

97 .
66

31

Fl

tc

Lane#:1234567891011

97.4 kDa
66.2 kDa

45.0 kDa

31.0 kDa

 

Figure 4-6. Silver stain of CEM and L363 nuclear extract IP samples.

Although, the Western blot analysis on the IP samples was successful, it
took a few tries in figuring out the exposure length, which seemed to vary for
each blot. This led to an effort to determine the optimal Western blotting
conditions for both antibody concentration as well as exposure time. It was
determined that the primary antibody required a 1:250 dilution in order to allow
for visualization by audioradiography for reasonable and reproducible exposure
lengths (11/2 - 2 hr).

As illustrated in Figure 4-6, the silver stained gel of the lP samples shows
a significant number of proteins present within the IP. Some of which could be
modifications of p65, or its subunit p50, and some of the bands could correspond
to proteins that bind to p65 or have some specificity towards the antibody used.
Due to the number of bands present, it makes it challenging to identify the band
which corresponds to p65 in the silver stained gel. This led to the idea of

233

 

preparing duplicate sets of the same samples and running them on one gel.
Once the gel has been run, the gel would be divided in half and on side would be
exposed to the silver stain protocol while the other half would be used to perform
western blot analysis.

This experiment was performed on a set of THP-1 nuclear extract
immunoprecipitations (Figure 4-7). The Western blot on the left included the
following samples in lanes 1-6: (1) Molecular weight ladder, (2) lgG-Protein NG
agarose, (3) p65 antibody-agarose, (4) THP-1 iP of 200 pg untreated NE, (5)
THP-1 IP of 1000 pg treated NE, (6) THP-1 IP of 200 pg treated NE. The silver
stained gel included the following samples in lanes 7-12: (7) THP-1 IP of 200 pg
treated NE, (8) THP-1 IP of 1000 pg treated NE, (9) THP-1 lP of 200 pg
untreated NE, (10) p65 antibody-agarose, (11) lgG-Protein NG agarose, (12)

Broad Range molecular weight standard.

Lane#: 6 5 4 3 2 1 7 8 9 10 11 12

 

Figure 4-7. Western blot and silver stain comparison of IP samples.

234

While the Western blot clearly illustrates that p65 is much more prevalent
in the treated nuclear extract lP samples, it is harder to elucidate that in the
corresponding silver stained gel. In the silver stained samples, we do see a band
that corresponds to the same molecular weight as p65, however, we also see
quite a few other bands as well. This could be from p105, different modified
forms of p65, IgG or additional proteins that either bind to p65 or have affinity for
the epitope of the antibody. Complications in identifying p65 could in part be
due to presence (in equal amounts in lanes 7-10) of the p65-antibody that
consists of a wide, faint band, which could be masking the p65. There are two
bands present at the very bottom of the p65 antibody band in the IP samples that
are more pronounced in the treated nuclear extract IP lanes than in the untreated
IP, which could be two differently modified forms of p65. Additionally, it is worth
commenting on that the two treated IP samples appear to have both more
proteins present as well as in higher concentrations. This could be attributed to
the activation of additional proteins as a result of the stimulation by TNF-a, which
could be carrier proteins for NF-KB. The relative increase could also be a result
of proteins that have affinity to NF-KB and since more NF-KB is present, more

associated proteins are pulled out as well in the immunoprecipitation.

4.7 Enrichment attempts made using diazo ketone solid supports
While some smaller amounts of the nuclear extract lP samples were

prepared to be run on a gel and Western blotted (200 pg of total protein) which

235

 

000‘

acti‘

lmn

<38“

pre

me

we

ral

thl

confirmed the presence of p65 and also illustrated the importance of NF-KB
activation. The next step was to use the scaled-up remaining
immunoprecipitated samples for phosphopeptide enrichment using our
developed diazo ketone solid support. These additional lP samples were
prepared using a larger amount of total protein (1000 pg of total protein), since
more is required for the enrichment and analysis. The larger scale lP samples
were digested off of the antibody-conjugate via in-solution digestion protocol
rather than being on run on a gel and then in-gel digestion, as recovery was
thought to be higher through in-solution means rather than from gel extraction.
To perform the in-solution digestion, first a 6 M urea-Tris buffer was
introduced to the IP pellets, which serves as a denaturing agent to unfold the
protein, enabling more complete digestion when introduced to the protease. The
disulfide bonds are reduced with dithiolthreitol, subsequently modified with
iodoacetamide to prevent reformation of the disulfide and then introduced to
trypsin to perform the proteolytic digestion. This in-solution tryptic digestion
protocol is performed for both the un-stimulated and stimulated nuclear extract lP
samples and upon digestion, both samples were then converted to their methyl
ester counterparts upon the introduction of methanolic hydrochloric acid.82 The
importance of this conversion was previously described in chapter three, and
since these samples will be subjected to enrichment, it was deemed that MALDI-
ion trap MSIMS analysis on the digests should be performed once they have
been converted to their corresponding methyl esters since that is the form they

will be in post-enrichment. Additionally, digestions were performed on the p65

236

 

antll

into

par
phi
of

dig

(I)

antibody as well as lgG, to be used as negative controls as well as conversion
into their corresponding methyl esters as well.

We were hopeful that upon MS and MSIMS analysis of the digests,
particularly the treated lP digests, we would identify peptide/s (either
phosphorylated or not) that belonged to p65. Unfortunately, the MS and MSIMS
of all the ions that had a relative intensity >10% of the untreated and treated IP
digests looked the same as the p65 antibody negative control digest (Figures 4-
8 thru 4-10). Not only do the main ions present in the digests not correspond to
p65 for either the stimulated or un-stimulated samples, they are all the same ions
that are present in the p65 antibody digest. This is most likely due to the
significant excess of p65 antibody present in the sample since the antibody:p65
ratio is probably close to 20:1 respectively, which results in those dominant ions.

To confirm the hypothesis that all the ions are in fact the same,
subsequent sequence analysis of all of the dominant ions present in the digests
was performed and it was found that all the ions coincided with the p65 antibody
ions. Additionally, none of the sequenced ions corresponded to any
phosphopeptide ions. This is not necessarily bad if the enrichment proved
successful, as this would illustrate the importance of performing phosphopeptide
enrichment, since no ions corresponding to phosphopeptides were found in the
pre-enrichment digest. Regardless of the fact that the p65 antibody, the
untreated IP and treated lP sample digests all appeared the same and there was
no indication of any phosphopeptides, i went on to try the enrichment using the

diazo ketone solid support, theorizing that the phosphopeptides present in the

237

 

digest are most likely to be in sub-stoicheometric levels relative to the p65
anfibody.

An aliquot of these methyl ester converted digests were pulled up in
tetrahydrofuran and ~20 mg of our optimal enrichment material, the diazo ketone
functionalized polystyrene, was added. The covalent coupling took place
overnight while being rocked gently at room temperature, the same enrichment
conditions as described for the commercially available phosphoprotein digests.
The resin was filtered and washed with THF, acetonitrile and methanol to ensure
removal of all unbound, non-phosphorylated peptides. After filtration, the resin
was exposed to 50% trifluoroacetic acid, 40% dichloromethane overnight to
hydrolyze the phosphoester-resin bond, liberating the phosphopeptide. Upon
exposure to the cleavage conditions, the eluent (containing the liberated
phosphopeptides) was collected by filtration, and dried down under nitrogen.
The dried enriched phosphopeptides were desalted with C18 zip tips and
subsequently analyzed by MALDI-ion trap MS.

The enrichment of the stimulated THP-1 nuclear extract lP proved to be
successful, as there were a few phosphopeptides present in the MS spectrum
(Figure 4-11). There was no enrichment identified in the un-stimulated nuclear
extract IP, which coincides with both the western and silver stain data as the
amount of NF-KB is significantly less in the un-stimulated lP. Additionally, there
was not any enriched phosphopeptides for the p65 antibody standard digest
either, which illustrates that the enriched peptides were from a nuclear protein

and not a result of an enriched peptide from a p65 antibody digest. The two

238

 

confirmed phosphopeptides were the 1738.1 and the 2302.3 ions. The MS/MS
data for the 1738.1 ion indicated a dominant loss of ammonia (1720.8 ion) and
the loss of methanol (1705.8). There was a smaller ion at 1640.1, indicating a
loss of -98, corresponding to -H3PO4 (Figure 4-12). Further confirmation that
the 1738 ion was a phosphopeptide was obtained by the MS3 data. The 1705.8
ion was further fragmented and the dominant ion was a -98 loss of -H3PO4
(1607.8), confirming the peptide was phosphorylated (Figure 4-13).

Additionally, the ion at m/z 2302.3 was also subjected to MSIMS analysis
and the resulting spectra contained a dominant ion at 2204, indicating a -98 loss
of -H3PO4 (Figure 4-14). The subsequent MS3 analysis did provide some
additional sequence information, as the peptide contained a C-terrninal arginine
(Figure 4-15). The other ions present in the enrichment did not exhibit a -98 loss
when fragmented, thus they cannot be confirmed (or ruled out) as being
phosphopepfides.

Although, this data is very promising, as we were able to successfully
enrich a few phosphopeptides from the stimulated nuclear extract lP, however,
the phosphopeptide ions present in the enrichment spectra, did not correspond to
the predicted p65 phosphopeptide ions from a tryptic digest (Table 4-3). There
are a few possible reasons for this outcome. These enriched phosphopeptides
could belong to one of the 15+ additional proteins that were visualized in the
silver stain of the IP sample. Additionally, the protein could be modified in a
plethora of different ways, which would also shift the monoisotopic mass,

resulting is a lack of lineage for the predicted peptide masses.

239

 

The subsequent MSIMS analysis then would prove to be a valuable tool in
determination if the phosphopeptide ions were in fact from the p65 subunit.
Sequence analysis proved to be difficult on the enriched samples, however, due
to the dominant H3PO4, and MeOH (since peptides were converted into methyl
esters) loss. As indicated previously, not much sequence information, other than
determining that the peptide was in fact a phosphopeptide, was determined from
MSIMS analysis (Figures 4-12, 4-14). There were a few amino acids that were
believed to be present given the MS/MS data, but the partial sequence data did
not coincide with any of the p65 protein sequence. The collection of MS3 data
did not aid in providing much more sequence information (Figures 4-14, 4-16).

Because if this difficultly, the enrichment samples were subjected to
hydrolysis under basic conditions, to remove the methyl esters, which could
reduce some of the complexity of the MSIMS and MS3 data. Additionally, the
fragmentation in the MS/MS and MS3 could be different for the hydrolyzed form
of the peptide, providing additional sequence information. I was able to obtain
the fully hydrolyzed form of the enrichment sample (the 1738.1 and 2303.0
correspond to the 1649.6 and 2231.1 respectively), but now the baseline appears
more noisy, potentially creating more difficulty in interpreting the sequence
(Figure 4-16). The ion at mlz 2232.09 corresponds to the methyl ester
converted peptide at m/z 2302, which indicates that there are at least four
carboxylic acid residues + the C-terrninus present in the peptide, as the
hydrolysis led to a 70 Da loss. The MS/MS analysis had an ion present at

2133.6, indicating a loss of -98 (H3PO4) and provided a few sequence ions.

240

 

 

Hc

58

0C

er

I!"

However, the ions present in the MSIMS data did not provide any protein
sequence hits in the database (Protein Prospector) (Figure 4-17).

The ion at m/z 1649.6 is believed to be the hydrolyzed version of the
methyl ester converted ion at 1738.1. While the difference in the masses does
not add up exactly to equal a number of methyl groups, this could be the result of
an additional elimination of water + hydrolysis of the methyl esters. If that is the
case, then there are at least four carboxylic acid residues present in this peptide
as well. The MSIMS analysis of the ion at m/z 1649.6 confirmed that this peptide
is also phosphorylated by the dominant ion at 1551.5 (-H3PO4) (Figure 4-18).
The additional sequence ions present in the MSIMS were also input into an MS
database to determine the sequence and the protein it is from. Unfortunately no
hits matched up well to either the methyl ester converted or fully hydrolyzed
sequence ions that were present in the enriched sample.

I changed a variety of parameters to see if I could obtain better sequence
coverage in the database, but I was never able to get any prospector hits with
MSIMS ions that l obtained from any of the enriched peaks. In an effort to obtain
part of the peptide sequence, l broadened the search by entering only the main
MSIMS ions to, which also proved unsuccessful. This is most likely due to the
absence of the protein of interest being in the database. The small sequence
information I did obtain from the MS data as well as the determination of the
number of acidic residues present, I was able to rule out the possibility that either

of the two enriched phosphopeptides belonged to the p65 protein.

241

 

Further confirmation that the enriched phosphopeptides were from a
nuclear extract protein and not from lgG or from the p65 antibody used, was
obtained by the enrichment of the negative control digests and there were no
phosphopeptides enriched in either of these two controls. Even when l analyzed
both the digests and the enriched samples in SIM (selected ion monitoring) for
the two phosphopeptides that were enriched in the treated samples, I was not
able to observe anything at the m/z value. This led me to conclude that the
enriched peptides were not a result of either the antibody or lgG, but from some
protein pulled out in the immunoprecipitation experiment.

While the success of the enrichment illustrates promise of this approach
(and the chosen enrichment material) in its application towards nuclear extract
phosphopeptide enrichment, the limitations/challenges that have been identified
raise some questions on how to modify this approach to effectively perform the
task it was designed for; to identify the phosphopeptides present in NF-KB as a

result of activation of its” pathway.

4.8.a Future project directions towards the application of enrichment on cellular
proteins

The problems that have arisen as a result of using this technique to purify
and enrich phosphopeptides from p65 are as follows: (1) the digests are
saturated with the p65 antibody, making it very difficult to confirm that p65

peptides are present in the digest, (2) the immunoprecipitation purification still

242

 

contains many additional proteins whose identity is largely unknown, (3) the
enriched phosphopeptides’ protein origin cannot be identified.

While these problems are evident there are options to circumnavigate
around them. The continuation of this project and its application in nuclear
protein enrichment is plausible, by implementing a few modifications. Elimination
of the significant excess of p65 antibody in the IP digest could provide very
valuable MS data, which would contain peptide ions that were purified by the IF.
Using this data, we would gain a better idea of which proteins were present in the
IP, provided the proteins were in the database. This could be accomplished one
of two ways, either by covalently coupling the antibody to the immobilized
support (ref), which could allow for liberation of the affinity bound protein/s by
changing the stringency of the buffer used without liberating the antibody as well.
Additionally, the immunoprecipitated sample could be separated by gel
electrophoresis and the bands of interest could be excised and subjected to in-
gel digestion, however, preparatory gel electrophoresis would be required to
obtain enough protein to use for phosphopeptide enrichment.

The use of gel electrophoresis to purify the proteins in the IP sample,
could also address the problem in the identification of the proteins present within
the sample. To reduce the tedious extraction, digestion and analysis of every
band present, the use of a phosphospecific stain, such as Pro-Diamond Q, could

be used to identify the phosphoproteins.89"‘35

The subsequent extraction,
digestion and analysis of only the detected phosphoproteins would reduce the

sample’s complexity.

243

 

 

Q)

n\

An additional potential complication that should not be overfooked is the
sequence coverage of phosphorylated peptides in a tryptic digest of p65. While it
is hypothesized that there are phosphorylation sites on NF-KB that are unknown,
which is why we are pursuing to enrich and analyze this protein, there are
already some known phosphorylation sites of p65 as a result of TNF-a
activation."'10 The known phosphorylation sites were included in the theoretical
tryptic digest of p65, however, additional post-translational modifications were not
taken into account. As illustrated in Table 4-3, the resulting known
phosphopeptides present in a tryptic digest would be challenging albeit
impossible to enrich and then observe by MALDI-ion trap MS. Either the
monoisotopic mass is too small to be observed relative to the matrix adducts
present in the spectrum, or the peptides are too large to even be observed by
MALDI-ion trap M8. Implementation of other proteases (chymotrypsin, GIuC,

and pepsin) could somewhat improve the sequence coverage in the digestion.

244

 

 

 

Table 4-3.

 

 

 

 

 

 

“an 'e.mzra".'1

 

 

 

 

 

 

 

 

 

Ref. Residues Sequence Missed Monoisotopic
cleavages mass
311-315 pSlMKK 1 686.4
20
18 274-278 RPpSDR 0 710.3
20 305-314 TYETFKpSIMK 1 1327.6
13 268-278 VSMQLRRPpSDR 1 1424.7
503-551 LVTGAQRPPDPAPAPLGAPGL 0 5052.4
27,30 PNGLLpSGDEDFSpSlADMDFS
ALLSQISS
337-502 SSASVPKPAPQPYPFTSSLSTI 0 17141.6
NYDEFPTMVFPSGQISQASALA
PAPPQVLPQAPAPAPAPAMVS
ALAQAPAPVPVLAPGPPQAVA
22-24 PPAPKPTQAGEGTLSEALLQL
QFDDEDLGALLGNSTDPAVFT
DLApSVDNSEFQQLLNQGlPVA
PH'ITEPMLMEYPEAITR
Table 4-3. Predicted masses for the known phosphorylated peptides of a

tryptic digest of the p65 subunit of NF-KB

245

 

4.8

SU

of

ti‘

4.8.b Future project direction towards the improvement in enrichment efficiency
While we have illustrated that the this solid-phase enrichment approach is
successful, further improvements have been hypothesized to increase the level
of enrichment using the diazo-phosphate covalent binding including the
development of a solution-phase enrichment and a diazo-functionalized support

that stabilizes the phosphate-reactive form.

4.8.b.i Solution-phase approach to improved efficiency

While solid supports clearly are advantageous for their ease in purification,
the reaction can be compromised when performing a solid-phase reaction on ‘in-
solution” materials. Additionally, solid supports are very solvent dependent as
their swelling properties vary significantly depending on the solvent used. in an
effort to improve the percentage of recovery of phosphorylated peptides, looking
to change to a solution-phase reaction might prove to be more efficient. By
utilizing the same diazo moiety that we know is reactive towards phosphate and
placing it on a ‘support’ that is soluble and then purifying the ‘support’ in another
manner (i.e. affinity). With that in mind, multiple groups have previously used
biotin to react with their modified peptide and then isolated the biotin-peptide by
manipulating biotin’s affinity for avidin, using an immobilized avidin column.

Specifically, diazo functionalizing a biotin-containing moiety to bind to
phosphopeptides and then utilizing its affinity for avidin is an approach worth
pursuing. in the introduction chapter l briefly mentioned a few groups who used

the biotin-avidin interaction to react with the phosphopeptidesaf”87 However, in

246

 

 

these cases the phosphate had been eliminated and the covalent coupling takes
place upon the Michael addition of the modified phosphopeptide and the biotin
denvafive.

In an effort to covalently label the phosphate backbone in nucleotides,
Bourget and co-workers used a diazo functionalized biotin material.88 The diazo
species was prepared following the synthetic route outline in Scheme 4-1. First,
biotin was coupled to 2-amino acetophenone in the presence of isobutyl
chloroformate and N-methyl morpholine to form compound 4-1. The coupled
biotin-amide was then subjected to conversion of the ketone moiety to its
corresponding hydrazone in the presence of hydrazine monohydrate under
refluxing conditions to obtain product 4-2. The prepared hydrazone was then
oxidized to form its corresponding diazo moiety upon the introduction of
manganese dioxide to complete the synthesis of 4-3. The manganese dioxide
was removed and the resulting pink solution was removed of solvent under
vacuum evaporation. Confirmation of the diazo-biotin product was achieved by

both NMR and lR.

247

 

NH2 0

JUL 1.©/u\ j:

 

 

HN NH HN NH O
2 S o 2 H
"I OH ‘1”
S r/V\Ir A S /\/\n/
O Y0 CI 0
4-1
N-methyl morpholine, in DMF
JHZN—NHZ
0 it
NH2
HN NH HN NH '
N2 Mnoz N \
n = H n
S "11W 8 "Ir/\/\"/
0 O
4-3 4-2

Scheme 4-1. The formation of the diazo-functionalized biotin derivative

Upon the successful preparation of the diazo biotin material, a
phosphopeptide, angiotensin II (MW 1154.5 when converted to corresponding
methyl ester peptide), was introduced to the diazo biotin reagent and the
coupling was accomplished using the optimized conditions cited by Bouget and
co-workers; heating the mixture in a 1:3:1 solution of DMSO:ACN:H20 in 2mM
Boric acid, pH 7.3 (Scheme 4-2). An aliquot from the coupling vessel was
removed, dried and desalted with a C18 zip tip prior to MALDI-ion trap MS
analysis. Confirmation of the success of the coupling was ascertained by the
presence of a molecular weight shift by 346 Da in the spectra (Figure 4-19),
corresponding to angiotensin ll-biotin coupled product. While the reaction did not

go to completion, as some unmodified angiotensin II peptide is present, the result

248

 

does illustrate that the coupling is occurring and as such, could be manipulated

under modified conditions to yield a higher percent conversion.

H o R o
H2N_§\ll’N\(lkfi/k§§AOMe
o

 

0
CH2 HNJLNH N2
’ U “
.,,I N
8 /\/\ll/
0‘ ,OH O
o’/P‘0H
1:3:1 DMSO:ACN:H20
Boric Acid
60°C
I
H O R O
Him-gm” NJ\§§JLOMe
H
0 CH2
0‘ ,OH
o”P‘o
0 HM
\>~NH
HN #0
\

8

Scheme 4-2. The coupling of angiotensin II to diazo biotin

The proof of concept of the diazo biotin coupling leads to potential other
avenues of study including the application of this approach followed by
phosphopeptide purification through the introduction of an immobilized avidin

column. Additionally, the phosphopeptide could be liberated from the avidin

249

 

 

 

 

nu

column under acidic conditions to eliminate the potential disassociation problems
associated with the avidin-biotin chromatography, which have already been
previously addressed. While biotin affinity chromatography is an attractive
approach because of its availability and well documented protocols, there are a
few limitations using biotin as an affinity tag. The biotin-avidin interaction is quite
strong, making elution of the biotin-peptide species sometimes difficult. As a
result, other researchers have utilized this established enrichment strategy but
developed a solution to the current issues associated with biotin affinity tags.
The incorporation of both the ethanedithiol and the maleimide was performed,
however, the maleimide was bound to an acid cleavable solid support instead of
biotin, thereby eliminating the problems associated with biotin.89 Additional work
maintained the use of biotin as an affinity label, but was first coupled to an acid
cleavable linker prior to its introduction to the modified peptide and then
subsequently liberated the modified peptide from the affinity column under acidic
conditions.87

It would also be of interest to compare the solution-phase phosphate
coupling of the phenylmethyldiazo biotin with a biotin derivative containing the
diazo ketone moiety which has shown to be the more efficient enrichment
material in the solid-phase reaction. The preparation of these types of materials
can be proposed by the application of the previously used synthetic route for
diazo ketone supports.

The coupling of an 0- or p- amino substituted benzoic acid in the same

manner as illustrated in Scheme 4-2, followed by the conversion for the acid to

250

 

de

VI

the acid chloride using thionyl chloride. Upon formation of the acid chloride biotin
derivative, the conversion into the diazo ketone can be obtained by the addition
of TMS-diazomethane. Thorough washing and subsequent solvent removal in
vacuo should remove the un-reacted TMS-diazo methane. Once the material is
in hand, it would be important to determine its relative enrichment efficiency
compared to the diazo ketone solid support as well as the other in-solution

reagent, the phenylmethyldiazo-biotin.

O O

o
HNJLNH DAG” HNJKNH
8

Scheme 4-3. Synthetic route to prepare diazo ketone functionalized biotin

denvafive
4.8.b.ii Preparation of a new diazo solid support to improve efficiency

Additional diazo-functionalized solid supports could be theorized as being

potential phosphopeptide enrichment reagents. As mentioned previously, the

251

 

basis of the reactivity of the diazo carbonyl moiety towards phosphopeptides is

the result of the interchanging resonance forms of the support (Scheme 4-4).

 

..._ O
H
O
O
HO’P‘o 2
2% H H2C o
MeO N
I \r 11*” aé-NHZ
R 0

Scheme 44. Mechanism for enrichment of phosphopeptides.

It can be hypothesized that implementation of an additional group to
stabilize the formation of the resonance form containing the carbanion, could shift
the equilibrium to favor that form. Since the reactivity of the diazo ketone species
towards phosphopeptides is a result of that resonance structure, the

incorporation of an additional carbonyl would help shift the equilibrium to the

252

 

negatively charged carbon resonance form, which is phosphopeptide-reactive

(Scheme 4-5).

VS.
0 O O O
W .__——-- W
4.
c3_ mi

Scheme 4-5. Comparison between diazo ketone solid support and a diazo

dicarbonyl solid support

Due to the stabilization of the diazo diketone moiety and subsequent
application of these materials to perform synthetic transformations, there are
numerous publications highlighting the synthesis and use of these types of diazo
diketone structures, particularly on solid supports.”93 The seemingly straight-
fonrvard preparation of these materials on solid supports also makes these
materials good candidates for pursuing their potential as phosphopeptide
enrichment supports. As we have already illustrated in Chapter 2, the various
reactivities of these diazo-functionalized species, we have not yet pursued

manipulating the structure to favor the desired resonance form.

253

4.9 Conclusions

The preliminary data illustrating the success of phosphopeptide
enrichment from a nuclear extract using our optimal solid support is encouraging
for the potential of this technique. The aforementioned problems in both the
purification of the protein and its corresponding analysis, limited the approach in
its current form. However, there are possible solutions to some of the issues
discussed previously and I believe the implementation of these solutions can
provide the ability to isolate, enrich and characterize the phosphorylation sites of
p65 (or one of its carrier proteins).

Additional to the implementation of modifications from the nuclear
extract/protein purification end, is the development of a more efficient enrichment
material, which could also improved the enrichment of phosphopeptides from a
nuclear extract IP sample. The concept of developing a solution-phase
enrichment support using a functionalized biotin is a possible approach, which
has shown promise in it preliminary data. The advantages in both reaction time
and efficiency by implementing solution-phase chemistry, instead of mediating
the enrichment through a solid-support, is an avenue worth pursuing. The use of
developing a material, which shifts the equilibrium to the reactive ‘carbanion’
resonance form, could also prove to be an additional material (or group of
materials) to analyze for the enrichment capabilities.

Lastly, i would like to briefly highlight the impact that phosphoproteomic
characterization and technologies have had on the identification of cancer

biomarkers and drug targets. The sheer number of publications that are relevant

254

to this area illustrate its importance and depth of research ongoing in the field.
While our phosphoproteomic technique and the other enrichment approaches
discussed herein, indicate that much progress in this area have been made in the
last two decades, there are still limitations with each technique and no ‘gold-
standard’ has yet to be found for phosphoprotein enrichment and
characterization. The merging of technologies seems to be on the rise, leading
toward a more encompassing approach that utilizes multiple methods, as the
limitations of one approach may not be limitations in another. This type of
cooperative analysis yields more complete phosphoproteomic analysis and
results. While complete global phosphorylation site elucidation is not yet routine,
publications everyday are identifying novel modification sites and including these
sites on protein databases, enabling more complete sequence information for
database analysis.

Additionally, new MS sequencing parameters are being renovated to
provide more complete phosphopeptide identification and sequence
analysis/interpretation. improving the MS automated capabilities allows for larger
datasets to be analyzed with a higher confidence and fewer ‘false positive’
results. All of the technologies developed for phosphoprotein identification and
quantification have increased the capabilities of these phosphoproteins to be
used as biomarkers. Specific elucidation of signal transduction pathways
pertaining to cancer improves the breadth of use of phosphoproteins as

predictive or diagnostic markers, and potential drug targets.

255

 

 

4.10 Experimental procedures

Materials

Sequencing-grade trypsin was purchased from Promega. Cell culture materials
were all purchased from Gibco. Biotin and additional synthetic reagents were all
purchased from Sigma Aldrich Chemical Co. All primary and secondary
antibodies and the antibody-agarose conjugates were all purchased from Santa

Cruz Biotechnology.

Nuclear Extraction of CEM cells

THP-1, CEM, L363, RPMI 8336 cells were all grown in RPMI-1640 media
supplemented with 10% fetal bovine serum, penicillin (614 ng/mL), streptomycin
(10 pg/mL), and HEPES buffer (pH 7.2) at 37°C, 5% C02. The activated cells (~
1.0 x 106 cells/mL) were subsequently stimulated with TNF-q (0.05 pg/mL) for 30
min. The cells (~ 4.0 x 107 total cells) were harvested by centrifugation, washed
in ice-cold PBS (1 mL), and the cellular pellets were lysed on ice in 1 mL
cytoplasmic extraction buffer (10 mM HEPES buffer, pH 7.9, 10 mM potassium
chloride, 10 mM sodium fluoride, 1.5 mM magnesium chloride, 1.0% Nonidet p-
40, 1 mM sodium orthovanadate, 1 mM B—glycerophosphate, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5pL leupeptin, 5pL aprotinin).
Nuclei were pelleted (10,000 RPM, 4°C, 1 minute), and maintained on ice and
the cytosolic extracts were extracted off using a pipet and stored at -80 °C . The

nuclei were resuspended by vortexing at 4°C for 20 minutes in 120 pL of nuclear

extraction buffer (20 pM HEPES buffer, pH 7.9, 0.42 M sodium chloride, 10 mM

256

 

 

sodium fluoride, 1.5 mM magnesium chloride, 25% glycerol, 1 mM sodium
orthovanadate, 1 mM B-glycerophosphate, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 0.2 mM EDTA, 5 pL leupeptin, 5pL aprotinin). The
nuclear extracts were cleared (14,000 RPM, 4 °C. 10 minutes), transferred to
fresh tubes and pulled up in 240 pL of nuclear protein storage buffer (20 mM
HEPES buffer, pH 7.9, 0.1 M potassium chloride, 10 mM sodium fluoride, 20%
glycerol, 1 mM sodium orthovanadate, 1 mM B-glycerophosphate, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.2 mM EDTA, 5 pL
leupeptin, 5 pL aprotinin). Protein concentrations were determined by the
Bradford assay. The nuclear extracts were all stored at -80 °C and thawed on ice
prior to their use for either the western blot analysis or for immunoprecipitation

punficafion.

Bradford Assay of Nuclear Extracts

A five point standard curve was obtained using lgG (0 pg/pL, 0.1 pg/pL, 0.2
pg/pL, 0.4 pg/pL, 0.8 pg/pL) in a 1:4 dilution of nuclear storage buffer (20 mM
HEPES buffer, pH 7.9, 0.1 M potassium chloride, 10 mM sodium fluoride, 20%
glycerol, 1 mM sodium orthovanadate, 1 mM B-glycerophosphate, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.2 mM EDTA, 5 pL
leupeptin, 5 pL aprotinin) in sterile water (40 pL total volume for each standard).
The standards were then pulled up in 500 pL Bradford Reagent Dye (diluted 1:4

in deionized water), and their absorbance was measured at a wavelength of 595

nm. Nuclear extracts were prepared as outlined above and an aliquot of each

257

 

 

nuclear extract sample (1 pL) was diluted in deionized water (39 pL) and then
further diluted in 500 pL Bradford Reagent Dye (diluted 1:4 in deionized water),
the absorbance measured at a wavelength of 595 nm and its corresponding

protein concentration was determined by comparison to the prepared standard

curve.

p65 Western Blot Analysis of Nuclear Extracts

Each nuclear extract samples (20 pg) diluted in deionized water (15 pL total
volume) and further diluted in 15pL of 2x sample buffer (10 mM tris pH 6.8, 0.2 M
sodium dodecyl sulfate, 20% glycerol, 0.01% bromophenol blue, 2% 2-
mercaptoethanol). The samples were boiled for 3 min, briefly centrifuged and an
aliquot (20 pL) of each sample was loaded onto a 10% Tris-SDS gel and run at
200 const. V until tracking dye ran off gel. The proteins were electroblotted to a
PVDF membrane in Towbin buffer (25 mM tris base, 192 mM glycine, 5%
methanol) at 60 V for 75 minutes. The membrane was blocked in 4% non-fat
dairy milk for 1 hour at room temperature and then washed for 10 minutes in
TBS-T buffer (50 mM tris base pH 7.6, 154 mM sodium chloride, 0.2% Tween-
20) and probed overnight at 4 °C with p65 antibody (Santa Cruz sc-372) that was
diluted 1:1000 in 4% non-fat dry milk (10 mL). The membrane was washed 3x10
minutes in TBS-T buffer and probed for 1 hr. with the 2° antibody (anti-rabbit
conjugated to horseradish peroxidase) that was diluted 1:3000 in 4% non-fat dry
milk (10 mL). The membrane was washed again 3x10 minutes in TBS-T buffer

and the detection reagent (GE Healthcare) was introduced for 1 min (2 mL) and

258

 

then exposed the film for detection. To confirm the same amount of protein was
loaded for each sample, the membrane was probed for actin for three hours at
room temperature with actin antibody that was diluted to 1:10000 in 4% non-fat
dry milk (10 mL). The membrane was washed 3x10 minutes in TBS-T buffer and
probed for 1 hr. with the 2° antibody (anti-mouse conjugated to horseradish
peroxidase) that was diluted 1:3000 in 4% non-fat dry milk (10 mL). The
membrane was washed again 3x10 minutes in TBS-T buffer and the detection
reagent (GE Healthcare) was introduced for 1 min (2 mL) and then exposed the

film for detection.

immunoprecipitation of p65 from Nuclear Extracts

Nuclear extract samples (200-1000 pg) were pre-cleared by adding them to lgG
(1 pg) and Protein A/G Plus Agarose (20 pL) and incubated at 4 °C for 30 min.
The samples were centrifuged at 10,000 RPM for 1 min at 4 °C. The eluent was
removed and placed in an eppendorf tube containing p65-antibody agarose
conjugate (Santa Cruz sc-372) (20 pL), pulled up in cytoplasmic extraction buffer
(10 mM HEPES buffer, pH 7.9, 10 mM potassium chloride, 10 mM sodium
fluoride, 1.5 mM magnesium chloride, 1.0% Nonidet p-40, 1 mM sodium
orthovanadate, 1 mM B-glycerophosphate, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 5pL leupeptin, 5pL aprotinin) (200 pL) and
incubated by rocking overnight at 4 °C. The immunoprecipitate was centrifuged
10,000 RPM for 1 min and the eluent was removed by a pipet. The protein-

antibody-agarose pellet was washed 2x in cytoplasmic extraction buffer (500 pL)

259

 

 

and 2x PBS (500 pL) and placed on ice until further use in either proteolytic

digestion or gel electrophoresis.

p65 Western Blot Analysis of lmmunoprecipitated Samples

The immuoprecipitatied sample pellet (200 pg total protein) was diluted in 30 pL
of 2x electrophoresis sample buffer (10 mM tris pH 6.8, 0.2 M sodium dodecyl
sulfate, 20% glycerol, 0.01 % bromophenol blue, 2% 2-mercaptoethanol) and
boiled at 100 °C for 3 minutes. An aliquot (20 pL) of sample was loaded onto a
10% Tris-SDS gel and run at 200 const. V until tracking dye ran off gel. The
proteins were electroblotted to a PVDF membrane in Towbin buffer (25 mM tris
base, 192 mM glycine, 5% methanol) at 60 V for 75 minutes. The membrane
was then blocked in 4% non-fat dairy milk for 1 hour at room temperature and
then washed for 10 minutes in TBS-T buffer (50 mM tris base pH 7.6, 154 mM
sodium chloride, 0.2% Tween-20) and probed overnight at 4°C with p65 antibody
(Santa Cruz sc-8008) diluted 1:250 in 4% non-fat dry milk (10 mL). The
membrane was washed 3x10 minutes in TBS-T buffer and probed for 1 hr. with
the 2° antibody (anti-mouse horseradish peroxidase conjugate) diluted 1:3000 in
4% non-fat dry milk (10 mL). The membrane was washed again 3x10 minutes in
TBS-T buffer and the detection reagent (GE Healthcare) was introduced for 1

min (2 mL) and then exposed to film for detection.

260

 

Tryptic Digestion of lmmunoprecipitated Samples and the Conversion of
the Peptides to Methyl Esters

The immuoprecipitatied sample pellet (1000 pg total protein) 20 pL of a
denaturing solution (6 M urea, 50 mM Tris-HCI) was added along with 5 pL of
dithiolthreitol (10 mM) and the sample was incubated at 65 °C for 1 hour. The
sample was cooled to room temperature and 160 pL of ammonium bicarbonate
(50 mM) and 10 pL of iodoacetamide (100 mM) was added and the sample was
incubated at room temperature in the dark for 1 hour. A 10 pL aliquot of
sequencing-grade trypsin (purchased from Promega) was then added (0.5 pg/
pL) and the sample was incubated overnight at 37 °C. The digested sample was
then dried down under nitrogen gas, pulled up in a solution of 30% acetonitrile in
water (200 pL), aliquoted out into 50 pL aliquots, then dried down under nitrogen
again. The dried digests were converted to their methyl esters upon the addition
of methanolic hydrochloric acid, which was prepared by the addition of acetyl
chloride (320 pL) to anhydrous methanol (2 mL). An aliquot (40 pL) of this
solution was added to dried digests and incubated at room temperature for 2 hr.
The methyl ester converted digest was then dried down under nitrogen and

subsequently stored at -20 °C until their use.

Enrichment of phosphorylated peptides from a digest of an
immunoprecipitated protein using diazo functionalized resins
Diazo functionalized material (~20 mg) and tetrahydrofuran (1 mL) was added to

dried methyl ester converted immunoprecipitated protein digest. The mixture

261

man:
.I
l

 

was rockedg
filtration and
and then met
was complete
10% water I
temperature,
solution. Th
combined 6
samples we
following ti
acetonitrile
and inlrod
liberatedt
In a ima\

MALDI-i

SV“~ht
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cook;
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and

was rocked gently overnight at room temperature. The resin was recovered by
filtration and washed with tetrahydrofuran (5x500 pL), acetonitrile (3x 500 pL)
and then methanol (3x 500 pL). Cleavage of the captured species from the resin
was completed by incubation in a solution 50% trifluoroacetic acid, 40% DCM,
10% water (500 pL). The mixture was rocked gently overnight at room
temperature, allowing for full interaction between the resin and the cleavage
solution. The resin was filtered and washed once with methanol (500 pL). The
combined eluents were concentrated under nitrogen. Prior to analysis, the
samples were diluted in water (20 pL) and desalted using Millipore C18 Zip Tips,
following the manufacturers protocol. Briefly, the zip tip was moistened with
acetonitrile (3x10 pL), then equilibrated with 0.1% trifluoroacetic acid (3x10 pL)
and introduced to the enrichment sample (18x10 pL). The bound peptides were
liberated from the zip tip with 50:50 0.1% trifluoroacetic acidzacetonitrile, resulting
in a final volume of 6 pL. All samples were subsequently stored at -20°C prior to

MALDI-ion trap MS analysis.

Synthesis of 4-1a. 2-(N-Biotinoylamino)acetophenone

To a solution of U-biotin (1.0 g, 4.1 mmol) in dry N,N—dimethylformamide (45 mL)
coole dat 0 °C under argon, N-methylmorpholine (590 pL, 5.33 mmol) and
isobutyl chloroformate (840 pL, 6.60 mmol) were added successively. The
solution was stirred for 30 min, and then 2-aminoacetophenone (824 mg, 6.10
mmol) was added. The solution was then stirred at room temperature for 3.5 h

and then the solvent was removed in vacuo. The residue was triturated with cold

262

 

 

 

water (50 mL) and the resulting precipitate was filtered, washed with water and
recrystallized from ethanol to obtain the coupled product 4-1a (0.8 mg, 53%). lR
(NaCl plate): 3238, 2920, 1699, 1653, 1450, 1247 cm". 1HNMR (500 MHz,
DMSO-d5): 6 = 1.38-1.66 (m, 6H), 2.38 (t, J = 7.0 Hz, 2H), 2.58 (d, J = 7.5, 1H),
2.61 (s, 3H), 2.82 (dd, J: 5.0, 12.5 Hz, 1H), 3.11 (m, 1H), 4.14 (m, 1H), 4.31 (m,
1H), 6.35 (br s, 1H), 6.44 (br s, 1H), 7.19 (t, J = 7.0 Hz, 1H), 7.58 (t, J = 7.5 Hz,
1H), 7.97 (d, J = 7.5 Hz, 1H), 8.33 (d, J = 8.5 Hz, 1H), 11.25 (br s, 1 H). 13cme
(500 MHz, DMSO-d6): 6 = 24.87, 28.03, 28.06, 28.69, 37.12, 55.33, 59.18,
61.00, 120.54, 122.78, 124.20, 131.48, 134.06, 139.03, 162.70, 171.49, 202.59.

MS (calc. for M+ = 361.15 ), found: 362.1 (M-H“).

Synthesis of 4-2. 2-(N-Biotinoylamino)acetophenone hydrazone

To a solution of the ketone 4-1a (0.40 g, 1.11 mmol) in absolute ethanol (8 mL)
and hydrazine monohydrate (570 pL, 11.0 mmol) was added. The solution was
refluxed for 50 min and then solvents were evaporated. The residue was
suspended in water, filtered, washed with water and with ether and dried in
vacuo to afford hydrazone 4-1b (0.30 g, 73 %). IR (NaCl plate): 3249 (br), 2921,
1697, 1608, 1530, 1446 cm". 1HNMR (500 MHz, DMSO-d6): 5 = 1.31-1.63 (m,
6H), 2.10 (s, 3H), 2.32 (t, J = 7.4 Hz, 2H), 2.57 (d, J = 12.5, 1H), 2.82 (dd, J =
5.0, 12.5 Hz, 1H), 3.10 (m, 1H), 4.14 (m, 1H), 4.30 (m, 1H), 6.36 (br s, 1H), 6.43
(br s, 1H), 6.61 (br s, 2H), 7.04 (t, J = 7.5 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.45
(d, J = 7.5 Hz, 1H), 8.36 (d, J = 8.0 Hz, 1H), 11.98 (br s, 1 H). ”CNMR (500

MHZ, DMSO-d6): 5 = 13.19, 13.23, 25.15, 28.06, 28.16, 37.41, 55.43, 59.18,

263

 

 

61.06, 120.11, 122.56, 125.55, 127.40, 127.63, 136.71, 145.36, 162.71, 170.90.

MS (calc. for M+ = 375.17), found: 375.2 (M*).

Synthesis of 4-3. 2-(N-Biotinoylamino)phenylmethyldiazomethane

To a solution of hydrazone (0.30 g, 0.80 mmol) in 10 mL N,N-dimethylformamide,
Mi’IOz (800 mg) was added and the suspension was stirred for 15 min at room
temperature. The mixture was suction filtered through Celite (~1 cm thickness).
The filtrate was placed into a clean 25 mL round bottom flask and the solvent
was evaporated in vacuo. The residue was then triturated with ether to obtain
product 4-1, as a pink powder (0.18 g, 60%). IR (NaCl plate): 3250, 2039, 1700,
1685 cm". 1HNMR (500 MHz, DMSO-d6): 5 = 1.39-1.64 (m, 6H), 2.13 (s, 3H),
2.26 (t, J = 7.0 Hz, 2H), 2.59 (d, J = 12.5, 1H), 2.83 (dd, J = 4.0, 12.5 Hz, 1H),
3.13 (m, 1H), 4.15 (m, 1H), 4.32 (m, 1H), 6.36 (br s, 1H), 6.44 (br s, 1H), 7.02-
7.21 (m, 4H), 9.37 (brs, 1 H). ”CNMR (500 MHz, DMSO- d6): 5 = 12.26, 24.96,
28.08, 28.28, 35.10, 55.39, 59.20, 61.03, 124.14, 124.80, 126.33, 127.63,
128.20, 132.76, 162.70, 171.09. MS (calc. for M+ = 373.47 ), found: 345.2 (M‘-

N2).

Biotin-phenylmethyldiazomethane-phosphopeptide coupling

In an eppendorf tube containing a dried aliquot of angiotensin II converted to its
methyl esters (100 pmol), a small amount of the compound 4-1 (~5 mg) was
added. The mixture was dissolved in 1:3:1 DMSO:acetonitrile:water solution

(100 pL total) containing 2 mM H3B03 (pH 7.3) and incubated at 60 °C for 2

264

hours. An aliquot of the solution (10 pL) was removed from the mixture, and was
dried under nitrogen. Prior to analysis, the samples were diluted in water (20 pL)
and desalted using Millipore C18 Zip Tips, following the manufacturers protocol.
Briefly, the zip tip was moistened with acetonitrile (3x10 pL), then equilibrated
with 0.1% trifluoroacetic acid (3x10 pL) and introduced to the enrichment sample
(18x10 pL). The bound peptides were liberated from the zip tip with 50:50 0.1%
trifluoroacetic acidzacetonitrile, resulting in a final volume of 6 pL. The samples
were subsequently stored at -20°C prior to MALDI-ion trap MS analysis.

MALDI-ion trap MS: ion at m/z 1154.5 (angiotensin II) and ion at m/z 1500.6

(angiotensin ll-coupled to biotin derivative)

Mass Spectrometry

M8 and CID-MSIMS analyses were performed using a linear quadrupole ion trap
mass spectrometer equipped with a Matrix Assisted Laser Desorption Ionization
(MALDI) source (model vMALDl-LTQ, Thermo, San Jose, CA). Desalted
enrichment samples were spotted on MALDI plate (0.5 pL) and an aliquot of DHB
matrix (10 mg/mL in a solution of 50:50 acetonitrile:1 % phosphoric acid) was
spotted over the sample. Additionally, another sample was spotted on the
MALDI plate (0.5 pL) and then overlaid with 1 pL a-cyano matrix (10 mglmL in a

solution of 50:50 acetonitrile: 0.1 % trifluoroacetic acid).

265

.f’iimmltt!
I _ - _ ,

 

Appendix

 

 

 

 

Figure 4-8.
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Figure 4-8. MALDI-ion trap MS of the p65 antibody standard tryptic digest.

266

Figure 4-9.

 

 

 

 

 

 

 

 

 

 
  
    
  
   

 

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Figure 4-9. MALDI-ion trap MS of the THP-1 untreated nuclear extract
immunoprecipitation tryptic digest.

267

Figure 4-10.

Relative intensity

 

 

 

 

 

 

 

_L
o 8 8
l I I l I I I [J
‘4
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Figure 4-10. MALDI-ion trap MS of the THP-1 treated nuclear extract
immunoprecipitation tryptic digest.

268

Figure 4-11.
Relative intensity

009 003i
OO'OQSI
60 891i

0002:

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000$

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00017

Figure 4-11. MALDI-ion trap MS of the THP-1 treated nuclear extract
immunoprecipitation tryptic digest enrichment.

269

Figure 4-12.
Relative intensity

 

 

 

 

 

o 8 8
8 l I I L l L l I i
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Figure 4-12. MALDI-ion trap CID MSIMS of ion 1738 from the THP-1 treated
nuclear extract IP enrichment

270

 

Figure 4-13.
Relative intensity

 

 

 

m .4
o o 8
8 llllIJJlf
0
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8
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Figure 4-13. MALDI-ion trap CID MS3 of ion 1738, 1705 from the THP-1 treated
nuclear extract lP enrichment

271

 

Figure 4-14.

Relative inten sity _.
o. 8
C) O

lLl'lllJ

     

008

003i

3

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0098.71.
‘Hls-

Figure 4-14. MALDI-ion trap CID MS/MS of ion 2302 from the THP—1 treated lP
tryptic digest enrichment.

272

 

Figure 4-15.

Relative intensity

OH
O

00L

lllllJl‘ll1

4w
09H 0031 096

001.1.

 

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096i

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Figure 4-15. MALDI-ion trap CID MS3 of the 2302, 2204 ion from the THP-1
treated enrichment

273

‘4___-2—. an?"

_ I‘
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Figure 4-16.
Relativeinhensily

0|
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Figure 4-16. MALDI-ion trap MS of the THP-1 treated nuclear extract IP tryptic
digest enrichment, NH4OH hydrolysis

274

 

 

Figure 4-1 7.

Relative intensity

 

.3
(II D
O o Q
g lLllllillJ
O
—_4
8.—
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Figure 4-17. MALDI-ion trap CID MSIMS of ion 2232 from THP-1 treated nuclear
extract IP tryptic digest enrichment, NH4OH hydrolysis

275

 

 

Figure 4-18.
Relative Intensity

006 099
8L'6178 L
— 0
~— 00l

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Figure 4-18. MALDI-ion trap CID MSIMS of ion 1649 from THP-1 treated nuclear
extract IP tryptic digest enrichment, NH4OH hydrolysis

276

Figure 4-19.

Relative in ten sity

 

 

 

 

o 8 8
<5 I I L! 1 1 J i I
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11
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8
Figure 4-19. MALDI-ion trap MS angiotensin II-diazo biotin binding.

 

277

10.

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