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This is to certify that the
dissertation entitled

DEVELOPMENT AND APPLICATION OF TANDEM MASS
SPECTROMETRY METHODS FOR PHOSPHOPROTEIN
ANALYSIS

presented by

AMANDA M. PALUMBO

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DEVELOPMENT AND APPLICATION OF TANDEM MASS SPECTROMETRY
METHODS FOR PHOSPHOPROTEIN ANALYSIS

By

Amanda M. Palumbo

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Chemistry

2009

 

ABSTRACT

DEVELOPMENT AND APPLICATION OF TANDEM MASS SPECTROMETRY
METHODS FOR PHOSPHOPROTEIN ANALYSIS

By
Amanda M. Palumbo

Protein phosphorylation is involved in nearly all essential biochemical
pathways and the deregulation of phosphorylation events has been related to the
onset and progression of numerous diseases. Mass spectrometry has been at
the forefront of phosphoproteomic characterization due to the plethora of different
tandem mass spectrometry (MS/MS) strategies that enable comprehensive
phosphoproteome analysis. However, each of these tandem mass spectrometry
strategies has a limited set of conditions under which phosphoproteome
information may be produced due to the difference in compatible precursor ion
charge states and the type (i.e., sequence vs. nonsequence), structure, and
abundance of the structurally-informative product ions formed. Accordingly,
understanding the underlying chemical principles involved in these techniques
that influence the gas-phase fragmentation reactions are critical, as the chemistry
directly influences their utility. Therefore, a comprehensive review regarding the
gas-phase fragmentation reactions of phosphopeptides and their influence on the
various tandem mass spectrometry strategies employed for phosphoproteome
analysis is discussed here. Based on this discussion it is deduced that much of
the gas-phase ion chemistry involved in the most commonly used tandem mass
spectrometry method for phosphoproteome analysis, collision-induced

dissociation (CID), has yet to be understood. To address this need, the studies

described here demonstrate that the combined role of precursor ion charge state
and peptide basic residue content (i.e., proton mobility) heavily influences the
gas-phase ion chemistry of phosphorylated peptide ions using CID. The
mechanisms and related product ion structures associated with the formation of
phosphate-specific reactions that occur under CID conditions are also elucidated
and invalidate previously established mechanisms. Additionally, it is shown that
intramolecular gas-phase phosphate migration and other competing
fragmentation reactions involving the phosphate occurs upon ClD-MS/MS, which
may cause ambiguous or erroneous phosphorylation site interpretation. As a
result of all of these studies, the applicability of CID for phosphopeptide
identification and characterization is discussed. Finally, the development and
application of a phosphoproteome analytical strategy aimed to determine the
phosphorylated proteins that associate with p65, a protein critically involved in
the immune response, from differentially treated monocytic leukemia cells are
described. Importantly, the affects of gas-phase ion chemistry of phosphorylated
peptides was considered during the downstream data interpretation. In this
study, five p65-associated proteins are identified, including two that have not

been previously reported to interact with p65.

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my graduate co-advisors,
Professors Gavin Reid and Jetze Tepe for allowing me to complete this
collaborative research project which required the expertise from each person's
lab. I am so proud of the research that we were able to complete and the results
that we achieved together. Gavin and Jetze, I want to thank you both for your
guidance and never-ending support, both in and out of the lab. I also must thank
all of the current and former members of the Reid and Tepe research groups who
have supported me, laughed with me, and encouraged me. Many thanks to all of
the collaborators who have contributed to this work. Dr. Henry Duewel at Sigma
Aldrich and Stacey Hoge at Sigma Genosys for synthesizing many of the
phosphorylated peptides for this work. Additionally, Professor Don Hunt and Dr.
Erin Jeffery at the University of Virginia and Professor Paul Stemmer at Wayne
State University for enabling the analysis of many phosphopeptides by electron
transfer dissociation. I would like to also thank Mr. Weihan Wang and Professor
Menin Bruening from Michigan State University for developing their
phosphopeptide enrichment strategy. In particular, I truly thank Weihan for
helping me with data acquisition. The results from this work could not have been
achieved had it not been for your dedication. Lastly, the completion of my
degree would not have been possible without the hospitality and kindness shown
to me by my many interim roommates: Nicole Torres, Troy Knight, Claire Laity,

Georgia, Angus, and Sylvester.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................ IX
LIST OF FIGURES ............................................................................................... X
LIST OF SCHEMES .......................................................................................... XVI
CHAPTER ONE .................................................................................................... 1
Mass Spectrometry Methods for Phosphoproteome Analysis ............................... 1
1.1 Introduction ................................................................................................. 1
1.2 Mass Spectrometry Methods Employed for Phosphoproteome Analysis ...5
1.2.1 Ionization of Phosphorylated Peptides ............................................... 5
1.2.2 MS Analysis of Phosphorylated Peptide Ions ..................................... 6
1.2.3 Tandem Mass Spectrometry and Gas-phase lon Chemistry of
Phosphorylated Peptides .................................................................... 7

1.2.3.1 Collision-Induced Dissociation (CID) ........................................ 13
1.2.3.1.1 Formation of 79 m/z (P03) and [M+nH-79]‘"‘1* Ions ....... 15

1.2.3.1.2 Formation of Phosphotyrosine-Specific Immonium Ions at

m/z 216.043 .................................................................... 16

1.2.3.1.3 Product Ions Formed From 80 or 98 Da Neutral Losses. 18

1.2.3.1.4 Chemical Derivatization and ClD-MS/MS ........................ 23

1.2.3.2 MALDI Post-Source Decay (PSD) ............................................ 25
1.2.3.3 Photodissociation ..................................................................... 28
1.2.3.3.1 Infrared Multiple Photon Dissociation (IRMPD) ............... 28

1.2.3.3.2 Ultraviolet Photodissociation (UVPD) .............................. 29

1.2.3.3.3 Femtosecond Laser-Induced Ionization/Dissociation

(stlD) ............................................................................. 35

1.2.3.4 Electron-Driven Dissociation .................................................... 36
1.2.3.4.1 Electron Capture Dissociation (ECD) .............................. 37

1.2.3.4.2 Electron Transfer Dissociation (ETD) .............................. 41

1.2.3.4.3 Electron Detachment Dissociation (EDD) ....................... 45

1.2.3.5 Metastable Atom-Activated Dissociation (MAD) ....................... 45
1.2.3.6 Comparison of Tandem Mass Spectrometry Techniques for
Phosphoproteome Analysis ...................................................... 47

1.3 Specific Aims ............................................................................................ 50
CHAPTER TWO .................................................................................................. 53
Mechanistic Insights into the Multistage Gas-Phase Fragmentation Behavior of
Phosphoserine- and Phosphothreonine-Containing Peptides ............................. 53
2.1 Introduction ............................................................................................... 53
2.2 Results and Discussion ............................................................................ 59

2.2.1 Evaluation of Charge-Remote and Charge-Directed Fragmentation
Mechanisms for the Loss of H3PO4 from Phosphoserine- and

V

Phosphothreonine-Containing Peptides via Multistage Tandem Mass
Spectrometry Analysis of Their Regioselectively and Uniformly
Deuterated Precursor Ions ............................................................... 59
2.2.2 Neutral Loss of H3PO4 from Both Phosphoserine- and
Phosphothreonine-Containing Peptides ls Highly Dependent on
Precursor Ion Charge State and Amino Acid Composition (i.e., Proton
Mobility) ............................................................................................ 76
2.2.3 Experimental Evidence for the Neutral Loss of H3PO4 from
Phosphoserine- and Phosphothreonine-Containing Peptides via an

8N2 Neighboring Group Participation Reaction Mechanism. ............ 90
2.3 Conclusions .............................................................................................. 95
CHAPTER THREE .............................................................................................. 96

Evaluation of Gas-Phase Rearrangement and Competing Fragmentation
Reactions on Protein Phosphorylation Site Assignment Using Collision-induced-

dissociation-MS/MS and MS3 .............................................................................. 96
3.1 Introduction ............................................................................................... 96
3.2 Results and Discussion .......................................................................... 104
3.2.1 ClD-MS/MS of Protonated Phosphopeptides Results in Gas-Phase
Phosphate Group Transfer Reactions. ........................................... 104

3.2.2 CID-MS3 of [M+nH-98]n+ Ions May Not Be Used for Unambiguous
Phosphorylation Site Localization. .................................................. 145

3.3 Conclusions ............................................................................................ 156
3.4 Other Recent Studies Demonstrating Intramolecular Gas-phase
Phosphate Group Transfer ..................................................................... 157

3.5 Additional Commentary .......................................................................... 159
CHAPTER FOUR .............................................................................................. 161
Experimental Methods for Gas-Phase Fragmentation Studies of
Phosphopeptides (Chapters Two and Three) ................................................... 161
4.1 Materials ................................................................................................. 161
4.2 Phosphopeptides .................................................................................... 163
4.2.1 Fmoc Solid—Phase Peptide Synthesis (SPPS) ................................ 164
4.2.1.1 Apparatuses ........................................................................... 164
4.2.1.2 Synthesis ................................................................................ 164
4.2.1.2.1 Fmoc Deprotection ........................................................ 165

4.2.1.2.2 Amino Acid Coupling ..................................................... 165

4.2.1.2.3 Resin Cleavage and Deprotection of Acid-Labile
Orthogonal Protecting Groups ....................................... 166

4.2.2 Synthesis of GAlL(d3-D,L)pSGAILK ............................................... 167
4.2.2.1 Synthesis of (d3-D,L)-Fmoc-Serine-OH (4.a) ......................... 168
4.2.2.2 Synthesis of (d3-D,L)-Fmoc-Serine(O-TBDMS)-OH (4.b) ...... 168
4.2.2.3 On-resin SPPS of Boc-GAIL(d3-D,L)pSGAlLK(Boc) (4.c) ...... 169

4.2.3 Peptide Purification ......................................................................... 170
4.3 Multistage Tandem Mass Spectrometry Analysis of Phosphopeptides ..171
4.3.1 Phosphopeptide Sample Preparation ............................................. 171
4.3.2 Mass Spectrometry ......................................................................... 172

vi

4.3 Quantitative Analysis of Percent Product Ion Abundances ..................... 174

CHAPTER FIVE ................................................................................................ 175
Application of Phosphoproteomic Analysis Strategy to Elucidate p65-Associated
Phosphoproteins in the NF-KB Signaling Pathway From Treated and Untreated

THP-1 Cell Nuclear Extracts ............................................................................. 175
5.1 Introduction ............................................................................................. 175
5.2 Results and Discussion .......................................................................... 185

5.2.1 Development of the Phosphoproteome Analysis Strategy Employed
for Identification of p65-Associated Phosphoproteins From

Differentially Treated THP-1 Cells .................................................. 185

5.2.1.1 Proteolytic Digestion ............................................................... 185
5.2.1.2 Phosphopeptide Enrichment .................................................. 186
5.2.1.3 Mass Spectrometry Analysis and Database Searching .......... 186
5.2.2 Differentially Treated THP-1 Cells and Resultant Differential Levels in
Nuclear p65 .................................................................................... 190

5.2.3 Identification of p65-Associated Phosphoproteins From TNF—o—
Treated THP-1 Cells ....................................................................... 192

5.2.4 Identification of p65-Associated Phosphoproteins From Untreated
THP-1 Cells .................................................................................... 215

5.2.5 Relevance of Identified Proteins Co-lmmunoprecipitated With p65220
5.2.5.1 Ribosomal Proteins: 6OS Acidic Ribosomal Protein from the P1

or P2 Isoforms and Ribosomal Protein L19 ............................ 220

5.2.5.2 TLS and TLS-Associated Serine-Arginine Protein (TASR) ..... 222
5.2.5.3 Actin ....................................................................................... 223

5.3 Conclusions and Future Directions ......................................................... 224
5.4 Supplemental Figures ............................................................................ 226
CHAPTER SIX .................................................................................................. 234
Experimental Methods for Applications of Mass Spectrometry Methods for
Phosphoproteomic Analysis (Chapter Five) ...................................................... 234
6.1 Materials ................................................................................................. 234
6.2 Cells ....................................................................................................... 235
6.3 Nuclear Extraction of THP-1 Cells .......................................................... 235
6.3.1 Bradford Protein Assay: Determination of Protein Concentration in
Cellular Extracts ............................................................................. 237

6.3.2 Western Blot Analysis of p65 from Nuclear Extracts ...................... 237
6.4 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
of lmmunoprecipitated Proteins from Nuclear Extracts .......................... 238

6.4.1 Preparation of Immunoprecipitation (IP) Matrix: Covalently Cross-
Linked p65 Primary Antibody to Protein A/G-Agarose .................... 238

6.4.2 Immunoprecipitation of Cellular Extracts Using Covalently Cross-
Linked p65 Primary Antibody to Protein A/G-Agarose .................... 240

6.4.3 Western Blot Analysis of p65 From p65-lmmunoprecipitated Nuclear
Extracts of THP-1 Cells .................................................................. 241

6.5 ln-Solution Tryptic Digestion and Subsequent Phosphopeptide Enrichment
of lmmunoprecipitated Proteins .............................................................. 242

vii

6.6 Synthesis of the lntemal Standard "D5” CDgCDzCO-LFTGHPEpSLEK..243
6.7 Matrix Assisted Laser Desorption/Ionization and Multistage Tandem Mass

Spectrometry .......................................................................................... 244
6.8 Peptide Identification by Database Searching ........................................ 245
REFERENCES .................................................................................................. 246

viii

LIST OF TABLES

Table 2.1 Summary of the MS/MS and MS3 neutral loss product ion abundances
from the gas-phase fragmentation reactions of protonated phosphgsebrine- and
phosphothreonine-containing peptides. a “Partially mobile” proton.9 b“Mobile”
proton. 93 ° “Nonmobile” proton. ‘93 d Results obtained by ClD-MS/MS of
uniformly deuterium labeled peptide ions. ° Note that these values differ from
those shown in Figure 2.1 for the regioselectively deuterated GAIL(d3-
D,L)pSGAILK peptide and are likely due to either their acquisition on a different
instrument platform (LCQ-DECA versus LTQ), the effect of the D, L- versus L-
phosphoamino acid, or deuterium isotope effects on the charge-remote versus
charge.directed fragmentation mechanisms. fResults obtained by CID-MS/MS of
protonated peptide ions. 9 Calculated as the ratio of % total product ion ,
abundances of the [M+nH—H3PO41" ions from isomer phosphopeptide Ions (e. g.,
GAILpSGAILK versus GAILpTGAILK). hResults obtained by CID-MS3 of [M+nH-
H3PO4-CH20]"+ (pSer) or [M+nH- -H3PO4-CH3CHO]"+ (pThr) product ions initially
formed by MS/MS. .............................................................................................. 70

Table 3.1 Summary of the gas-phase phosphate group transfer product ions
observed following ESI- and/or MALDI-ClD-MS/MS of independently synthesized
tryptic phosphopeptides. a Proton mobility classifications defined by Kapp, et

Non-mobile: number of ionizing protons 5 number of arginine residues;
Partially-mobile: number of arginine residues < number of ionizing protons 5
combined number of arginine, lysine and histidine residues; Mobile: number of
ionizing protons > combined number of arginine, lysine and histidine residues. b
.......................................................................................................................... 133

Table 4.1 Common amino acid building blocks used for solid-phase peptide
synthesis. .......................................................................................................... 163

LIST OF FIGURES

Figure 1.1 Structures of phosphorylated serine, threonine and tyrosine residues.3

Figure 1.2 Flow chart of previously demonstrated tandem mass spectrometry
strategies for phosphopeptide analysis. The phosphopeptide precursor ion
charge state (n) requirement for each technique is specified. Some techniques,
namely ClD-, PSD-, and IRMPD-MS/MS, may involve formation of “non-
sequence” product ions that diagnostically indicate the presence of
phosphorylated peptide ions. The observation of these diagnostic product ions
has been used to trigger the acquisition of CID-MS3 spectra of the neutral loss
product ion, or to trigger the acquisition of MS/MS spectra of the original
precursor ion by other methods (e.g., ECD-MS/MS). *Phosphopeptide analysis
by UVPD of deprotonated precursor ions has been performed on phosphate-
derivatized peptides. ............................................................................................. 9

Figure 1.3 Nomenclature of peptide backbone fragment ions or sequence ions.
............................................................................................................................ 12

Figure 1.4 General structure of an immonium ion, where R is the amino acid
side-chain and the phosphotyrosine-specific immonium ion, m/z 216.043 .......... 17

Figure 2.1 Multistage tandem mass spectrometry of regioselectively deuterated
GAIL(d3-D,L)pSGAILK. CID-MS/MS of the (A) [M+H]+ and (B) [M+2H]2+
precursor ions. CID-MS3 of the (C) singly and (D) doubly protonated [M+nH-
H3PO4]"” neutral loss product ions from panels A and B, respectively. CID-M83
of the (E) singly and (F) doubly protonated [M+nH-HZDPO41'” neutral loss product
ions from panels A and B, respectively. A = -H3PO4, ° = -H20, * = -NH;;. ........... 61

Figure 2.2 Multistage tandem mass spectrometry of GAILpSGAILK. CID-MS/MS
product ion spectra of the (A) [M+H]+ and (B) [M+2H]2+ precursor ions. CID-MS3
product ion spectra of the (C) singly and (D) doubly protonated [M+nH-H3PO4]n+
neutral loss product ions from panels A and B, respectively. A =-H3PO4, ° = -
H20, * = -NH3. ..................................................................................................... 64

Figure 2.3 ClD-MS/MS product ion spectra of protonated and deuterated
LRRApSLG. (A) [M+H]*, (B) [M+2H]2*, (C) [M(D19)+D]", and (D) [M(D19)+ZD]2+
IONS. A = -H3PO4. ............................................................................................... 73

Figure 2.4 Multistage tandem mass spectrometry of LRRApTLG. ClD—MS/MS
product ion spectra of the (A) [M+H]+ and (B) [M+2H]2+ precursor ions. CID-MS3
product ion spectra of the (C) singly and (D) doubly protonated [M+nH-H3POII]n+
neutral loss product ions from panels A and B, respectively. A = -H3PO4, * = -
NH3. .................................................................................................................... 74

Figure 2.5 Multistage tandem mass spectrometry of GAILpSGAILR. CID-
MS/MS product ion spectra of the (A) [M+H]+ and (B) the [M+2H]2+ precursor
ions. CID-M83 product ion spectra of the (C) singly and (D) doubly protonated
[M+nH-H3PO4]"" neutral loss product ions from panels A and B, respectively. A
=-I"I3PO4, * = -NI"I3. .............................................................................................. 78

Figure 2.6 Multistage tandem mass spectrometry of GAILpTGAILR. CID-MS/MS
product ion spectra of the (A) [M+H]+ and (B) [M+2H]2+ precursor ions. CID-M83
product ion spectra of the (C) singly and (D) doubly protonated [M+nH-H3P04]n+
neutral loss product ions from panels A and B, respectively. A =-H3PO4, * = -NH3.
............................................................................................................................ 80

Figure 2.7 Multistage tandem mass spectrometry of GAILpTGAILK. ClD-MS/MS
product ion spectra of the (A) [M+H]+ and (B) [M+2H]2+ precursor ions. CID-MS3
product ion spectra of the (C) singly and (D) doubly protonated [M+nH-H3P04]n+
neutral loss product ions from panels A and B, respectively. A =-H3PO4, ° = -
H20, * = -NH3. ..................................................................................................... 82

Figure 2.8 Multistage tandem mass spectrometry of LFTGHPEpSLEK. CID-
MS/MS 3product ion spectra of the (A) [M+H]+ and (B) [M+2H]2+ precursor ions.
CID-MS product ion spectra of the (C) singly and (D) doubly protonated [M+nH-
H3PO4]n+ neutral loss product ions from panels A and B, respectively. A =-H3PO4,
o = -H20. .............................................................................................................. 84

Figure 2.9 Multistage tandem mass spectrometry of LFTGHPEpTLEK. CID-
MS/MS :product ion spectra of the (A) [M-I-H]+ and (B) [M+2H]2+ precursor ions.
CID-MS product ion spectra of the (C) singly and (D) doubly protonated [M+nH-
H3PO4]"“ neutral loss product ions from panels A and B, respectively. A =-
H3PO4, * = -NH3. ................................................................................................. 86

Figure 2.10 CID-MS3 product ion spectra of the (A) singly and (B) doubly
protonated [M+nH-H3,PO.I]n+ neutral loss product ions of LRRApSLG from Figure
2.3A and B, respectively. * = -NH3. .................................................................... 93

Figure 3.1 Ion trap CID—MS/MS product ion spectra of the (A) singly, (B) doubly,
and (C) triply protonated precursor ions of the model synthetic phosphopeptide
LFTGHPEpSLER (TpSR). The spectra are labeled according to the known or
expected Ser phosphorylation site. Ions labeled with bold text are
unambiguously indicative of the expected site of phosphorylation at the Ser
residue. A = —98 Da (-H3PO4 or —(H20+HPO3)); CI = —80 Da (—HP03); ° = -18
Da (-HZO); I = +80 Da (+HP03). ...................................................................... 106

Figure 3.2 lon trap ClD—MS/MS product ion spectra of the (A) singly, (B) doubly
and (C) triply protonated precursor ions of the model synthetic phosphopeptide
LFpTGHPESLER (pTSR). The spectra are labeled according to the known or
expected Thr phosphorylation site. Ions labeled with bold text are
unambiguously indicative of either the expected or correct site of phosphorylation

xi

at the Thr residue, or the unexpected or incorrect site of phosphorylation at the
Ser residue. A = -98 Da (—H3PO4 or —(H20+HP03)); CI = —80 Da (—HP03); ° = —
18 Da (—H20); I = +80 Da (+HP03). ................................................................. 109

Figure 3.3 Ion trap CID—MS/MS product ion spectra of the (A) singly, (B) doubly
and (C) triply protonated precursor ions of the model synthetic phosphopeptide
LFpTGHPESLER (pTSR). The spectra are labeled according to the unexpected
(i.e., incorrect) site of phosphorylation at the serine residue. A = —98 Da (—H3PO4
or -(H20+HPO3)); c1 = —80 Da (—HP03); ° = —18 Da (—HzO); I = +80 Da (+HP03).
.......................................................................................................................... 115

Figure 3.4 Ion trap CID—MS3 product ion spectra of (A) the y6+80 Da (y6+ HPO3)
product ion formed from ClD-MS/MS of the singly protonated pTSR precursor ion
in Figure 3.2A and (B) the ye product ion formed from ClD—MS/MS of the singly
protonated TpSR precursor ion in Figure 3.1A .................................................. 120

Figure 3.5 lon trap ClD—MS/MS product ion spectra of the singly protonated
precursor ion of the model synthetic phosphopeptide LFpTGHPESLER (pTSR)
using (A) 10 msec and (B) 2000 msec ion activation times at an activation q
value of 0.25. The spectra are labeled according to the known (i.e., expected)
site of phosphorylation (i.e, Thr). Ions labeled with bold text are unambiguously
indicative of the unexpected site of phosphorylation (i.e, Ser), resulting from the
gas-phase rearrangement reaction. A = -98 Da (—H3PO4 or —(HZO+HP03)); CI =
—80 Da (-HP03); ° = —18 Da (—H20); I = +80 Da (+HP03); RA = relative
abundance. ....................................................................................................... 124

Figure 3.6 Triple quadrupole CID-MS/MS product ion spectra of the (A) singly,
(B) doubly, and (C) triply protonated precursor ions of the model synthetic
phosphopeptide LFpTGHPESLER (pTSR). The spectra are labeled according to
the known (i.e., expected) site of phosphorylation (i.e, Thr). Ions labeled with
bold text are unambiguously indicative of the expected site of phosphorylation at
the threonine residue. A = —98 Da (—H3PO4 or -(HZO+HP03)); CI = —80 Da (-
HPOa); ° = —18 Da (—H20). ................................................................................ 126

Figure 3.7 Expanded regions of the product ion spectra obtained by (A) ion trap
(Figure 3.2A) and (B) triple quadrupole (Figure 3.6A) ClD-MS/MS of the singly
protonated precursor ion of the model synthetic phosphopeptide
LFpTGHPESLER (pTSR). The spectra are labeled according to the known (i.e.,
expected) site of phosphorylation (i.e, Thr). Ions labeled with bold text are
unambiguously indicative of the unexpected site of phosphorylation (i.e, Ser),
resulting from the gas-phase rearrangement reaction. a = —80 Da (—HP03); ° = —
18 Da (-HzO); I = +80 Da (+HPO3). ................................................................. 129

Figure 3.8 lon trap CID—M83 product ion spectra of the (A) singly, (B) doubly,
and (C) triply protonated ([M+nH—98]"*) neutral loss product ions from the TpSR
peptide in Figure 3.1A—C, respectively. Ions labeled with bold text correspond to
the presence of an unmodified serine residue rather than the expected

xii

 

dehydrated residue. The inset in panel B shows the expanded m/z region from
600-630. ° = —18 Da (—H20); . = +18 Da (+H20). ............................................. 149

Figure 3.9 lon trap CID—M83 product ion spectra of the (A) singly, (B) doubly,
and (C) triply protonated ([M+nH—98]"+) neutral loss product ions from the pTSR
peptide in Figure 3.2A—C, respectively. Ions labeled with bold text correspond to
the presence of an unmodified threonine residue rather than the expected
dehydrated residue. The inset in panel B shows the expanded m/z region from
600-630. ° = -18 Da (—HZO); - = +18 Da (+H20). ............................................. 153

2[figure 4.1 Flowchart to determine cleavage solution composition. Adapted from
. ..................................................................................................................... 167

Figure 5.1 The NF-kB pathway. Adapted from 254. .......................................... 178

Figure 5.2 Summary of the known phosphorylation sites of p65 and the
corresponding [kinases] and [phosphatases*]. The kinases responsible for
Ser205, T254, and Thr435 phosphorylation are at present unknown, indicated
with [?]. This information was compiled from 256' 257; and adapted from 256. RHD
is the Rel homology domain and TAD is the transactivation domain ................. 181

Figure 5.3 Schematic of the phosphoproteome analytical strategy employed for
identification and phosphorylation site determination of p65-interacting proteins
from the nuclear fractions of untreated and TNF-o-treated THP-1 cells. ........... 184

Figure 5.4 Western blot detection of p65 (total, C-terminal epitope) and actin
from replicate untreated and TNF-a-treated THP-1 nuclear extracts. Western
blot of the actin loading control indicates that the untreated and TNF-cr-treated
THP-1 nuclear extracts contain comparable amounts of protein; and therefore,
that the amount of nuclear p65 is greater for the TNF-a-treated nuclear extract
than the untreated nuclear extract. This gel was edited to enable facile
comparison between the untreated and treated samples. The unaltered image is
shown in Figure 5.15. ........................................................................................ 191

Figure 5.5 Western blot detection of p65 (total, N-tenninal epitope) from p65-
immunoprecipated from untreated (IPT) and TNF-u-treated (lP‘) THP-1 nuclear
extracts. The immunoprecipitation of p65 from the untreated and treated
samples were completed on separate occasions. For this reason the
experiments are differentiated here. Control experiments involving
immunoprecipitation of p65 from phosphate-buffered saline are also shown (*).
Higher amounts of nuclear p65 is present in the treated than the untreated
samples after p65-immunoprecipitation, consistent with the results shown in
Figure 5.4 for the samples prior to p65-immunoprecipitation. The unaltered
image is shown in Figure 5.16 ........................................................................... 192

Figure 5.6 Representative MALDI-mass spectrum of the samples resulting from
phosphopeptide enrichment of the trypsin digested p65-lmmunoprecipitated
nuclear extracts from TNF-a-treated THP-1 cells, where * = identified

xiii

phosphorylated peptides; o = identified nonphosphorylated peptides; ? =
unidentified peptides; and m = matrix ions. ....................................................... 194

Figure 5.7 CID-MS/MS product ion spectrum of m/z 2061 (from the MS spectrum
shown in Figure 5.6). This peptide was identified as KEEpSEEpSDDDM(cam-
105)GFGLFD from the P1 or P2 isoforms of the 608 acidic ribosomal protein,
and resulting from the gas-phase neutral loss of 2-(methylthio)acetamide (105
Da) from the S-carbamidomethylmethionine (M(cam))-containing species (m/z
2166). A = —98 Da (—H3PO4 or —(H20+HP03)); CI = —80 Da (—HP03); ° = —18 Da
(—H20). .............................................................................................................. 196

Figure 5.8 ClD-MS/MS product ion spectra of m/z 2109 and 2125 (from the MS
spectrum shown in Figure 5.6). These peptides were identified as the
unmodified methionine— and oxidized methionine-containing species of
KEEpSEEpSDDDMGFGLFD from the P1 or P2 isoforms of the 608 acidic
ribosomal protein, respectively. A = -98 Da (—H3PO4 or —(HZO+HP03)); CI = —80
Da (—HP03); ° = -18 Da (-H20); :1: = —64 Da (—CH3$OH or methane sulfenic
acid). ................................................................................................................. 199

Figure 5.9 CID-MS/MS product ion spectrum of m/z 2166 (from the MS spectrum
shown in Figure 5.6). This peptide was identified as the S-
carbamidomethylmethionine-containing species of KEEpSEEpSDDDM(cam)-
GFGLF D from the P1 or P2 isoforms of the 608 acidic ribosomal protein. ....... 203

Figure 5.10 Residue structures of methionine, oxidized methionine (M(ox)). and
S-carbamidomethylmethionine (M(cam)). ......................................................... 204

Figure 5.11 CID-MS/MS product ion spectra of m/z (A) 1677, (B) 1693, (C) 1629,
and (D) 1734 (from the MS spectrum shown in Figure 5.6). These peptides were
identified as various isoforms of KEEpSEEpSDDDMGF, including the unmodified
methionine (m/z 1677), oxidized methionine (m/z 1693), product ion resulting
from the gas-phase neutral loss of 2-(methylthio)acetamide (105 Da) from the S-
carbamidomethylmethionine (m/z 2166), and S-carbamidomethylmethionine—
containing species (m/z 1734) from the P1 or P2 isoforms of the 608 acidic
ribosomal protein. A = —98 Da (—H3PO4 or —(H20+HP03)); CI = -80 Da (-HP03);
° = -18 Da (—H20); :I: = —64 Da (—CH3$OH or methane sulfenic acid). ............. 207

Figure 5.12 CID-MS/MS product ion spectrum of m/z 1367 (from the MS
spectrum shown in Figure 5.6). This peptide was identified as pSRpSFDYNYR
from the translocation liposarcoma (TLS)-associated serine-arginine protein
(TASR). A = —98 Da (-H3PO4 or —(H20+HP03)); I] = —80 Da (—HP03); ° = -18
Da (—H20). ........................................................................................................ 212

Figure 5.13 Representative MALDI-mass spectrum of the samples resulting from

phosphopeptide enrichment of the trypsin digested p65-immunoprecipitated
nuclear extracts from untreated THP-1 cells, where * = identified phosphorylated

xiv

peptides; o = identified nonphosphorylated peptides; ? = unidentified peptides;
and m = matrix ions. .......................................................................................... 217

Figure 5.14 MALDI-mass spectra of the (A) TNF-d-treated THP-1 cells and (B)
untreated THP-1 samples resulting from p65 immunoprecipitation, trypsin
digestion, phosphopeptide enrichment, and addition of 100 amol of an internal
standard C03C02CO-LFTGHPEpSLEK (“05”, m/z 1398), where * = identified
phosphorylated peptides; o = identified nonphosphorylated peptides; ? =
unidentified peptides; and m = matrix ions. ....................................................... 219

Figure 5.15 Unedited Western blot of p65 (total, C-terminal epitope) and actin
from replicate untreated and TNF-a-treated THP-1 nuclear extracts. Replicate
samples labeled with * were prepared previously (1 year prior to the preparation
of replicates 1 and 2) by another laboratory member and had been stored at -80
°C. Due to their similar amount of p65 in the untreated and treated previously
prepared samples, they were not used for any of the experiments described in
Chapter Five nor were they included in Figure 5.4. ........................................... 226

Figure 5.16 Unedited Western blot of p65 (total, N-terrninal epitope) from
untreated and TNF-q-treated THP-1 nuclear extracts (THP—1 NE); and p65-
immunoprecipated untreated (IP*) and TNF-a-treated (IPI) THP-1 nuclear
extracts. The immunoprecipitation of p65 from the untreated and treated
samples were completed on separate occasions. For this reason they are
differentiated here. Control experiments including immunoprecipitation of p65
from phosphate-buffered saline are also shown (*). An additional protein band
(lPa) is present in the samples resulting from immunoprecipitation of the treated
sample (IP‘). However, because this band is also present in the PBS control
sample, it lPal results from the immunoprecipitation process. ............................ 227

Figure 5.17 ClD-MS/MS product ion spectrum of m/z 1191 (from the MS
spectrum shown in Figure 5.6). This peptide was identified as HMYHSLYLK
from ribosomal protein L19. ° = —18 Da (—HzO) ................................................ 228

Figure 5.18 ClD-MS/MS product ion spectrum of m/z 1022 (from the MS
spectrum shown in Figure 5.6). This peptide was identified as AAIDWFDGK
from translocation liposarcoma (TLS) protein. ° = -18 Da (—HzO); * = —17 Da (—
NH3). ................................................................................................................. 230

Figure 5.19 CID-MS/MS product ion spectrum of m/z 1515 (from the MS
spectrum shown in Figure 5.6). This peptide was identified as IWHHTFYNELR
from actin (B—isoform). ° = -18 Da (—H20). ....................................................... 232

XV

LIST OF SCHEMES

Scheme 1.1 Previously proposed mechanism for cleavage of the P-O
phosphoester bond of negatively charged phosphorylated peptides forming [M-
nH-79]<""* and P03‘(m/z 79). .............................................................................. 15

Scheme 1.2 Fragmentation pathways involving the phosphate group upon CID-
MS", where dS and dT represent dehydrated serine and threonine residues,
respectively. ........................................................................................................ 23

Scheme 1.3 Analysis of napthyl chromophore-derivatized phosphorylated serine
and threonine peptides by UVPD at 266 nm. B-elimination of phosphoserine and
phosphothreonine peptides followed by Michael addition to form the napthyl-
derivatized labeled peptides. UVPD of the derivatized peptides results in
selective cleavage of the chromophore and radical-induced cleavage allows for
the selective formation of sequence ions adjacent to the formerly phosphorylated
residue. ............................................................................................................... 32

Scheme 1.4 Proposed site-specific an+1-97 ion formation of singly protonated,
phosphorylated peptides that contain N-terminal basic residues upon UVPD at
193 nm, where R1 is the side chain for any amino acid ....................................... 35

Scheme 1.5 Previously proposed fragmentation mechanism for the production of
c- and z-type product sequence ions for the reaction between a low-energy
electron with a multiply protonated peptide ion. .................................................. 39

Scheme 1.6 Penning ionization of a protonated peptide (M) ion, where n 2 1, by
interaction with a metastable noble gas atom (m*) .............................................. 47

Scheme 2.1 Potential mechanisms for the neutral loss of H3PO4 from
phosphoserine- and phosphothreonine-containing peptides via (A) a charge-
remote B—elimination reaction, (B) a charge-directed E2 reaction, and (C) a
charge-directed 8N2 neighboring group participation reaction. For clarity, the
mechanisms are presented for phosphoserine-containing peptides only. ........... 58

Scheme 2.2 Proposed mechanisms for the dominant loss of H3PO4 via a charge-
directed SN2 neighboring group participation reaction (Pathway 1) and for the
loss of formaldehyde (CHzO, 30 Da) or acetaldehyde (CH3CI‘IO, 44 Da) from the
[M+H-H3PO4]" product ions (Pathway 2) of protonated phosphoserine- and
phosphothreonine-containing peptide ions .......................................................... 89

Scheme 3.1 Previously proposed mechanisms for the “charge directed” neutral
loss of H3PO4 and RCHO from protonated phosphoserine- or phosphothreonine-
(where R = H or CH3, respectively) containing peptides during ClD-MS/MS.206
(A) Intramolecular E2 mechanism and (B) intramolecular SN2 neighboring group
participation mechanism. .................................................................................. 100

xvi

Scheme 3.2 Competing pathways for the formation of isobaric product ions via
the loss of 98 Da (—H3PO4 or —(HPO3+H20)) from a protonated phosphopeptide
containing multiple potential phosphorylation sites. R1 = H or CH3 for
phosphorylated serine or phosphorylated threonine, respectively; R2 = any amino
acid side chain; R3 = H or CH3 for non-phosphorylated serine or non-
phosphorylated threonine, respectively. ............................................................ 101

Scheme 3.3 Proposed mechanism for the intramolecular gas-phase phosphate
group transfer facilitated by hydrogen bonding to a protonated basic residue. .144

Scheme 3.4 Proposed mechanism for intramolecular phosphate group transfer
for deprotonated peptide ions. .......................................................................... 159

xvii

CHAPTER ONE

Mass Spectrometry Methods for Phosphoproteome Analysis

1.1 Introduction
Protein phosphorylation is perhaps the most pervasive post translational

modification, playing an essential role in the coordination and regulation of

. 1, 2 . .
vrrtually all cellular processes. SInce the genome does not encode for protein

phosphorylation directly, phosphorylation events are dynamic and reflect the
current physiological needs of a cell. Accordingly, protein phosphorylation is
reversible, whereby phosphorylation by kinases and dephosphorylation by
phosphatases serves to regulate protein activity, expression levels, sub-cellular
localization, and protein—protein interactions. Due to its broad biological
importance, it is not surprising that deregulation of phosphorylation events has

been associated with the onset and progression of many pathological

conditions}8 Therefore, understanding protein phosphorylation on a molecular

level has been of great interest towards understanding the difference between

“healthy" and “abnormal” cellular states, as well as for opening new avenues of

 

The concepts discussed in Chapter One have been submitted for publication in
Mass Spectrometry Reviews: Palumbo, A. M.; Smith, S. A.; Stemmer, P. M.;
Kalcic, C. L.; Dantus, M.; Reid, G. E. Tandem Mass Spectrometry Strategies for
Phosphoproteome Analysis.

disease therapy.8.12

Due to the elaborate nature of protein phosphorylation, its analysis
presents a formidable challenge. For example, since phosphorylation is a
transient modification, the lifetime of a phosphorylated site may be short and
therefore the associated structure may be difficult to “capture” for analysis.
Furthermore, a particular phosphorylated form of a protein may be
substoichiometric, meaning that only a small fraction of the population of a
particular protein may be phosphorylated at any given time. Moreover,
differentially phosphorylated isoforms (i.e., different sites of phosphorylation) of a
given protein may also exist simultaneously in this substoichiometric population.
The sheer number of potential phosphorylation sites also complicates their
analysis. In eukaryotes, phosphorylation predominantly occurs on serine,

threonine, and tyrosine residues (Figure 1.1), which represent approximately
17% of the total amino acid content in an average protein.13 Based on this fact it
has been calculated that in an average eukaryotic cell, there exists nearly

700,000 different potential phosphorylation sites.14

OH OH ,‘p’OH
I I O \\
HO-P=O HO-P=O o
o o
a,” /['( KE'IIK H. N
o o H o

Phosphoserine, pS Phosphothreonine, pT Phosphotyrosine, pY

Figure 1.1 Structures of phosphorylated serine, threonine and tyrosine residues.

To overcome these inherent biological challenges, numerous mass
spectrometry (MS) based approaches have been developed, and have now
become the methods of choice for the identification and characterization of an
individual phosphoprotein or for the comprehensive examination of an entire

“phosphoproteome” from a given tissue, cell or sub-cellular fraction under a given

set of physiological or pathological conditions.15'21 Phosphoproteome analysis

methods are generally based on the interrogation of peptides derived from
enzymatic digestion of intact phosphoproteins, i.e., the “bottom-up approach”.
For complex biological mixtures, peptide digests are typically fractioned off-line

by chromatographic separation techniques (e.g., Reversed Phase High

Performance Liquid Chromatography, RP-HPLC).22 To address challenges

associated with the analysis of low abundance phosphoproteins or
substoichiometric phosphorylation, phosphopeptides are typically enriched prior
to on-line RP-HPLC and introduction to the mass spectrometer. Many different

enrichment techniques are available for this purpose, including immunoaffinity

chromatography,23 ion exchange chromatography,24'27 Immobilized Metal ion

Affinity Chromatography (IMAC),28'31 Metal Oxide Affinity Chromatography

(MOAC),32'36 and chemical derivatization.37'46 Furthermore, enrichment on the

phosphoprotein level may be employed to decrease sample complexity prior to

enzymatic digestionfw.49 Comprehensive reviews describing the development

and application of these phosphopeptide and phosphoprotein enrichment

methods have recently appeared elsewhere in the Iiterature,5o' 51 and so will not

be further described here. Although these sample preparation methods have
undoubtedly improved phosphoproteomic analysis strategies, significant
challenges remain associated with the mass spectrometry-based aspects of the
phosphoproteome characterization process. In particular, the presence of the
phosphate group can significantly alter the gas-phase chemistries of peptide
ions, particularly with regard to phosphopeptide structural analysis using tandem
mass spectrometry (MS/MS). This ultimately impacts the effectiveness of MS/MS
strategies used for phosphopeptide identification and characterization (i.e.,
phosphorylation site assignment). For this reason, this chapter focuses on
providing an overview of both traditional and newly emerging tandem mass
spectrometry strategies employed for phosphopeptide identification and
characterization. Particular attention is given to highlighting the underlying
chemical principles, mechanisms and other factors that influence the gas-phase
fragmentation reactions associated with each of these strategies, as these issues
directly influence their utility. Furthermore, several of the analytical challenges

that remain to be overcome in order for this important sub-discipline of the

maturing field of proteomics to be used more efficiently and effectively are

discussed.

1.2 Mass Spectrometry Methods Employed for Phosphoproteome
Analysis

The major goals of mass spectrometry-based phosphoproteomic analysis
are: (i) identify the presence of phosphorylated peptides, (ii) determine the
identity of phosphopeptides by their amino acid sequences, (iii) localize the
specific sites of phosphorylation, and (iv) quantitatively determine any temporal
dependence to the phosphorylation site occupancies, under the specific
biological conditions from which the phosphoprotein was isolated. These aims
may be achieved by “soft" ionization of phosphopeptides followed by the use of

mass spectrometry (MS), tandem mass spectrometry (MS/MS), and multistage

tandem mass spectrometry (MSn) techniques. Each of these mass spectrometry

approaches are described in detail below for phosphoproteome characterization

1.2.1 Ionization of Phosphorylated Peptides

Phosphopeptide ionization is typically achieved by electrospray ionization
(ESI) or matrix-assisted laser desorption/ionization (MALDI). The use of these
two sources often allow complementary MS and MS/MS information to be
obtained, as they produce different types of ions, i.e., multiply charged ions are
typically formed by ESI whereas singly charged ions are formed by MALDI. The

ionization, subsequent detection and/or structural analysis of phosphopeptides

by these sources may, however, be limited by the presence of extraneous
peptides. It has previously been proposed that the ionization efficiency of

phosphorylated peptides may be lower than their nonmodified peptide analogs

52, 53

using MALDI. For these reasons, the use of alternative MALDI matrices

such as 2’,4’,6’-trihydroxyacetophenone, and a range of matrix additives such as

ammonium salts and phosphoric/phosphonic acids, have been employed to

enhance the phosphopeptide ionization yields.54'57 The potential for decreased

ionization efficiency of phosphorylated peptides versus their nonphosphorylated

analogs has also been examined for ESI. However, the results of this study did

not indicate selective suppression.58 Rather, it was concluded that

phosphopeptide ions produced by ESI may be nonspecifically suppressed owing

to their low stoichiometry.58 These results further indicate a necessity for

phosphopeptide enrichment prior to MS analysis.

1.2.2 MS Analysis of Phosphorylated Peptide Ions
Perhaps the simplest method for identification of the presence of a

phosphopeptide by MS is via analysis of a sample before and after phosphatase

treatment.53’ 59 The observed reduction in mass by n multiples of 80 Da of a

peptide is indicative of the presence of n phosphorylation sites. Additionally,

derivatization of phosphopeptides with mass tags, either in solution or in the gas-

37-41, 46, 60

phase (via ion-molecule reactions), and subsequent MS analysis may

be used to determine the presence of phosphopeptides from a mixture.
Derivatization techniques have also been used for the incorporation of differential

labeling tags, allowing quantitative analysis of the occupancies of

38, 46, 61

phosphorylation sites. All of these techniques are, however, limited to

relatively simple mixtures, where the mass shift is readily observable. Thus, the
majority of methods for phosphoproteome analysis rely on the use of tandem

mass spectrometry techniques.

1.2.3 Tandem Mass Spectrometry and Gas-phase Ion Chemistry of
Phosphorylated Peptides
Tandem mass spectrometry (MS/MS) involves isolation of a selected
precursor (reactant) ion, followed by a gas-phase reaction (typically
fragmentation) and analysis of the resultant product ions. In certain types of
mass spectrometry instrumentation, product ions formed from a previous stage of

reaction can be subsequently isolated and then subjected to further reaction and

mass analysis. This is termed MSn analysis, where n represents the total

number of reaction stages. A plethora of tandem mass spectrometry methods

have been employed for the characterization of peptide ions, including collision-

)(reviewed in 62) (reviewed in

induced dissociation (CID post-source decay (PSD),

63)

(reviewed in 64)

infrared multiple photon dissociation (IRMPD), ultraviolet

in 65)

photodissociation (UVPD),(reViewed femtosecond laser induced

ionization/dissociation (stID),66 electron capture dissociation (ECD),(rev'ewed m

7

67)

(reviewed in 68)

electron transfer dissociation (ETD), electron ionization

dissociation (EID),69 electronic excitation dissociation (EED),(revlewed m 67)

(reviewed in 67)

electron-detachment dissociation (EDD), and metastable atom-

activated dissociation (MAD).70 Many of these tandem mass spectrometry

methods have been applied to phosphoproteome analysis, and the demonstrated
strategies are summarized in Figure 1.2. These tandem mass spectrometry
strategies have allowed for the identification of the presence of phosphorylated
peptides, the identification of the phosphopeptide by its amino acid sequence,
the determination of phosphorylation sites, and quantitative analyses of

phosphorylation site occupancies.

Figure 1.2 Flow chart of previously demonstrated tandem mass spectrometry
strategies for phosphopeptide analysis. The phosphopeptide precursor ion
charge state (n) requirement for each technique is specified. Some techniques,
namely ClD-, PSD-, and lRMPD-MS/MS, may involve formation of “non-
sequence” product ions that diagnostically indicate the presence of
phosphorylated peptide ions. The observation of these diagnostic product ions

has been used to trigger the acquisition of CID-MS3 spectra of the neutral loss
product ion, or to trigger the acquisition of MS/MS spectra of the original
precursor ion by other methods (e.g., ECD-MS/MS). *Phosphopeptide analysis
by UVPD of deprotonated precursor ions has been performed on phosphate-
derivatized peptides.

Figure 1.2 (cont'd)

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The ability to achieve such a wealth of phosphoproteome information from
these MS/MS techniques is ultimately dependent on the types of product ions
formed. The possible product ions resulting from the dissociation of a peptide ion

along its backbone have been defined previously and are summarized in Figure

71, 72

1.3 Briefly, fragmentation can occur at the Ca-Ccarbonyl. Ccarbony|-N (i.e.,

amide), or N-Ca bonds to give rise to a-, b-, or c-ions if the charge is retained on

the N-terminal segment and x-, y-, or z-ions if the charge is retained on the C-
tenninal segment. Therefore, if a population of precursor peptide ions undergo
fragmentation to produce a series of consecutive a-, b-, c-, x-, y-, or z-type
“sequence” ions, the amino acid sequence of the peptide could be determined
from the difference between these consecutive ions. Importantly, since the
residue masses of phosphorylated serine (167 Da), phosphorylated threonine
(181 Da), and phosphorylated tyrosine (243 Da) are unique, the product
sequence ions of phosphorylated peptides are indicative of the presence of

phosphopeptides and the location of the phosphorylation sites within the peptide.

11

......
OOOOOOOOOOOOOOOOOO

OOOOOOOOOOOOOOOOOO

r r . ..

O R' O R'"

 

 

 

 

 

 

 

 

 

Figure 1.3 Nomenclature of peptide backbone fragment ions or sequence ions.

In addition to backbone fragmentation reactions, fragmentation of certain
amino acid side chains may occur. Although these ions are not indicative of the
amino acid sequence or the location of the phosphorylation site within the
peptide sequence, and are therefore termed “nonsequence” ions, they may
provide information regarding the amino acid composition. In particular, some
ion dissociation techniques induce selective cleavage of the phosphoester bond
of phosphopeptides, resulting in the observation of nonsequence product ions
which indicate the presence of a phosphorylated peptide (Figure 1.2-Diagnostic
Ions). This is in contrast to ion dissociation techniques that give rise only to
sequence ions, where interpretation of the sequence is required to determine
whether or not a peptide is phosphorylated

Importantly, the specific method by which the ion is subjected to

fragmentation dictates the type (i.e., sequence vs. nonsequence) and abundance

12

of the product ions that are observed following an MS/MS experiment. The
observed sequence and/or non-sequence ions formed are also heavily
dependant on the properties of the precursor ion (e.g., precursor ion polarity and
charge state). As a result, the success of any given tandem mass spectrometry

strategy employed for phosphoproteome identification and characterization is

critically dependant on the underlying chemistry of these techniques.21 The

remainder of Chapter One therefore focuses on describing the current state of
knowledge regarding the gas-phase ion chemistry associated with the various
MS/MS methods employed to date for phosphopeptide identification and
phosphorylation site assignment (i.e., characterization), and highlights the
influence of gas-phase ion chemistry on the applicability of the analytical
strategies used. Reviews describing the development and application of
quantitative tandem mass spectrometry based phosphoproteomic analysis

strategies have recently been discussed elsewhere, and therefore will not be

discussed further herezo’ 73

1.2.3.1 Collision-Induced Dissociation (CID)

Collision-induced dissociation, CID, (also known as collision activated
dissociation, CAD) is the most widely used tandem mass spectrometry method
available for peptide sequencing. Many different mass analyzer platforms are
compatible with CID, including quadrupole ion trap (QIT) and Fourier transform-
ion cyclotron resonance (FT-ICR) mass spectrometers. Additionally, utilization of

quadrupole-based collision cells allow for CID-tandem mass spectrometry in

13

triple quadrupole (QqQ), quadrupole time of flight (qTOF), and TOF/T OF
instruments.

Ion activation by CID occurs by conversion of ion translational energy into
internal (vibrational) energy upon collision with a neutral inert target gas (e.g., He

or Ar). The acquired vibrational energy is then quickly redistributed throughout

the entire ion (510'12 s) and ergodic fragmentation of a particular bond occurs

once the vibrational energy exceeds the activation barrier of that bond.74 Since
CID is under kinetic control and is generally a “slow heating” process (i.e., the
vibrational energy deposition occurs over many collision events), the most labile
bonds fragment preferentially.74 CID of nonphosphorylated peptides generally

leads to non-selective sequence ion fragmentation, forming b- and y-sequence

ions relating to fragmentation of amide bonds (activation energy barrier of amide

bond cleavage ~ 40 kcal/mol cleavage).75' 76 Phosphorylated peptides,

however, often undergo preferential nonsequence ion fragmentation of the

77, 78

phosphoester bond (activation energy barrier ~12 kcal/mol). Selective

cleavage of labile phosphoester bonds may give rise to neutral losses of 80 Da

(HPO3) and 98 Da (H3PO4 or H20+HPO3) or charged losses of 63 Da (P02)
79 Da (P03) and 97 Da (H2PO4') forming either nonsequence product ions
(e.g., [M+nH-981n'1) or sequence product ions resulting from sequential

fragmentation reactions (e.g., bn-98),24' 26' 79‘89

14

ClD-MS/MS has been used for the identification of phosphopeptides and
localization of phosphorylation sites in both negative and positive ion modes
(Figure 1.2-1 and 1.2-2, respectively). The gas-phase ion chemistry governing
these ClD-MS/MS techniques, as well as its impact on the utility of these

methods, is described in detail below.

1.2.3.1.1 Formation of 79 mlz (P03) and [M+nH-79](n'1)' Ions

CID of negatively charged phospho-serine, -threonine, and -tyrosine

peptide precursor ions commonly result in the cleavage of the phosphoester
bond giving rise to P03' ions (mlz 79). When the precursor ion is multiply de-

101-1)-

protonated, a charge reduced counterpart ion ([M-nH-79 ) may also be

observed. A mechanism for the formation of these ions has been previously

proposed, and is shown in Scheme 1.1 for a doubly deprotonated precursor

. 85-88
IOD.

 

(190
0Q:— o'
o H ClD-MS/MS_ i H + PO'
FJLN N.“ r u N.“ 3
H o o
[M-nH-79]("'1" m/z 79

Scheme 1.1 Previously proposed mechanism for cleavage of the P-O
phosphoester bond of negatively charged phosphorylated peptides forming [M-

nit-791""1 * and Poe'(m/z 79).

15

Observation of the PO3' nonsequence product ion (mlz 79) by CID in

negative ion mode has been used to indicate the presence of phosphorylated

85. 86

peptides (Figure 1.2-1 a). Since m/z 79 may be too low to be observed in

ion trap platforms under typical peptide analysis conditions, the [M+nH-79](n'1)'

product ion may alternatively be used for phosphopeptide detection (Figure 1.2-

1a).88 Additionally, the observation of these nonsequence product ions has been

used to trigger the acquisition of a positive ion mode ClD-MS/MS spectrum of the
corresponding protonated precursor ion for phosphopeptide sequence analysis
(Figure 1.2-1a followed by Figure 1.2-2). This has been achieved by either

switching the polarity during sample analysis or performing a second round of

. . . . . 83, 84, 90
analySIs In posrtive Ion mode.

1.2.3.1.2 Formation of Phosphotyrosine-Specific Immonium Ions at mlz
216.043
In positive ion mode, CID-MS/MS of protonated phosphotyrosine-

containing peptide ions can give rise to nonsequence phosphotyrosine-specific

91, 92

immonium ions at m/z 216.043 (Figure 1.4). Immonium ions provide

composition-specific information and may result from cleavage of the N-terminal

residue or from two backbone cleavages surrounding a particular internal

residue.93 It has been previously shown that the abundance of the

16

phosphotyrosine (pY) immonium ion is dependent upon the sequence of the
peptide,94 and the immonium ion abundance is highest when the
phosphotyrosine residue is located at the first or second residue from the N-

ten'ninus.94 This is in accordance with the observation that N-tenninal residues
generally favor the formation of immonium ions.93 Additionally, since immonium
ions may result via secondary fragmentation of bn sequence ions (bn-)an+CO)
and b2 ion formation is favored, localization of the phosphotyrosine residue at the

second position from the N-terminus may also preferentially yield these ions?3

Furthermore, a fragmentation study on a series of synthetic phosphopeptides
with the general sequence DQQDFFPK where pY was placed in positions 19

demonstrated that the closer the pY residue was to the C-terminal lysine, the less

likely the corresponding immonium ion was to form.94

OH
I
HN=CH-R HN=CH-CH2©—O-fi-OH

O

Figure 1.4 General structure of an immonium ion, where R is the amino acid
side-chain and the phosphotyrosine-specific immonium ion, m/z 216.043.

Precursor ion scanning of the pY immonium ion has been used for

selective detection of phosphotyrosine-containing peptides from within complex

91, 92, 95

mixtures (Figure 1.2-2a). Since this diagnostic ion may have the same

17

nominal mass as other ions (216 Da), this technique has proven most effective

when using high resolution/high mass accuracy instruments such as a qTOF.91'

92' 95 However, considering that this method is reliant upon the formation of this

ion, this technique may not allow identification of the presence of
phosphotyrosine-containing peptides when the residue is located near the C-

terminus.

1.2.3.1.3 Product Ions Formed From 80 or 98 Da Neutral Losses
The analysis of peptides containing phospho-serine, -threonine, and/or -

tyrosine residues by ClD-MS/MS in either positive or negative ion mode may give

rise to product ions corresponding to the neutral losses of 80 Da (HP03) and 98

24, 26, 80-83, 87, 89

Da (commonly assigned as H3PO4). These neutral loss

processes yield nonsequence product ions (i.e., [M-nH—80]n', [M-nH-981n',

[M+nH-80]n+, and [M+nH-80]n+) that diagnostically indicate the presence of

phosphorylated peptide ion (Figure 1.2-1b, 1.2-2b). Furthermore, product ions

formed via sequential nonsequence and sequence type fragmentation reactions
(e.g., bn-80, bn-98, yn-80, and yn-98), may also be observed.96

Competition between the formation of nonsequence ions, which may be

used to determine the presence of phosphopeptides, and the formation of

sequence ions,97 which may be used for peptide identification and localization of

18

the phosphorylation site, critically affects the utility of a particular ClD-MS/MS
approach for phosphoproteome characterization.

When the formation of b- and y- sequence product ions is suppressed by
the dominant formation of a 98 Da neutral loss nonsequence product ion,
alternative methods have been used to obtain the required sequence information.

These include automatically subjecting the initial 98 Da neutral loss product ion

to a further stage of ClD-MS/MS (i.e., M83) (Figure 1.2-2b.i) or “pseudo-M83”

(Figure 1.2-2b.ii) in ion trap mass spectrometers in a data dependent mode of

24, 98-101

operation. CID-MS3 entails isolation and further fragmentation of the

. 3 . . . .
neutral loss speCIes, whereas pseudo-MS Involves Simultaneous activation of

the precursor ion and the resultant 98 Da neutral loss product ion during a single

CID-MS/MS event.101 Therefore, the pseudo-MS3 product ion spectrum

contains a “composite” of the product ions generated from fragmentation of both
the precursor peptide and the initial neutral loss product ion. Alternatively,
observation of these 98 Da neutral loss product ions has been used to trigger
ECD-MS/MS analysis of the original precursor ion (Figure 1.2-2b followed by

Figure 1.2-7), under which conditions the phosphate group remains intact

102, 103

(described further in its respective section below). These data-dependent

strategies have been used for peptide identification and phosphorylation site
determination. The utility of these methods are, however, highly dependent upon

the formation of these neutral loss ions as abundant species. Additionally, the

appearance of the product ion spectrum resulting from CID-MS3 of the 98 Da

19

neutral loss product ion is dependent on the structure of the initial neutral loss ion
and the mechanism(s) by which it is formed.

For these reasons, it is important that the factors that govern the
_ abundance and structures of these neutral loss ions, as well as the mechanisms
by which they are formed, are determined. Previous studies have noted that

these neutral loss product ions are formed at higher abundance at higher

. . . 8 4 .
colhsron energies, and In mass spectrometers that employ more energy

transferred in short activation times (microsecond), like triple quadrupole

instruments.105 The structure of the phosphorylated amino acid also affects the

extent of the formation of these neutral loss product ions .by CID, whereby

phosphotyrosine undergoes the neutral loss of 80 Da more frequently than 98

87. 89
a.

D while phosphoserine and phosphothreonine undergo the neutral loss of

87. 89

98 Da more frequently than the loss of 80 Da. Woods and coworkers have

also shown that the neutral loss of 98 Da from phosphotyrosine-comaining
peptides is highly dependent upon the presence of basic residues, where the

neutral loss of 98 Da was observed only when arginine and lysine residues were

present within the peptide};2 This has been hypothesized as requiring the

presence of strong hydrogen-bonding interactions between the phosphate group

. . . . . 82.106-110
and a protonated arginine or lysrne Side chain.

It is generally agreed that the precursor ion charge state also influences

the abundance of the 98 Da neutral loss ion, where higher charge states result in

20

87,89,111

lower phosphate neutral loss product ion abundance. Another factor

that may influence the abundance of these nonsequence neutral loss ions is the
combined effect of precursor ion charge state and basic residue content (i.e.,
proton mobility). The “mobile proton” model is a central concept to rationalize the

appearance of CID product ion spectra with the features of the protonated

precursor peptide ion.112'114 In particular, this model considers the fact that the

ionizing proton may be directly involved in the fragmentation reaction (charge-
directed), or sequestered to a region of high proton affinity (e.g., side chains of
basic residues) and therefore unavailable for involvement in fragmentation
(charge-remote). Therefore, the proton mobility of a precursor ion, which is
influenced by both the precursor ion charge state and the amino acid
composition, rather than the charge state alone, is expected to dictate the type
and abundance of product ions observed and the mechanisms by which they are
formed. A body of evidence exists in the literature that demonstrates the
important role of proton mobility on the gas-phase fragmentation reactions of

protonated peptide ions containing other post-translational and process-induced

115,116

modifications. Consequently, it is critical to obtain detailed insights into

the role of proton mobility on the multistage gas-phase fragmentation reactions of
protonated phosphopeptides, as this will ultimately dictate the utility of CID
tandem mass spectrometry methods for phosphoproteome analysis. The work

described in Chapter Two aims to elucidate the relationship between proton

21

mobility and the gas-phase fragmentation of phosphorylated peptides under CID
conditions.

Many mechanisms for fragmentation of the phosphate group have been

previously proposed. These include the direct loss of H3PO4 from the

78, 87, 89, 117

phosphorylated residues, the losses of HPO3 and H20 from a

phosphorylated and a nonphosphorylated hydroxyl (or carboxyl)-containing

89, 118

amino acid residue, and the potential for intramolecular gas-phase

phosphate group transfer upon CID (Scheme 1.2).89 It is clear from Scheme 1.2

that these specific fragmentation pathways involving the phosphate group will
dictate the utility of CID tandem mass spectrometry methods for accurate and
unambiguous phosphorylation site assignment, since differentially
phosphorylated isomers may give rise to the same product ion structures. In

particular, the presence of sequence ions that have undergone a phosphate
neutral loss (e.g., bn-80 or bn-98) complicate phosphate site interpretation since
they are isostructural to nonphosphorylated ions; while sequence ions resulting

from the neutral loss of H20 from a nonphosphorylated residue may be confused
with sequence ions resulting from the losses of H3PO4 from a phosphorylated

residue. Additionally, sequence ions resulting from neutral losses of HPO3 are

indistinguishable from nonphosphorylated residues, and as a result, the reliability
of phosphate-intact sequence ions may be compromised. Consequently, the

work described in Chapters Two and Three aims to reveal the potential for these

22

fragmentation pathways, elucidate the specific mechanisms involved, determine
the resulting product ion structures, and establish the factors that govern these
processes. Importantly, discussion of the implications of these processes on CID

strategies for phosphoproteome analyses is also included in these chapters.

13
999009990
I “20(3).

-H3P0.(pT) C'Dlv's" -HP03<pT>

I i I
OGGODOGOO Pfisfisfle GGOODOGOB
t I I

41200): CIDIMS" 'H3P04IPS)
-HP03(pS)

eeeoooege

Scheme 1.2 Fragmentation pathways involving the phosphate group upon CID-

MS", where dS and dT represent dehydrated serine and threonine residues,
respectively.

 

1.2.3.1.4 Chemical Derivatization and CID-MSIMS

To circumvent the potential limitations associated with the Iability of the
phosphate group upon CID-MS/MS, other phosphopeptide characterization
strategies involving chemical derivatization or conversion of the phosphopeptides
prior to CID-MS/MS have been developed. One of the simplest derivatization
techniques involves B-elimination of phosphate groups to form the dehydrated
species (dehydroalanine for phosphoserine and dehydroaminobutyric acid for

phosphothreonine) under alkaline conditions followed by Michael addition with a

23

nucleophile.37 This method has been adapted such that dithiol nucleophiles may

act as crosslinkers between modified peptides and affinity materials such as

biotin or solid phase resins allowing for both phosphopeptide enrichment and

altered CID gas-phase fragmentation behavior.38.41 This approach has also

been used to transform phosphorylated serine to aminoethyl cysteine, a lysine

mimic.42 By this method, the formerly phosphorylated sites can be targeted for

proteolysis by trypsin then subsequently identified by analysis of their CID-
MS/MS spectra. Another method involves conversion of the phosphate group to

2-dimethylaminoethanesulfoxide followed by CID to give rise to the characteristic

neutral loss of sulfenic acid (mlz 122.06).119 Alternatively, others have

converted phosphate groups to S-pyridylethyl groups, which fragment to produce

characteristic ions at m/z 106 upon CID.120 The abundance of each of these

characteristic product ions is, however, dependent upon the proton mobility of the
peptide precursor ion and as a result, they may not be universally detected.
Furthermore, due to their low m/z, these methods may not be applicable for
detection in ion traps under typical CID conditions. Drawbacks for all of these
techniques also exist since (i) O-linked sugars and nonphosphorylated serine
and threonine residues may undergo elimination, (ii) phosphotyrosine does not
undergo elimination, and (iii) precautions that may be taken to reduce elimination

of nonphosphorylated residues may cause limited conversion efficiency of the

phosphorylated residues.40

24

Since the gas-phase interactions between phosphate groups and side

107,108,121

chains of basic residues has been demonstrated, other derivatization

approaches that block these interactions may also be used to decrease the
Iability of the phosphate group upon CID. In fact, malondialdehyde has been
used to decrease the basicity of arginine in phosphorylated peptides and CID-
MS/MS of these derivatized phosphopeptides has been shown to result in an

increase of phosphate-containing b- and y-fragment ions and a decrease in the

98 Da neutral loss species.122 Gronert et al. have shown that protonated

phosphopeptides may react in the gas-phase with alkoxy-boron species in an

78,123

ion-molecule reaction. ClD-MS/MS of the resulting product ions that

correspond to alkoxy moiety loss gives rise to the boron derivatized peptide ions

(e.g., [M+B-H]+). Further CID activation of the boron derivatized ions results in

phosphopeptide backbone fragmentation. Production of phosphate-containing

sequence ions from these boron derivatized ions is greater in abundance than for

their underivatized counterparts-’8’ 123 decreasing the ambiguity of

phosphorylation site assignment by CID.

1.2.3.2 MALDI Post-Source Decay (PSD)
Post-source decay (PSD) has also been used for phosphopeptide
characterization (Figure 1.2-3). PSD involves metastable decomposition of a

precursor ion after leaving the MALDI source in a field free region of a TOF

instrument.124 In essence, this process relies on ions that are stable enough to

25

exit the source, but are not stable enough to survive the flight to the detector, and
decompose. Ion activation for PSD is thought to originate from different

mechanisms, where those of major influence occur in the source (photon-

. . . . . 63
molecule Interactions, matnx-molecule Interactions, temperature effects, etc.).

The resulting product ions are then resolved by their difference in kinetic energy

(as a result of their difference in mass) in a reflector TOF.125

Similar to CID, PSD of peptides gives rise to sequence information as b-

and y-product ions.126 PSD product ion spectra of phosphorylated peptides are

also similar to that of CID, where the phosphotyrosine-specific immonium ion has

been observed and may be used to indicate the presence of phosphotyrosine-

containing peptides (Figure 1.2-3a)127 and where nonsequence product ions
resulting from the neutral losses of 98 Da (H3PO4 or HP03+H20) and 80 Da

(HPO3) may be used as diagnostic indicators of the presence of

phosphopeptides (Figure 1.2-3b).127'129

The effects of the identity of the phosphorylated amino acid on the
abundance of the phosphate neutral loss product ions formed by PSD have been
previously reported. Similar to CID, the loss of 98 Da is more prevalent for PSD

of phosphoserine- and phosphothreonine-containing peptides compared to

phosphotyrosine-containing peptides.127 It has also been determined that the

loss of 80 Da occurs more readily for phosphorylated tyrosine-containing

peptides than for those containing phosphoserine or phosphothreonine.127

26

Furthermore, the relative abundance of phosphate-intact product sequence ions
vs. their “dephosphorylated” counterparts (e.g., bn-80 and bn-98) is high for
phosphotyrosine-containing peptides and varies for phosphoserine- and

phosphothreonine-containing peptides.127'129

Due to the similarities in CID and PSD product ion spectra, it is likely that
the mechanisms by which the phosphate neutral loss product ions are formed
are similar. Accordingly, it has been previously hypothesized that the neutral

loss of 98 Da upon PSD of phosphorylated serine- and threonine-containing

peptides may result from the direct loss of H3PO4 (98 Da) or from the

consecutive losses of HPO3 from a phosphorylated residue and H20 from a

nonphosphorylated residue (combined mass of 98 Da) (Scheme 1.2).127' 130

Additionally, intramolecular phosphate group transfer from phosphorylated

tyrosine to a formerly unmodified residue, followed by the loss of H3PO4, has
also been proposed for PSD (Scheme 1.2).128 In that study, it was

demonstrated that the neutral loss of H3PO4 depended on the presence of

aspartic acid and arginine residues. By this observation it was proposed that the

HPO3 group was transferred from tyrosine to aspartic acid followed by cleavage

to yield a succinamide-containing product ion.128 Consequently, the same

possible ambiguities regarding phosphorylation site determination by CID can

occur with PSD.

27

1.2.3.3 Photodissociation

Ions may also be excited and subsequently dissociated by absorption of
photons. Photodissociation (PD) techniques may be selective in comparison to
the aforementioned techniques because only ions that absorb at the wavelength
of the light used are activated. These techniques are often used with ion
trapping mass spectrometers, because they allow for ions to be confined to the

area irradiated by the light.

1.2.3.3.1 Infrared Multiple Photon Dissociation (IRMPD)

64, 131

Ion activation may be achieved using infrared lasers. Due to its

relatively low energy (0.1 eV/photon), the absorption of multiple IR photons (tens

to hundreds) are required for ion dissociation. Ions are typically irradiated for

tens to hundreds of milliseconds for a continuous-wave 002 (10.6 um) laser.

Like CID, IRMPD is a slow heating method and allows for intramolecular energy
redistribution over all of the vibrational degrees of freedom prior to the next
photon absorption event. As a result, ergodic dissociation of low-energy
pathways predominates and the resulting spectra are often comparable with
those of CID.

Since the P-O stretch (9.6-11 pm) is in resonance with 10.6 um light, the

phosphate groups of phosphorylated peptides provide strong chromophores for

77, 132

efficient dissociation by IRMPD; hence, IRMPD has been used for

phosphopeptide characterization (Figure 1.2-4). Similar sequence coverage,

28

types of product ions formed (b- and y-sequence ions; nonsequence and
sequence product ions resulting from phosphate neutral loss), product ion
abundance (including the predominant formation of the nonsequence phosphate
neutral loss ions) are formed by IRMPD as those formed by CID. Accordingly,
selective detection of phosphorylated peptides from mixtures has been achieved

by the observation of 80 and 98 Da neutral loss species upon IRMPD (Figure

1.2-4b).133‘136 However, due to the various mechanisms that may be involved

in the cleavage of the phosphate group (Scheme 1.2), the same complications

for phosphorylation site determination by CID and PSD, exist for IRMPD.

1.2.3.3.2 Ultraviolet Photodissociation (UVPD)
Photodissociation of peptides in the UV range has targeted common
chromophores such as amide bonds (193 and 157 nm) as well as residue-

specific chromophores such as aromatic amino acids (220, 266, and 280

reviewed in 65

nm)

Phosphopeptide analysis using ultraviolet photodissociation

1

(UVPD) has been performed using 220,137 157, 38 266 (after derivatization with

a chromophore),139 and 193 nm light (Figure 1.2-5).140

One study by Dugourd and coworkers demonstrated that UVPD of
peptides containing unmodified tyrosine (chromophore) and phosphorylated
serine residues at 220 nm (5.64 eV/photon) result in the predominant production

of radical product ions formed by homolytic cleavage and subsequent loss of

twosine side chains.137 The formation of radical ions at this wavelength was to

29

some extent expected since nonphosphorylated tyrosine-containing peptides

. . . . 141 . . .
also give nse to Similar cleavages. When this Side chain cleavage was

suppressed, the 98 Da neutral loss product ion was also formed

predominantly.137

Kim and Reilly have recently shown that UVPD of singly protonated
phosphoserine- and phosphothreonine-containing peptide ions at 157 nm (7.90
eV/photon) results in dominant formation of the neutral loss of 98 Da from the

precursor ion, while phosphotyrosine-containing peptides largely retain the

phosphate group.138 Interestingly, time-resolved detection of the UVPD product

ions demonstrated that the nonsequence product ion resulting from the neutral
loss of 98 Da was formed within 300 ns (the measurable limit) post

photoexcitation, while product sequence ions (a-, b-, x-, y-, and z-ions) retained
the phosphate group up to 1 us post photoexcitation.138 Additionally, a complete

series of phosphate-intact sequence ions was formed for all of the model

phosphopeptides examined under these conditions.138 Collectively, these

observations demonstrate the potential of this strategy for phosphoproteome
analysis since the observed 98 Da neutral loss nonsequence product ion may be
used as a diagnostic indicator of the presence of phosphorylated peptides while
the complete series of sequence ions retaining the intact phosphate group may
be used to unambiguously assign the site of phosphorylation.

Diedrich and Julian have developed an alternative UVPD approach

involving conversion of the phosphate group to a UV-absorbing chromophore.139

30

This method involves B-elimination of the phosphate group followed by

introduction of sulfhydryl-containing napthyl chromophores by Michael addition

(Scheme 1.3).139 Upon UVPD at 266 nm of singly deprotonated or multiply

protonated ions, the chromophore allows for enhanced absorbance of the
formerly phosphorylated, labeled peptides. Furthermore, the carbon-sulfur bond
connecting the label to the peptide is susceptible to direct photodissociation

resulting in homolytic cleavage and subsequent loss of a napthylsulfide radical

(Scheme 1.3).139 This resulting ion could be used as a diagnostic indicator of

the presence of phosphopeptides. Sequence ions resulting from consecutive

cleavage of the napthylsulfide radical and the peptide backbone adjacent to the

former position of the label were also observed.139 Interestingly these ions were

the only sequence ions observed in the product ion Spectrum. The production of
these site-specific sequence ions was proposed to occur by the mechanism

shown in Scheme 1.3, where the radical in the B-position (post napthylsulfide

cleavage) induces backbone fragmentation adjacent to the labeled residue.139
Such Site-specific sequence ions could allow for unambiguous assignment of the
phosphorylation Site within a grouping of potential phosphorylation sites.

However, this method would incur the drawbacks of other previously described 8-

elimination strategies.

31

Scheme 1.3 Analysis of napthyl chromophore-derivatized phosphorylated serine
and threonine peptides by UVPD at 266 nm. B-elimination of phosphoserine and
phosphothreonine peptides followed by Michael addition to form the napthyl-
derivatized labeled peptides. UVPD of the derivatized peptides results in
selective cleavage of the chromophore and radical-induced cleavage allows for
the selective formation of sequence ions adjacent to the formerly phosphorylated
residue.

32

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33

Site-specific dissociation of phosphopeptides (non-derivatized) at the

phosphorylated residues has also been recently demonstrated by Shin et al.
using UVPD at 193 nm.140 In that study, phosphorylated peptide ions containing
a basic residue (arginine, lysine, and histidine) at the N-terminus of the peptide

resulted in the selective formation of intense an-97 ions C-terrninal to the location

of the phosphorylated residues.140 This was demonstrated for singly protonated

ions containing one, two, or three phosphorylations. Hydrogen—deuterium

exchange experiments were completed and demonstrated that the an-97 ions
formed from H3PO4 loss from the an+1 radical cation, which, in turn, is formed by

Cot-Ccarbonyl cleavage of the peptide backbone (Scheme 1.4).140 Importantly,

since these ions form selectively at the phosphorylation Sites, they could be used
to localize the phosphorylation sites of peptides with multiple serine, threonine,
and tyrosine residues. Additional product ions were also formed upon UVPD at
193 nm of these phosphorylated peptides, including nonsequence product ions
resulting from the neutral loss of 98, 80, and 18 Da, and combinations thereof, as
well as other sequence ions. Despite the potential for this technique for
phosphorylation Site determination, this strategy may be limited by the choice of

proteolytic enzyme available that would produce N-terminal basic residues.

34

 

NH O:é—OH NH Ozé—OH
’ O UVPD 193 nm / ‘~..O
(CI-bk“ O Homolytic cleavage (CHZI3N O J‘\
H2N \(IkN .. F H2N j/IKN '
0 R1 H o 0 R1 H
Protonated phosphopeptide an+1

Scheme 1.4 Proposed site-specific an+1-97 ion formation of singly protonated,
phosphorylated peptides that contain N-temiinal basic residues upon UVPD at

193 nm, where R1 is the side chain for any amino acid.

1.2.3.3.3 Femtosecond Laser-Induced Ionization/Dissociation (stID)
Another photodissociation technique which has been applied to the

analysis of phosphopeptides is femtosecond laser-induCed ionization/dissociation

(stID; Figure 1.2-6).66 In stlD, the photons of a near-IR laser (A = 798i30 nm

(~1.55 eV/photon)) are concentrated into ultra-short pulses (~35 fS duration) to

enable very high photon intensities (~1014 W/cmz) for ion activation. With such a

high-power femtosecond laser, novel activation processes may be accessible by
stID. Additionally, since fSLID is capable of overcoming the threshold energy for

ion oxidation (electron removal) on a timescale similar to or faster than that of

bond vibrations (commonly 10-100 fs),142 it is just'rfiable to hypothesize that the
technique can achieve non-ergodic dissociation enabling nonselective
fragmentation and retention of labile functionalities, such as phosphate groups.

In a recent study, Kalcic et al. have shown that stlD-MS/MS of singly and

multiply protonated phosphorylated peptides (and nonmodified tryptic peptides)

35

results in the formation of ample product sequence ions, including those of the a-

, b-, c-, x-, y-, and z-type.66 It was determined that many of these sequence ions

were formed as a result of radical-driven dissociation of the photo-oxidized

precursor.66 Importantly, this study showed that stlD-MS/MS analysis of

phosphorylated peptides produces sufficient sequence information to enable
unambiguous phosphorylation site localization.

Although the mechanisms associated with both ionization and dissociation
have yet to be elucidated, plausible ionization mechanisms for stID include
multi-photon ionization, or field ionization where the electric field produced by the
photon packet warps the ion potential energy surface to an extent that the ions

may incur the loss of an electron.

1.2.3.4 Electron-Driven Dissociation

Tandem mass spectrometry methods that involve dissociation by electron-
ion reactions (ECD, EDD) or ion-ion reactions (ETD) as the “activation”
component of the MS/MS experiment have also been used for phosphoproteomic
analyses. These techniques largely maintain the phosphate group upon
dissociation of the precursor ion and therefore overcome the potential problems
associated with CID, PSD, and IRMPD. However, because most of these
techniques involve charge state reduction by the addition or loss of an electron,

multiply charged precursor ions are typically required.

36

1.2.3.4.1 Electron Capture Dissociation (ECD)
Electron capture dissociation (ECD) involves exothermic capture of a low

energy electron (<0.2 eV) by a positively charged species followed by

dissociation of the resultant odd electron ion.143 Since the electron capture

process requires low energy electrons (<10 eV) and long interaction times, ECD
has traditionally been confined to instruments that employ static electromagnetic
fields that avoid energizing or heating electrons, such as FT-ICRs. In particular,
ECD was initially precluded for use in quadrupole ion traps since they utilize
radio frequency (rf) electric fields with amplitudes of hundreds of volts that can
either accelerate electrons greater than 10 eV or repel them from the region
where the trapped ions are located. However, recently, the addition of magnetic

fields to ion traps have allowed for ECD in such electrodynamic trapping

instruments.144’146 Additionally, the use of ECD in a digital ion trap mass

spectrometer has also been reported, where the application of a magnetic field
was not necessary since this instrument employs a relatively constant electric

field by rapidly switching between two dc voltage levels rather than using an rf

tieiti.147

Protonated precursor ions (i.e., M+nH]n+) where n 2 2 are required for

ECD-MS/MS because charge reduction of a singly protonated precursor ion
would result in an undetectable neutral species. Furthermore, multiply

protonated precursor ions formed by ESI are favored for ECD since the electron

capture cross section is proportional to the square of the ion charge.148

37

Additionally, there have been reports of limited dissociation of doubly protonated

precursor ions despite the expected high efficiency of electron capture.149’ 150

This has been rationalized by the potential for noncovalent interactions (e.g., salt

bridges) that prevent the separation of the product ion pairs.149 To overcome

such noncovalent interactions and increase the extent of dissociation, McLafferty
and coworkers have introduced activated ion-ECD (Al-ECD) which involves ion
heating before, during, or after the ECD process.151 Additionally, CID
supplemental activation of the newly formed charge-reduced product ion may be

performed simultaneous to the ECD experiment, thereby allowing for increased

fragmentation efficiency and a reduction in the extent of multiple electron capture
events that may otherwise be observed while performing ECD.152

The mechanism for bond dissociation by ECD is dictated by radical
reaction chemistry. For peptides, this largely results in the formation of c- and 2-

type product ions. A mechanism for this N-Ca bond cleavage has been proposed

and is shown in Scheme 1.5,153 where a H. from the initial electron capture site

is transferred to a backbone carbonyl group followed by cleavage to form c- and
z-product ions. Importantly, ECD is a nonergodic process, occurring in a
timescale faster than that of internal energy redistribution. Therefore,
nonselective fragmentation is achieved rather than kinetically controlled
fragmentation (like in CID, PSD, and IRMPD). For this reason, ECD of multiply

protonated phosphopeptide ions results in retention of the phosphate group upon

38

154, 155

fragmentation. As a result, ECD-MS/MS has been widely used for

phosphopeptide identification and unambiguous phosphorylation site assignment

(Figure 1.2-7).156’ 157
+ o
H2N H250
H e- HfW
, o __.. . (o
H3N\'/II\N HaNj/IKN
H
R1 Fl C) R1 C)
H2N H2N
+ OH + 0H
m
H3N\(§NH + H . ‘ H3N ,NV
R1 0 1 H o

c 2'
Scheme 1.5 Previously proposed fragmentation mechanism for the production
of c- and z-type product sequence ions for the reaction between a low-energy
electron with a multiply protonated peptide ion.
Since the phosphate group is maintained upon performing ECD and is lost
upon performing CID, Cooper and coworkers have proposed and demonstrated
the combined utility of these processes through automatic triggering of an ECD-

MS/MS spectrum of a protonated precursor ion following the observation of an

initial neutral loss of 98 Da upon performing CID (Figure 1.2-2b followed by

39

102,157

Figure 1.2-7). The success of this method is highly dependent on the

abundant formation of the phosphate neutral loss product ion formed by CID to

initiate the triggering event.157 Consequently, the factors that govern the

abundance of the nonsequence 98 Da neutral loss product ion will greatly affect
the utility of this method. Therefore, the work described in Chapter Two
elucidates these factors.

ECD of peptide polycations may also be achieved using high energy

electrons (3-13 eV) in a method termed “hot” ECD (HECD).158 Similar to

conventional ECD, capture of a hot electron by a peptide polycation induces N-

CO. bond cleavage, producing c- and z-type ions (similar to that shown in Scheme

1.5). However, due to the excess energy of the captured electron, the resultant
product ions may undergo secondary fragmentation. This method has been
successfully used to distinguish leucine (Leu) and isoleucine (Ile), due to the

abundant secondary fragmentation of their side chains from He or Leu-terminated

158.159

sequence product ions. An initial study demonstrated that HECD (11 eV

electrons) of a phosphopeptide resulted in phosphate-intact c- and z-type product
ions. In fact, the only product ions resulting from secondary fragmentation were

product ions resulting from the neutral loss of 98 Da from amide bond cleavage

ions (i.e., b and y-type ions).158 It was hypothesized that those product ions

resulting from secondary neutral loss 98 Da formed via inelastic ion-electron

collisions, Similar to collisions with neutral molecules for CID.

40

1.2.3.4.2 Electron Transfer Dissociation (ETD)

Electron transfer dissociation (ETD) is similar to ECD in that it also

induces nonergodic cleavage of the N-Ce bond on a peptide’s backbone to

produce c- and z-type product ions, while maintaining phosphate groups and

other potentially labile modifications.160 However, rather than the direct capture

of an electron, ETD involves transfer of an electron from a radical anion reagent
to a multiply protonated precursor ion. The use of reagent anions as electron
donors makes ETD amenable for use in quadrupole ion trap mass
spectrometers. Various anions with relatively low electron affinities (<60-70

kcal/mol), such as anthracene, fluoranthene, and azobenzene, have been

successfully used as electron donors for ETD.16O'163 After electron transfer to a

protonated peptide, the mechanism for dissociation of the N-Ce bond is thought

to be the same as that for electron capture (Scheme 1.5).160 For these reasons,

ETD has increasingly been used for phosphoproteome analysis (Figure 1.2-8),

enabling phosphoprotein identification and unambiguous phosphorylation site

. . 161,164-166

localization.
The performance factors associated with ETD have been previously

discussed by Good et al.162 In that study, the most Significant factor governing

ETD peptide fragmentation efficiency was determined to be the charge density or
the charge/residue ratio of a precursor peptide ion, where low precursor ion

charge density gave rise to low fragmentation efficiency. Accordingly, less than

41

1% of the unmodified peptides identified using ETD were derived from doubly

. 1 .
protonated precursor ions. 62 In another large-scale analySIs, Swaney et al.

similarly demonstrated that ETD is most effective for precursor ions with low m/z

(high charge density).167 This apparent trend of decreased ETD fragmentation

efficiency for low charge density precursor ions has been attributed to the

formation of the non-dissociated (intact) charge-reduced radical cation (e.g.,

[M+2H]2+ -> [M+2H]+.).162’ 168 In order to increase the ETD efficiency of

doubly protonated precursor ions and other peptide ions with low charge density,

application of supplemental low-energy CID of these radical cations has been

performed.168 By using this hybrid technique termed ETcaD, Molina et al.

demonstrated that 60% of identified tryptic peptides were from doubly protonated

precursor ions.169 Furthermore, it has been shown that ETcaD provides an

improvement for nonmodified peptide identification (89%) over CID (77%) or ETD

(63%) alone.168 In that study, ETcaD of doubly protonated phosphorylated

peptide ions was also completed. Not surprisingly, the product ion formed from

the neutral loss of 98 Da was the most abundant ion present and neutral losses

of 98 Da from c- and z-product ions were also observed.168 As a result, ETcaD
may incur the same limitations for phosphorylation site assignment as for
conventional CID.

Since both the charge and length of the peptide ion affects ETD efficiency,

Molina et al. have studied how the chosen proteolytic enzyme affects ETD for

42

phosphoproteome analyses.164 In particular, they compared trypsin (cleaves C-

terrninal to arginine and lysine), Lys-C (cleaves C-terminal to lysine), and Glu-C
(cleaves C-tenninal to glutamic acid and to a lesser extent aspartic acid). Due to
the cleavage specificity, it is expected that Lys-C would produce peptides with
more basic residues (and therefore higher charge) than their trypsin

counterparts, increasing the number of phosphopeptides identified using ETD.

However, Molina et al. showed that Lys-C and trypsin performed similarly.164

This was attributed to inefficient trypsin digestion near phosphorylated residues,

resulting in phosphopeptides with similar peptide lengths and protonation states

as Lys-C peptides.164 Molina et al. also studied the effects of Glu-C for ETD

analysis of phosphopeptides, since the average peptide length produced by Glu-

C digestion is thought to be between that of LyS-C and trypsin and therefore

produce peptides suitable for ETD.164 However, due to poor cleavage specificity

and efficiency, an order of magnitude less phosphopeptides were identified using

Glu-C as compared with LyS-C and trypsin.164

Heck and coworkers have shown that Lys-N may also be an appropriate

protease for use in conjunction with ETD and ETcaD analysis of phosphorylated

170, 171

peptides. As the name suggests, Lys-N cleaves N-terminal to lysine

residues and therefore produces peptides that are similar to Lys-C, but differs in
the position of the lysine residue. Taouatas, et al. have showed that ETcaD of

phosphopeptides produced by Lys-N digestion readily produce (N-terminal) c-ion

43

product ion series, corresponding to the retention of the ionizing protons on the

N-tenninal basic residue,170'173 enabling facile phosphopeptide

170, 171

identification. Additionally, a recent study by Gauci, et al. showed that

Lys-N and trypsin may provide complementary identification of phosphopeptides
(albeit using CID), since Lys—N produces lysine-containing phosphopeptides,

which were poorly represented in the phosphopeptides identified by the

analogous trypsin digest.174

One limitation of ETD (as well as ECD) is that it is not amenable to the
analysis of peptide anions. Moreover, phosphopeptides tend to ionize better in

negative ion mode than in positive ion mode, due to their relatively low isoelectric

points.175 Considering these seemingly opposing points, Gunawardena et al.

have demonstrated charge inversion of singly deprotonated phosphopeptide

anions with multiply protonated amino-terminated dendrimerS, to form

polycationic precursors in the gas-phase for ETD analysis.176 This strategy was

particularly beneficial because the phosphopeptides analyzed were either not

present or present in low abundance in the corresponding positive ion mode MS

1 76
spectra.

44

1.2.3.4.3 Electron Detachment Dissociation (EDD)

Electron detachment dissociation (EDD), another technique for electron-
induced ion activation/dissociation, is particularly notable in that it allows for
fragmentation of polyanions directly. EDD involves reaction of a polyanion with

an electron (>10 eV), inducing electron detachment and resulting in a charge-

reduced radical anion.177 Fragmentation of the radical anion is governed by

radical reaction chemistry and produces a- and x-type product ions resulting from

150, 177

cleavage of the Ca-Ccarbonyl bond. To date, and Similar to conventional

ECD, the application of EDD has been confined to FT-ICR instruments, where
magnetic rather than electrodynamic fields are used. Like the other electron-
driven methods, EDD is a nonergodic process, and phosphorylated peptides

largely retain their phosphate group upon dissociation, allowing for sequence

determination and accurate phosphorylation site assignment (Figure 1.2-9).150'

178 Kweon and Hakansson have demonstrated that despite its lower

fragmentation efficiency, EDD may give rise to complementary sequence

information to ECD for singly phosphorylated peptides.150

1.2.3.5 Metastable Atom-Activated Dissociation (MAD)
_ Metastable atoms produced by either fast atom bombardment or a glow
discharge source have also been used to impart energy to precursor peptide ions

for fragmentation in a technique termed metastable atom-activated dissociation

70, 179-181

(MAD) or metastable-induced dissociation of ions (MIDI). Since the

45

beam of metastable atoms is neutral, MAD may be completed in quadrupole ion
traps without causing problems relating to charge capacity. Misharin et al.
showed that fragmentation of polycationic peptide ions with metastable helium or
argon atoms in an ion trap gives rise to c- and z-sequence ions, similar to ECD

and ETD, while fragmentation of polyanionic peptide ions by similar methods

gives rise to a- and x-sequence ions, similar to EDD.7O As a result, two

mechanisms for MAD were proposed: for cations, electron transfer from the
metastable atoms; and for anions, electron detachment and de-excitation of the

metastable atom (i.e., Penning ionization; Scheme 1.6), each giving rise to

radical-induced non-ergodic peptide backbone fragmentation.70 Berkout and

Doroshenko have demonstrated that singly protonated peptides also undergo

fragmentation by interaction with metastable atoms, substantiating the

contribution of a Penning ionization mechanism.180 Similarly, Cook et al. have

recently demonstrated extensive backbone fragmentation may be achieved using

MAD of both singly and doubly protonated peptides.181 Notably, product ions

including a-, b-, c-, x-, y-, z-types, consecutive side-chain and backbone

cleavage ions, and product ions resulting from through-ring cleavage of proline

were observed, despite its relatively low fragmentation efficiency.181 Importantly,

fragmentation of phosphorylated peptides by MAD results in the formation of
sequence ions that maintain the phosphate groups at their native sites,

demonstrating that MAD could be employed for phosphoproteome analysis

(Figure 1.2-10).18°' ‘81

46

[M+nH]’.’* + m' -> [M+nH]("*1)"' + m +e'

Scheme 1.6 Penning ionization of a protonated peptide (M) ion, where n 2 1, by
interaction with a metastable noble gas atom (m*).
1.2.3.6 Comparison of Tandem Mass Spectrometry Techniques for
Phosphoproteome Analysis

Each of the tandem mass spectrometry methods described above differs
in their effectiveness for the analysis of phosphopeptides. In particular, they
differ by the instrument platform which may be used, the precursor ions for which
they are amenable, and the types of product ions that are formed (sequence vs.
nonsequence, phosphate neutral loss, phosphate group transfer, etc.).
Consequently, not one technique has emerged as a universal method for
phosphopeptide characterization. In fact, many studies comparing these tandem
mass spectrometry methods have been completed and have shown that many of
these techniques produce complementary information.

For example, studies of nonphosphorylated peptides have reported that
CID and ECD provide complementary sequence information, and that the

application of both provides a higher confidence in sequence assignment than

154, 155

can be attained with either technique alone. Moreover, a recent large-

scale comparison of ECD and CID of tryptic phosphorylated peptides by Sweet et

al. Showed that although ECD gives rise to more sequence coverage, it is
complementary to the information made available by CID.157 However, for the
purpose of unique phosphopeptide identification, it was also found that CID

47

(34%) outperformed ECD (13%).157 This was attributed to predominant

production of doubly protonated precursor ions from the analysis of tryptic
peptides, which are poor targets for electron dissociation techniques but are

generally considered to be optimal for CID. The ECD Spectra, however, gave

rise to more confident phosphorylation site localization than the CID spectra.157

The results of a small scale study on synthetic “tryptic” phosphopeptides

comparing CID and ECD using a hybrid LTQ-FT-ICR instrument also agreed with

the study by Sweet, et al.182 Accordingly, combination techniques that use the

strengths of both ECD and CID have proven successful for phosphoproteomic

. . 25. 102. 156, 162
applications.

Other efforts toward comprehensive analyses have employed the
combination of IRMPD and ECD since, as with the CID/ECD pairing, they also

give rise to complementary structural information and hence allow for higher

confidence in peptide identification.183 Additionally, similar to the Al-ECD,

CID+ECD, and ETcaD methods, IRMPD of the non-dissociated charge-reduced

radical cation species formed by ECD can also be performed in order to improve

sequence coverage.183 This technique has further been applied to a tryptic

phosphorylated peptide from protein kinase A.149 Interestingly, no product ions

resulting from the neutral loss of 98 Da were observed and, as a result, the

PhOSphorylation site was easily identified.149

48

The complementary nature of CID and ETD has also been demonstrated
in a large-scale study on unmodified peptides by Good et al., where there was a
12% overlap in peptide identification between CID and ETD spectra. Swaney et

al. have Shown that more peptides are identified using CID, but with lower

sequence coverage as compared with ETD.167 Additionally, a large-scale

comparison of ETD and CID for phosphoproteomic profiling of human embryonic

kidney 293T cells completed by Molina et al. resulted in the identification of 60%

of the 312 phosphopeptides by ETD and only 27% by Cit).164 It should also be

noted that although the product ions corresponding to the non-dissociated
charge-reduced cations were typically much more intense than the c— and z-ions
in the resultant ETD spectra, ETD was still better for phosphopeptide
identification than CID. Interestingly, this same study resulted in the identification
of more nonphosphorylated peptides by CID than ETD. Importantly, due to the
low amount of overlap between the two methods (13%), the combination of ETD
and CID data resulted in a greater number of phosphopeptides identified than

could be determined from the individual techniques and 80% of the known

phosphorylation sites were observed from the sample.164

Other complementary sequence information that may be achieved from
CID, ECD/ETD, and MAD relates to peptide backbone cleavage adjacent to
proline residues. For CID of protonated peptide ions, amide bond cleavage N-

terminal to Pro may be a preferred cleavage site, while cleavage C-terminal to

184. 185

Pro is often not observed. For ECD/ETD, cleavage to produce the

49

corresponding c- and z-ions on the N-terminal Side of Pro requires scission of

two bonds and its observation has, therefore, remained rare.186 This double

cleavage has, however, been observed in other strategies such as MAD.181

Spectral information available from newly emerging techniques for
phosphopeptide analysis such as UVPD, stlD, EDD, and MAD may also be
complementary to that obtained by the more established techniques,
ClD/PSD/IRMPD and ECD/ETD. This is particularly true when considering the
analysis of precursor ions with low protonation states (e.g., 1 or 2). In particular,
ClD/PSD/IRMPD of precursor ions with low charge states exhibit the
predominant neutral loss of the phosphate group, limiting the amount of
sequence information that may be observed; and limited dissociation efficiency of
precursor ions with low charge state is achieved using ECD/ETD. UVPD, stlD,
and MAD, on the other hand, are capable of ionizing low charge state peptides.
Additionally, UVPD, MAD, and EDD enable the analysis of anionic
phosphopeptides, which are not accessible by ETD or ECD due to a low reaction
cross-section, resulting in sequence information and/or phosphorylation-site

specific information.

1.3 Specific Aims

A hypothetical ideal technique for phosphoproteome analysis could be
described as: (i) globally applicable with regard to precursor ion composition,
sequence or charge state, (ii) enables facile differentiation between

phosphorylated and nonphosphorylated peptides, and (iii) provides unambiguous

50

sequence information for peptide identification and phosphorylation site
assignment. Unfortunately, no existing technique has been demonstrated to
fulfill all of these criteria Simultaneously. As a result, efforts to provide an
improved understanding of the gas-phase ion chemistry involved in these
existing methods, would enhance the utility of these techniques. For this reason,
the major goals of the research described in this dissertation were: (i) to provide
insights into the gas-phase ion chemistry of phosphorylated peptides, (ii) to
consider the effect of these chemistries on the applicability of tandem mass
spectrometry methods for phosphopeptide identification and characterization,
and (iii) to apply those methods towards the phosphoproteome analysis of
biologically relevant systems. These goals are discussed in the remaining

chapters with regard to these specific aims:

1. Obtain insights into the role of proton mobility on the multistage gas-phase
fragmentation reactions of protonated phosphoserine- and
phosphothreonine-containing peptides under CID conditions.

2. Determine the mechanisms and product ion structures associated with the
formation of nonsequence neutral loss product ions resulting from phosphate

bond cleavage and to examine the influence of these structures on the

appearance of the Spectra obtained by CID-MS3 of the neutral loss product

ions formed by CID-MS/MS.

3. Evaluate the potential competing fragmentation pathways involving the

phosphate group upon ClD—MS/MS, including the direct loss of H3PO4;

51

II

consecutive losses of HPO3 and H20; and intramolecular phosphate group

transfer.

Determine the effect of these potential competing fragmentation pathways on
phosphoproteome analysis by tandem mass spectrometry strategies.
Determine the phosphorylation status of proteins involved in the NF-KB
pathway, specifically p65 and its interacting proteins, in a differentially treated

leukemia cell line.

52

CHAPTER TWO

Mechanistic Insights into the Multistage Gas-Phase Fragmentation

Behavior of Phosphoserine- and Phosphothreonine-Containing Peptides

2.1 Introduction
The most common tandem mass spectrometry method used for

phosphopeptide analysis is collision-induced dissociation (CID) tandem mass

15, 26, 80

Spectrometry (MS/MS). Under these conditions, phosphoserine- and

phosphothreonine-containing peptides often undergo the facile neutral loss of

Phosphoric acid (H3PO4, 98 Da) from their protonated precursor ions.81’ 83’ 127

The formation of these “nonsequence” ions can be particularly useful in providing
diagnostic information regarding the presence of a phosphorylated peptide within

a complex peptide mixture and may therefore be attractive for use as a

24. 98-102

diagnostic “target” for selective phosphoproteome analysis, as well as

to improve the specificity of subsequent database search analysis strategies.187

188 However, when these ions are observed as the dominant species in the

product ion spectrum, the formation of b- and y-type “sequence" ions may be

 

The results described in Chapter Two have been published in: Palumbo, A.M.;
Tepe, J. J. Reid, G.E. Mechanistic Insights into the Multistage Gas-Phase
Fragmentation Behavior of Phosphoserine- and Phosphothreonine-Containing
Peptides. J. Proteome Res. 2008, 7, 771-779.

53

suppressed. This can make it difficult to unambiguously identify the sequence of
the protein from which the phosphopeptide originated or to localize the Site of
phosphorylation to a Specific amino acid residue within the modified peptide. In
such cases, alternate analysis strategies have been employed to obtain the
required sequence information, involving automatically subjecting the initial
neutral loss product ion to multistage CID-MS/MS (M83) or “pseudo M83“ in a

24. 98-101

data-dependent mode of operation. Alternatively, the loss of H3PO4

during ClD-MS/MS can be used to trigger MS/MS analysis of the original
precursor ion by electron capture dissociation (ECD) or electron transfer

dissociation (ETD), under which conditions the phosphate group remains

. 102.160.161.164
intact.

The utility of these data-dependent acquisition methods is highly

dependent on the ability to form the initial [M+nH-H3PO4]n+ neutral loss product

ion as an abundant species.24 Therefore, studies aimed at the development of

an improved understanding of the mechanisms and other factors that influence

the relative abundance of these ions, as well as determination of the structure(s)

of the initial [M+nH-H3PO4jm product ions subjected to further dissociation by

CID-M83, would enable the development of improved analytical mass

spectrometry approaches for comprehensive phosphoproteome analyses. The

results from these studies would also enhance ongoing efforts to develop in silico

54

spectral intensity prediction algorithms for use in enhanced database search

. . 189-192
analySIS strategies.

To date, it has been widely accepted that under low-energy CID-MS/MS

conditions, the loss of H3PO4 from protonated phosphoserine- and

phosphothreonine-containing peptides occurs via a B-elimination mechanism

87, 89

(Pathway A, Scheme 2.1). In this mechanism, the ionizing proton is distal

from the site of fragmentation and is therefore referred to here as “charge-
remote”. This reaction pathway would involve transfer of the hydrogen atom from
the a-carbon on the phosphoamino acid to the phosphate oxygen on the modified
amino acid Side chain via a six-centered transition state, resulting in the
formation of dehydroalanine- or dehydroaminobutyric acid-containing product
ions from phosphoserine- or phosphothreonine- containing precursor ions,
respectively. Support for this mechanism, including the formation of product ions

corresponding to cleavage on either side of the dehydroalanine (69 Da) or

dehydroaminobutyric acid (83 Da) residues during MS3 of the initial [M+nH-
H3PO4]n+ neutral loss products.89 Additionally, observation of the loss of
HD2PO4 (100 Da) following CID-MS/MS of a deuterated phosphopeptide

precursor ion,87 has been presented by several groups. However, the former

observation could be accounted for by an alternate mechanism (see below),

while the latter result must be considered inconclusive due to incomplete

55

hydrogen/deuterium exchange of the precursor ion and the relatively low

resolution of the mass analyzer employed at that time.

The former study also reported that the loss of H3PO4 from

phosphothreonine-containing peptide ions was highly dependent on the Charge

state of the precursor ion (i.e., that low charge states underwent a dominant loss

of H3PO4, while the highest observed charge state tended to retain the

phosphate moiety and instead undergo extensive fragmentation along the

peptide backbone), while the same loss from phosphoserine-containing peptides

was independent of the precursor ion charge state.89 However, this conclusion

was reached based on consideration of the effect of charge state alone and
therefore did not account for the combined effect of precursor ion charge state

and the number and composition of basic amino acids in the peptide sequence

(i.e., proton mobility) on the observed product ion abundances.112' 193

Two alternate mechanisms have Since been proposed for the side chain

neutral loss of H3PO4, based on results obtained by molecular orbital

calculations and from the gas-phase fragmentation reactions of uniformly

deuterated phosphoserine and model phosphoserine peptide analogues.78’ 117

The first of these involves an E2 elimination reaction, via intramolecular transfer
of the hydrogen atom from the a—carbon on the phosphoamino acid to an
adjacent nucleophile, resulting in the formation of either dehydroalanine- or

dehydroaminobutyric acid-containing product ions (Pathway B, Scheme 2.1).

56

(Note that the site of protonation for the product ions formed by this pathway

would differ from that for the B-elimination mechanism shown in Pathway A of

Scheme 2.1.) The second mechanism involves an SN2 neighboring group

participation reaction, involving intramolecular nucleophilic attack by the N-
terminal amide carbonyl group on the [3-0arbon of the phosphoamino acid side
chain, resulting in formation of a cyclic five-membered oxazoline product ion
(Pathway C, Scheme 2.1). In both of these mechanisms, the ionizing proton is
directly involved in the fragmentation reaction, and they are therefore referred to

here as “charge-directed".

57

(A) HO\ ,OH H-

(B) +OH

+9”
HO-P-OH
R (‘o
0‘, H
1,.an N
DI “h
H

O

(C)

 

 

H+
CID MS/MS ‘ o
> H
-H3PO4 pJLNJk’rN.“
H o
R
CID MS/Ms> 0“ I H
-I'I3PO4 ' rpkfi NH"!
H o

 

 

CID MS/MS R
> O
-H3PO4

 

R = H (p8). CH3 (pTl

Scheme 2.1

Potential mechanisms for the neutral loss of H3PO4 from
phosphoserine- and phosphothreonine-containing peptides via (A) a charge-
remote B-elimination reaction, (B) a charge-directed E2 reaction, and (C) a

charge-directed SN2 neighboring group participation reaction.

mechanisms are presented for phosphoserine-containing peptides only.

Several reports in the literature indicate that proton mobility plays an

important role in the gas-phase fragmentation reactions of protonated peptide

ions containing posttranslational or process-induced modifications.

Furthermore, the appearance of product ion spectra obtained by MS3

58

For clarity, the

15, 116

dissociation of the neutral loss product ions formed by MS/MS is expected to be
highly dependent on both the proton mobility and the structure(s) of the neutral
loss product ion. The purpose of the work described herein, therefore, was to
obtain insights into the role of proton mobility on the multistage gas-phase
fragmentation reactions of protonated phosphoserine- and phosphothreonine-
containing peptide ions, to determine the mechanisms and product ion structures

associated with their formation and to examine the influence of these structures

on the appearance of the Spectra obtained by MS3 of the neutral loss product

ions formed by MS/MS.

2.2 Results and Discussion

2.2.1 Evaluation of Charge-Remote and Charge-Directed Fragmentation

Mechanisms for the Loss of H3PO4 from Phosphoserine- and

Phosphothreonine-Containing Peptides via Multistage Tandem Mass

Spectrometry Analysis of Their Regioselectively and Uniformly

Deuterated Precursor Ions

Differentiation between charge-remote and charge-directed fragmentation
pathways for neutral loss of the side chains from modified peptide ions can be

readily achieved via examination of the CID-MS/MS product ion spectra of

regioselectively or uniformly deuterium-labeled peptide ions.115'117 For

example, the product ion spectra obtained by dissociation of the singly and

doubly protonated precursor ions of the regioselectively Side chain deuterated

59

phosphoserine-containing peptide, GAIL(d3-D,L)pSGAILK, are shown in Figure
2.1A and B, respectively. The product ion spectra obtained from the
nondeuterated form of this peptide, containing all L-amino acids, are shown in
Figure 2.2. Loss of the phosphorylated side chain from the regioselectively
deuterated peptide via the previously proposed charge-remote B-elimination

mechanism shown in Scheme 2.1, Pathway A, would result in the loss of

HzDPO4 (99 Da), whereas the loss of H3PO4 (98 Da) would result from the

charge-directed mechanisms (Scheme 2.1, Pathways B and C). Clearly, it can

be seen from Figure 2.1A and B that the ion corresponding to the neutral loss of

H3PO4 (98 Da) was the major product observed from dissociation of both the

singly and doubly protonated precursor ions, indicating that the fragmentation
process had occurred predominantly via a charge-directed mechanism in each
case (approximately 97% and 98.5% of the neutral loss product ion abundance,
respectively). An expanded region of the product ion spectrum is shown in the

inset Figure 2.1A and reveals that a product ion resulting from fragmentation via

the charge-remote pathway (loss of HzDPO4, 99 Da) was also observed, albeit at

a level representing only 3% of the neutral loss product ion abundance. Although
the presence of a similar charge-remote product ion could not be observed by
direct inspection of the doubly protonated product ion spectrum (presumably due
to limited mass analyzer resolution caused by local space charge effects),

evidence for the formation of this product at a level of approximately 1.5% of the

neutral loss product ion abundance was obtained by MS3 (see below).

60

Figure 2.1 Multistage tandem mass spectrometry of regioselectively deuterated
GAIL(d3-D,L)pSGAILK. CID-MS/MS of the (A) [M+H]+ and (B) [M+2H]2+
precursor ions. CID-MS3 of the (C) singly and (D) doubly protonated [M+nH-
H3PO4]I|+ neutral loss product ions from panels A and B, respectively. CID-MS3
of the (E) singly and (F) doubly protonated [M+nH-H2DPO4]n+ neutral loss
product ions from panels A and B, respectively. A = -H3PO4, o = -H20, * = -NH3.

61

Figure 2.1 (cont’d)

 

 

 

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62

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63

Figure 2.1 (cont’d)

 

 

 

 

 

ooN _. ooo F oom ooo oov ooN ooN _. ooo_.
_ _ — _ I— _ F — _ _ _ _ _ _ _

. i —tl at I I q a i a a . 0
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.. F I.
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Figure 2.2 Multistage tandem mass spectrometry of GAILpSGAILK. CID-
MS/MS product ion spectra of the (A) [M+H] and (B) [M+2H]2 precursor ions.
CID-MS3 product ion spectra of the (C) singly and (D) doubly protonated [M+nH-
H3PO4]'1+ neutral loss product ions from panels A and B, respectively. A =-
H3PO4, ° = -H20, * = -NH3.

64

 

 

 

 

 

 

 

 

 

Figure 2.2 (cont'd)

 

 

 

 

 

 

 

 

 

 

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65

Importantly, regioselective stable isotope labeling allows for the gas-phase
separation of isobaric product ions formed from different reaction mechanisms,

thereby allowing differences in the structures of these MS/MS product ions to be

independently interrogated using M83. Isolation and MS3 dissociation of the

[M+nH-H3PO4]n+ product ions from both the singly and doubly protonated

product ion spectra in Figure 2.1A and B resulted in the product ion spectra

shown in Figure 2.10 and D, while the product ion spectra obtained by M83

dissociation of the [M+nH-HzDPO4]n+ ions are shown in Figure 2.1E and F.

Clearly, the product ions formed in each case, and their relative abundances, are
quite different, indicative of the different structures of their precursor ions.

Notably, the product ion spectra shown in Figure 2.1E and F are essentially

identical to those previously obtained by dissociation of the [M+nH-CH3SOH]n+

product ions of an S-alkyl cysteine sulfoxide containing peptide

GAILC(Sme(ox))GAILK, where the charge-remote B-elimination mechanism was

shown to be the dominant process.112

Interestingly, MS/MS dissociation of the singly and doubly protonated
dehydroalanine (dA)-containing precursor ions of GAIL(dA)GAlLK, produced in
solution via B—elimination from its phosphoserine analogue (data not shown),

gave rise to significantly different product ion spectra than those observed by

MS3 of the [M+nH-H2DPO4]|n+ product ions formed in the gas-phase. As the

sites of protonation in these ions are expected to be similar, the differences in

66

fragmentation may be due to differences in the conformations of the precursor
ions formed in solution versus in the gas-phase. This result therefore highlights

the potential limitations associated with obtaining evidence for gas-phase

fragmentation mechanisms by comparison of MS3 fragmentation behaviors with

the MS/MS fragmentation behavior of proposed product ions formed from

solution. The differences in the MS3 data obtained from dissociation of the

[M+nH-H3PO4]n+ and [M+nH-HZDPO4]n+ product ions from the regioselectively

deuterated peptide also rule out the possibility that the charge-remote

fragmentation pathway was the dominant mechanistic process occurring, but that

the resultant [M+nH-HzDPO4]n+ product ion and the neutral HzDPO4 molecule

had undergone deuterium exchange prior to their separation from the ion—

molecule complex initially formed upon MS/MS, thereby resulting in formation of

the [M+nH-H3PC)4]n+ ion. Furthermore, ClD-MS/MS of the singly protonated

GAIL(d3-D,L)pSGAlLK peptide ion using a range of activation times (30 ms to 30

us at an activation Q of 0.75) was observed to result in no significant change in

the ratio of the [M+H-H3PO4]+ and [M+H-H20PO4]+ neutral loss products (data

not shown), again suggesting that no deuterium exchange had taken place.
Further evidence for the neutral loss of H3PO4 predominantly via a

charge-directed mechanism was obtained by ClD-MS/MS of various uniformly
deuterated phosphoserine- and phosphothreonine-containing peptides, including

some that had previously been examined in mechanistic studies of

67

phosphopeptide fragmentation161’ 164 (Table 2.1). Under these conditions, the

losses of D3PO4 (101 Da) and HD2PO4 (100 Da) would be expected for the

charge-directed and charge-remote mechanisms, respectively. The product ion
spectra obtained by ClD-MS/MS of the singly and doubly protonated precursor
ions of the phosphoserine-containing Kemptide peptide, LRRApSLG, and the
phosphothreonine-containing Kemptide analogue, LRRApTLG, are shown in

Figure 2.3 and Figure 2.4, respectively. Monoisotopic isolation and dissociation

of the uniformly deuterated singly [M(D19)+D]+ and doubly [M(|;)19)+ZD]2+

charged precursor ions of LRRApSLG both resulted in the dominant loss of

D3PO4 (80% and 82% neutral loss product ion abundance, respectively). This is

indicative of fragmentation occurring via the charge-directed fragmentation

mechanisms, whereas the abundances of product ions resulting from the charge-

remote fragmentation mechanism, via the loss of HDzPO4, were observed at

only 20% and 18%, respectively. Importantly, ClD-MS/MS of the singly and

doubly charged precursor ions from all the uniformly deuterium labeled peptide

ions studied to date have resulted in the dominant loss of 03PO4, unequivocally

demonstrating that the loss of H3PO4 from protonated peptide ions occurs

predominantly (>80%) via charge-directed mechanisms (Table 2.1). As
mentioned in the Introduction section (2.1) above, previous molecular orbital
calculations have predicted that the activation energy of the transition state

structure associated with the charge-remote B—elimi'nation mechanism is higher

68

(i.e., less thermodynamically favored) compared to those for the charge-directed

78, 11

8N2 or E2 processes, 7 thereby supporting the experimental results

observed here. Finally, no significant differences in the ratio of charge-directed
versus charge-remote fragmentation product ions were observed between
phosphoserine- and phosphothreonine-containing peptides or as a function of the
identity or number of basic amino acid residues within the peptide sequence

(arginine, lysine, and histidine).

69

Table 2.1 Summary of the MS/MS and MS3 neutral loss product ion
abundances from the gas-phase fragmentation reactions of protonated

phosphoserine- and phosphothreonine-containing peptides. a “Partially mobile”

b c d
proton.193 “Mobile” proton.193 “Nonmobile” proton.193 Results obtained

by ClD-MS/MS of uniformly deuterium labeled peptide ions. e Note that these

values differ from those shown in Figure 2.1 for the regioselectively deuterated
GAIL(d3-D,L)pSGAILK peptide and are likely due to either their acquisition on a
different instrument platform (LCQ-DECA versus LTQ), the effect of the D,L-
versus L-phosphoamino acid, or deuterium isotope effects on the charge-remote

versus charge-directed fragmentation mechanisms. Results obtained by ClD-
MS/MS of protonated peptide ions. 9 Calculated as the ratio of % total product
ion abundances of the [M+nH-H3PO4]n+ ions from isomer phosphopeptide ions
(e.g., GAILpSGAILK versus GAILpTGAILK). h Results obtained by CID-MS3 of

[M+nH-H3PO4-CHZO]n+ (pSer) or [M+nH-H3PO4-CH30HO]n+ (pThr) product
ions initially formed by MS/MS.

70

Table 2.1 (cont’d)

 

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71

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Figure 2.3 ClD—MS/MS product ion spectra of protonated and deuterated

2+

, (o) [M(019)+D]+, and (o) [M(D19)+ZD]

2+

LRRApSLG. (A) [M+H]+, (B) [M+2H]

ions. A = -H3PO4.

73

Figure 2.4 Multistage tandem mass spectrometry of LRRApTLG. CID-MS/MS
product ion spectra of the (A) [M+H]+ and (B) [M+2H]2+ precursor ions. CID-M83
product ion spectra of the (C) singly and (D) doubly protonated [M+nH-H3PO4]n+
neutral loss product ions from panels A and B, respectively. A = -H3PO4, * = -
NH3.

74

Figure 2.4 (cont’d)

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75

2.2.2 Neutral Loss of H3PO4 from Both Phosphoserine- and

Phosphothreonine-Containing Peptides ls Highly Dependent on

Precursor Ion Charge State and Amino Acid Composition (i.e.,

Proton Mobility)

Previous mechanistic and statistical analysis studies have demonstrated a
strong proton mobility dependence on the fragmentation reactions of peptide ions

containing common oxidatively modified amino acid residues, including

115,116

methionine and S-alkyl cysteine. These studies revealed that loss of the

modified amino acid side chains occurs via charge-remote B—elimination reaction
pathways, analogous to that shown in Pathway A of Scheme 2.1 above, and is
increasingly dominant under conditions of decreasing proton mobility. These

findings are consistent with the expectation that under conditions of low proton

112.193

mobility the ionizing proton would be “sequester ” at the most basic site

(e.g., the guanidine group of the arginine side chain), thereby enabling the
charge-remote mechanism to proceed as the energetically favored fragmentation
reaction.

In contrast, the results described above clearly indicate that the neutral

loss of H3PO4 from phosphoserine- and phosphothreonine-comaining peptides

does not predominantly occur via the charge-remote fragmentation mechanism.

However, a strong dependence on the magnitude of the side chain H3PO4

neutral loss product ion abundance as a function of decreasing proton mobility

76

was clearly observed (Table 2.1). For example, the magnitudes of the neutral
loss under nonmobile proton conditions, such as for the singly and doubly
protonated LRRApSLG (Figure 2.3) and LRRApTLG (Figure 2.4) peptide ions
and for the singly protonated GAILpSGAILR (Figure 2.5A) and GAILpTGAlLR
(Figure 2.6A) peptide ions, were found to range from 54 to 89% of the total
product ion abundance. Under mobile proton conditions, however, the
magnitudes of the neutral loss were significantly lower, ranging from 7 to 15% of
the total product ion abundance for the doubly protonated GAILpSGAILK (Figure
2.2B), GAILpSGAILR (Figure 2.58), GAILpTGAlLR (Figure 2.6B), and
GAILpTGAlLK (Figure 2.7B) peptide ions. For precursor ions with partial proton
mobility, i.e., the singly protonated GAILpSGAILK (Figure 2.2A) and
GAILpTGAlLK (Figure 2.7A) peptide ions and the singly and doubly protonated
LFTGHPEpSLEK (Figure 2.8) and LFTGHPEpTLEK (Figure 2.9) peptide ions,
the magnitudes of the total product ion abundance for the neutral losses were

found to be intermediate, ranging from 48 to 75%.

77

Figure 2.5 Multistage tandem mass spectrometry of GAILpSGAILR. CID-
MS/MS product ion spectra of the (A) [M+H]+ and (B) the [M+2H]2+ precursor
ions. CID-MS3 product ion spectra of the (C) singly and (D) doubly protonated
[M+nH-H3PO4]n+ neutral loss product ions from panels A and B, respectively. A
=-H3PO4, * = -NH3.

78

Figure 2.5 (cont’d)

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Figure 2.6 Multistage tandem mass spectrometry of GAllipTGAILR. ClD-
+ +
MS/MS product ion spectra of the (A) [M+H] and (B) [M+2H] precursor ions.

CID-MS3 product ion spectra of the (C) singly and (D) doubly protonated [M+nH-

H3PO4]n+ neutral loss product ions from panels A and B, respectively. A =-
H3PO4, * = -NH3.

80

 

 

 

 

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81

Figure 2.7 Multistage tandem mass spectrometry of GAILpTGAILK. CID-
MS/MS product ion spectra of the (A) [M+H]+ and (B) [M+2H]2+ precursor ions.
CID-MS3 product ion spectra of the (C) singly and (D) doubly protonated [M+nH-
H3PO4]'1+ neutral loss product ions from panels A and B, respectively. A =-
H3PO4, ° = -H20, * = -NH3.

82

Figure 2.7 (cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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83

Figure 2.8 Multistage tandem mass spectrometry of LFTGHPEpSLEK. CID-
MS/MS product ion spectra of the (A) [M+H]+ and (B) [M+2H]2+ precursor ions.
CID-MS3 product ion spectra of the (C) singly and (D) doubly protonated [M+nH-
H3PO4]n+ neutral loss product ions from panels A and B, respectively. A =-
H3PO4, ° = -H20.

84

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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85

Figure 2.9 Multistage tandem mass spectrometry of LFTGHPEpTLEK. CID-
MS/MS product ion spectra of the (A) [M+H]+ and (B) [M+2H]2+ precursor ions.
CID-MS3 product ion spectra of the (C) singly and (D) doubly protonated [M+nH-
H3PO4]r|+ neutral loss product ions from panels A and B, respectively. A =-
H3PO4, * = -NH3.

86

  

 

 

    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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87

(Li
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r 1

Sc

Considering that the loss of H3PO4 proceeds predominantly via charge-

directed mechanisms (where an ionizing proton is directly involved in the

fragmentation reaction), we propose that the neutral loss of H3PO4 as an

increasingly dominant product ion under nonmobile proton conditions is due to
the formation of a strong hydrogen bonding interaction between the phosphate

group and the guanidine side chain of a protonated arginine residue (shown in

Scheme 2.2, Pathway 1 for the 8N2 neighboring group participation reaction

mechanism). The presence of this hydrogen bonding interaction would be
expected to lower the barrier for transfer of the ionizing proton from its initial site

of protonation (the side chain guanidine group of the arginine residue, proton

affinity 251.2 kcal/mol)194 to the side chain of the phosphoserine or

phosphothreonine residues (proton affinity 224.2 and 224.8 kcal/mol,

respectively)195 either prior to or following collisional activation of the ion, thereby

facilitating cleavage of the B—methylene-phosphate oxygen side chain bond. This
hypothesis is consistent with a recent report describing the important role of

phosphate-arginine side chain interactions in the stabilization and gas-phase

. . . 107, 108, 196
fragmentation reactions of noncovalent peptide complexes.

88

 

 

H H
11+“ A. H
H’ (TN H’
Hc‘)“ f‘,- H’ ,N H (Pathway 1) NH
HO- p: 0' OH CID MS/MS_ OH
RG3 HN -H po 7 R HN
3 4 o
A O H A O
i'; NM‘PEPTIDE o HAW Nmpspnoe o
L U
0 HF
(Pathway 2)lH:n’N Transfer
MT: 0 < -RCHO o ”N 00
\J
ANN N-MPEPTIDE o #1:th 7” NMPEPTIDE'JL
" OH

R = H (pS), CH3 (pT)
Scheme 2.2 Proposed mechanisms for the dominant loss of H3PO4 via a
charge-directed 8N2 neighboring group participation reaction (Pathway 1) and for
the loss of formaldehyde (CH20, 30 Da) or acetaldehyde (CH30HO, 44 Da) from

the [M+H-H3PO4]+ product ions (Pathway 2) of protonated phosphoserine- and
phosphothreonine-containing peptide ions.

Although the peptides described here represent only a very limited data

set, the results described above indicate that the magnitudes of the H3PO4

neutral loss from phosphoserine-containing peptide ions are consistently higher
compared to those from their analogous phosphothreonine—containing peptide
ions (with an average ratio of 1.28) (Table 2.1). These results therefore highlight
the subtle but appreciable effect of the fi-methyl group on the phosphothreonine
residue, compared to the B—hydrogen atom on the phosphoserine side chain, on

the specificity of the gas-phase fragmentation reactions. This is presumably due

89

to steric hindrance effects on the transition state activation barriers for the

respective fragmentation reactions.

2.2.3 Experimental Evidence for the Neutral Loss of H3PO4 from

Phosphoserine- and Phosphothreonine-Containing Peptides via an

8N2 Neighboring Group Participation Reaction Mechanism.

Experimental evidence for loss of the phosphoserine and

phosphothreonine side chains via the 8N2 neighboring group participation

reaction mechanism (Scheme 2.1, Pathway C) was obtained by inspection of the
product ion spectra resulting from CID-MS3 of the [M+nH-H3PO4]n+ ions initially
formed by MS/MS. From these spectra (Figure 2.1 to Figure 2.10), product ions
corresponding to the loss of 30 Da from the phosphoserine-containing peptide
ions or the loss of 44 Da from the phosphothreonine-containing peptide ions
were observed. As the pairs of analogous phosphoserine and phosphothreonine
peptides examined in this study (e.g., GAILpSGAILK and GAILpTGAILK) differ
only by the side chain of the phosphorylated amino acid (a 14 Da difference), the
losses of 30 and 44 Da (a 14 Da difference) are likely to involve the side chains

of the serine and threonine amino acids. Thus, we have assigned the loss of 30

Da from the [M+H-H3PO4]+ ions of the phosphoserine-containing peptides as
corresponding to formaldehyde (CH20) and have assigned the loss of 44 Da

from the [M+H-H3PO4]Jr ions of the phosphothreonine-containing peptides as

90

corresponding to acetaldehyde (CH3CHO). Note that the loss of 32 Da (CD20)

was observed upon MS3 dissociation of the [M+H-H3PO4]+ product ion from the

regioselectively deuterated peptide GAIL(d3-D,L)pSGAlLK (Figure 2.1C),
consistent with this proposal. A proposed mechanism for these aldehyde losses,
via intramolecular proton transfer and subsequent fragmentation resulting in the
formation of a nitrilium product ion, is shown in Scheme 2.2, Pathway 2.
Evidence can be found in the literature indicating that nitrilium product ions can
be formed as stable product ions in the gas-phase, via dehydration involving the

carbonyl oxygen atoms of amide bonds contained within protonated peptide

. 197 . . . .
Ions. Despite extensrve consuderatlon, we were unable to propose a

reasonable mechanism for these losses from the [M+H-H3PO4]+ ions initially

formed via the E2 elimination pathway. We note that the ion trap instrumentation

employed in this study does not have the requisite mass resolution and mass

accuracy to distinguish between the losses of CH3CHO (44.05256 Da) and 002

(44.0095 Da), so we can not rule out that the loss of 002 does not also occur to

some extent from the phosphothreonine-containing peptides. However, as this
loss would also be expected from the analogous phosphoserine-containing

peptides, we consider that this process was unlikely. Interestingly, the proposed

8N2 mechanism for the loss of H3PO4 that results in the subsequent loss of the
aldehyde upon M83 (Scheme 2.2) may also be used to rationalize the

consecutive side-chain losses of H20 (18 Da) and CzH4O (44 Da) recently

91

observed for the fragmentation of protonated tryptic peptides containing N-

terrninal Thr-Thr or Thr-Ser residues under mobile proton conditions in a triple

quadrupole mass spectrometer.198 Additional fragmentation processes

observed upon MS3 dissociation of the [M+nH-H3PO4]n+ product ions from the
LFTGHPEpSLEK and LFTGHPEpTLEK peptides (Figure 2.8 and Figure 2.9)
included the formation of unexpected product ions corresponding to bn+H20.

Notably, these “hydrolysis"-type product ions have also been observed previously

from the M33 fragmentation reactions of both nonphosphorylated and

phosphorylated peptide ions.4o’ 99’ 199' 200

92

_s
C
C

% Relative Abundance

_L
O
O

% Relative Abundance

Figure 2.10

Similar to that described above for the neutral loss of H3PO4 upon MS/MS

1.63E6

 

 

 

 

   

 

 

-NH3
' -CH20(0._2%)
_ 5 b6
I l . I '1; 1‘].
I l I I I I I I
300 400 500 600 700 800 900 1000
1.44E6
*2+
_ b6
2+
b 2+
- 5. $9.6
b433, -CH202*<1%>
b3
‘ 1 Al A “‘3'; if]. _-
—l—_l— I I I I I I I
200 300 400 500 600 700 800 900 1000
m/z

CID-MS3 product ion spectra of the (A) singly and (B) doubly
protonated [M+nH-H3PO4]n+ neutral loss product ions of LRRApSLG from Figure
2.3A and B, respectively. * = -NH3.

of the protonated phosphoserine- and phosphothreonine-containing precursor

ions, the abundance of the CH20 or CH3CHO neutral loss product ions upon

93

MS3 of the [M+nH-H3PO4]n+ ions was observed to increase with decreasing
proton mobility (Table 2.1). However, the abundance of the CH3CHO neutral
loss product ions from the [M+nH-H3PO4]n+ ions of phosphothreonine-containing

peptide ions was consistently higher than that for the loss of CH20 from the

analogous phosphoserine-containing ions (Table 2.1).
Note that the observation of low-abundance product ions corresponding to

fragmentation of the amide bond on the N-terminal side of the phosphoamino

acid side chain upon MS3 of the [M+nH-H3PO4]n+ product ions from the

GAIL(d3-D,L)pSGAlLK peptide (e.g., the ya and b4 product ions in Figure 2.1C

and D, respectively) is suggestive of the presence of an acyclic dehydroalanine

residue and could be used as evidence for formation of the [M+nH—H3PO4]n+

neutral loss product ions during MS/MS (at least to some extent) via a

competitive E2 elimination pathway. However, the observation of these ions

could also be rationalized by ring opening of the 8N2 directed oxazoline ring-
containing product by intramolecular proton transfer, yielding the acyclic
dehydroalanine-containing product, prior to its further dissociation.

Unfortunately, we can not experimentally differentiate between these mechanistic

possibilities from the results obtained here.

94

2.3 Conclusions

Multiple competing charge-directed and charge-remote mechanisms can

contribute to the overall appearance of the resultant MS/MS and MS3 product ion

spectra formed via gas-phase fragmentation of protonated peptide ions.

However, in contrast to the widely accepted charge-remote B-elimination

mechanism for the gas-phase neutral loss of H3PO4 from phosphoserine- and

phosphothreonine-containing peptide ions, the results obtained from this study
unequivocally demonstrate that this loss occurs predominantly via charge-

directed mechanisms. Experimental evidence for formation of the neutral loss

product ion via a charge-directed 8N2 neighboring group participation reaction

mechanism under conditions of low proton mobility was obtained by the

observation of novel diagnostic product ions corresponding to the loss of

formaldehyde (CH20, 30 Da) or acetaldehyde (CH3CHO, 44 Da) upon CID-MS3

of the [M+nH-H3PO4]n+ product ions from phosphoserine- and

phosphothreonine-containing peptide ions, respectively. Further studies are

undenNay to examine the potential value of these diagnostic MS3 neutral loss

product ions for enhancing the utility of data-dependent MSn methods for the

characterization of phosphopeptides containing multiple potential

phosphorylation sites.

95

CHAPTER THREE

Evaluation of Gas-Phase Rearrangement and Competing Fragmentation

Reactions on Protein Phosphorylation Site Assignment Using Collision-

induced-dissociation-MS/MS and M83

3.1 Introduction
Due to the vital role that reversible protein phosphorylation plays in the

regulation of a large number of cellular processes and in the development of

various pathological conditions,1’ 3' 23 significant effort has been directed in

recent years toward the development and application of a range of mass

spectrometry (MS)—based analytical strategies for comprehensive

phosphoproteome analysis.15'18 In a typical approach, phosphopeptides

produced by enzymatic digestion are first enriched by ion exchange

chromatography, immobilized metal affinity chromatography (IMAC) or metal

17, 24, 79

oxide (e.g., TiOz and Zr02) resins, then introduced to the mass

spectrometer by either electrospray ionization (ESI) or matrix assisted laser

desorption/ionization (MALDI). The resultant protonated ions are then isolated

 

The results described in Chapter Three have been published in: Palumbo, A.M.;
Reid, G.E. Evaluation of Gas-Phase Rearrangement and Competing
Fragmentation Reactions on Protein Phosphorylation Site Assignment using

ClD—MS/MS and M83. Anal. Chem. 2008, 80, 9735-9747.

96

and subjected to collision-induced dissociation (CID)- tandem mass spectrometry
(MS/MS), where the presence of phosphorylated tyrosine, serine or threonine
residues within these peptides are commonly recognized by the formation of

characteristic “non-sequence” product ions corresponding to the losses of 80 Da

24. 26, 79-82. 127

(HPO3) or 98 Da (H3PO4) from the initial precursor ions. When

these product ions are observed as abundant fragmentation pathways,

subsequent isolation and M83 dissociation is often employed to obtain additional

24, 79, 98—100. 201

structural information. “Pseudo M33” methods may also be

employed, whereby activation of the precursor ion and the initial neutral loss

product ions are simultaneously carried out in a single step during CID-

MS/MS.101 Alternatively, observation of the characteristic neutral loss product

ions may be employed to trigger electron capture dissociation (ECD)- or electron

transfer dissociation (ETD)-MS/MS of the original precursor ion, under which

102,160,161,164

conditions the phosphate group remains intact. Identification of

the phosphopeptide sequence and assignment of the site of phosphorylation are

then performed via algorithm based interpretation or manual interrogation of the

24, 187, 202-204

MS/MS or MS3 spectra. Despite significant advances made in

each of these areas and the subsequent emergence of experimental datasets

containing thousands of protein phosphorylation site assignments,24’ 79' 205

various analytical obstacles remain that may hinder efforts to unambiguously

localize the sites of phosphorylation to specific residues within an identified

97

phosphopeptide of interest. For example, when multiple potential sites of

phosphorylation exist within a given peptide, the utility of ClD-MS/MS or MS3

analysis strategies for phosphorylation site assignment are ultimately dependent
on the observation of sufficient “sequence” ions (i.e., b— and y-type product ions)
from which the sequence of the peptide may be determined, and from which the

site of phosphorylation may be unambiguously assigned. It is rare however, that

complete “sequence” ion information is obtained upon ClD-MS/MS or MS3 of

protonated peptides, thereby often limiting the ability to localize the modification
to within only a region of the peptide sequence.

The observation of “non-sequence” fragmentation pathways following CID-

MS/MS or MS3 may also complicate phosphorylation site assignment. For

example, CID-MS/MS product ions formed via the non-sequence loss of 98 Da

from protonated phosphopeptides are typically assigned as corresponding to the

81, 83

loss of H3PO4. Due to the potentially limiting effects on phosphorylation

site assignment when this product ion is observed at high abundance, we have

recently evaluated the mechanisms and other factors that influence the formation

and abundance of H3PO4 neutral loss product ions.206 In that study, it was

determined that the loss is observed as a dominant process under conditions of
limited proton mobility due to the formation of a strong hydrogen bonding
interaction between the phosphate group and the side chain of a protonated
arginine or lysine residue. Furthermore, it was proposed that the 98 Da loss

primarily occurs via either a charge directed intramolecular E2 reaction, resulting

98

in the formation of either dehydroalanine- or dehydroaminobutyric acid-containing

product ions from phosphoserine- or phosphothreonine, respectively (Scheme

3.1A), or via an 8N2 neighboring group participation reaction mechanism to yield

cyclic five-membered oxazoline containing product ions (Scheme 3.18).206

Experimental evidence for the 8N2 mechanism was provided by the observation
of characteristic products formed via the losses of 30 Da (CHzO) and 44 Da

(CH3CHO) upon CID-MS3 dissociation of the initial [M+H-98]+ products formed
from phosphoserine- and phosphothreonine-containing model peptides,
respectively.206 However, it has also been previously recognized that the
product ions formed by the loss of 98 Da could also be formed via the sequential
non-sequence losses of HPO3 and H20 from a phosphorylated amino acid
residue and a non-phosphorylated hydroxyl (or carboxyl) containing amino acid

89, 118

residue, respectively (Scheme 3.2). Thus, CID-MS3 and subsequent

analysis of the product ions formed from this competing fragmentation process

could result in erroneous assignment of the phosphorylation site.

99

H

N+N
,‘H
HO’I’ €Nr
Ho pfo‘ H H
" OH
(‘5 HN

0 H
J H
%j<ENMPEPTIDE
H

O

>—

O
O

I
+

{Mfr
H

,NH

N

OJ
H O
’R‘KNMPEPTIDEAOOH
H

DU
6‘0)
I

HN

AoOH

N-M PEPTIDE O
OH

NN\

 

 

 

N ll
H’ ‘1’
NH
CID MS/MS_ OH
-H3PO4 R HN
0” l H A
\fi NMPEPTIDE o
H o
H' T
NH
CID MS/Ms_ OH
-H3PO4 O R HN
H A o
\+ NMPEPTIDE 0
LII] r V
H o
lH+ Transfer
H’ ‘11”
NH
3
< MS R HN OH
-RCHO O H A O
0
AN 3 erPEPTlDE o
4.))! 0
OH

R = H (pS), CH3 (pT)

Scheme 3.1 Previously proposed mechanisms for the “charge directed” neutral
loss of H3PO4 and RCHO from protonated phosphoserine- or phosphothreonine-

(where R = H or CH3, respectively) containing peptides during ClD-MS/MS.

206

(A) Intramolecular E2 mechanism and (B) intramolecular SN2 neighboring group

participation mechanism.

100

OOHO H' JHHEF

R10 OH

O HN
NHL 0
|N| PEPTIDE];k

CID MS/MS

-HPO3, -H20
(98 Da)

 

(44-IL H \ + E O
H H PEPTID O
0 R2 0

Scheme 3.2 Competing pathways for the formation of isobaric product ions via
the loss of 98 Da (-H3PO4 or —(HPO3+H20)) from a protonated phosphopeptide
containing multiple potential phosphorylation sites. R1 = H or CH3 for
phosphorylated serine or phosphorylated threonine, respectively; R2 = any amino

acid side chain; R3 = H or CH3 for non-phosphorylated serine or non-
phosphorylated threonine, respectively.

A further critical requirement for unambiguous phosphorylation site

assignment is that the information obtained by interrogation of the MS/MS or

MS3 product ion spectra must reflect the structure (i.e., the sequence and

101

phosphorylation site) of the phosphopeptide that was originally introduced to the
mass spectrometer. Therefore, any rearrangement of the peptide sequence or
the site of phosphorylation in the gas-phase, either prior to, during, or following
the dissociation reaction would interfere with both sequence determination and
phosphorylation site assignment. It is well established that a range of complex

rearrangement reactions can occur in the gas-phase under typical ClD-MS/MS

199, 207-218

conditions involving both protonated precursor and product ions. In

particular, the observation of “scrambled” sequence ions resulting from the
formation and subsequent fragmentation of an isomeric population of b-type
product ion structures following cyclization and subsequent ring opening of an

initial oxazolone containing b—type product ion formed upon ClD—MS/MS has

217,218

recently been reported. Notably, another more recent study performed to

evaluate the fragmentation reactions of several hundred synthetic peptide
sequences of variable composition and length has determined that half the

peptides included in the study exhibited evidence for such rearrangement

. 219
reactions.

Evidence for gas-phase rearrangement reactions have also been reported
previously for post-translationally modified peptide ions. For example, it has
been shown that formyl and acetyl groups from N-formyl- or N-acetyl- N-hydroxy

omithine residues may transfer in the gas-phase under ClD-MS/MS conditions to

unmodified lysine residues contained within the peptide sequence.220 Under

these circumstances, interpretation of the resultant product ion spectra resulted

102

in erroneous assignment of the known site of modification. The possibility of gas-
phase rearrangement reactions have also been hypothesized in order to

rationalize the observed loss of 98 Da from phosphotyrosine-containing peptides,

89, 128

since phosphotyrosine may not undergo the direct loss of H3PO4. For

example, Metzger and Hoffmann previously proposed that the loss of H3PO4

observed upon post-source decay of a phosphotyrosine-containing peptide may
have occurred via initial transfer of the phosphate group from the tyrosine to an

aspartic acid residue located within the peptide sequence, resulting in formation

of a succinamide containing product.128 DeGnore and Qin hypothesized that the

lengthy timescale (millisecond) for activation in an ion trap mass spectrometer

may allow for loss of H3PO4 following transfer of the tyrosine-phosphate group to

another residue within the peptide.89

Due to the potential limiting effect of gas-phase rearrangement reactions
on the assignment of post-translational modifications involving phosphorylation to
specific sites within a given phosphopeptide sequence, an improved
understanding of the conditions under which rearrangement reactions may be
observed is clearly required. Unfortunately, it is not generally possible to extract

this information via the interrogation of phosphopeptide datasets of MS/MS or

MS3 spectra obtained from proteins isolated from biological sources, as the

specific sites of modification (with the exception of those containing known

205

kinase motifs)24’ and the potential for rearrangement in these peptides are

103

not known a priori. However, it is expected that detailed insights may be

obtained via analysis of the MS/MS and MS3 spectra obtained from the

dissociation of individual synthetic phosphopeptide sequences or synthetic
phosphopeptide datasets, where the site of phosphorylation in each case is

unequivocally known.

3.2 Results and Discussion
3.2.1 ClD-MSIMS of Protonated Phosphopeptides Results in Gas-Phase

Phosphate Group Transfer Reactions.

In order to determine whether rearrangement reactions could occur during
the ClD-MS/MS of protonated phosphopeptides containing multiple potential
phosphorylation sites, resulting in the formation of product ions whose inclusion
in subsequent data interrogation strategies could lead to erroneous or ambiguous
phosphorylation site assignments, we have examined the gas-phase
fragmentation behavior of a series of singly and doubly phosphorylated peptides
(Figure 3.1, Figure 3.2 and Table 3.1). The product ion spectra obtained by ESI-
ClD-MS/MS of the singly, doubly, and triply protonated precursor ions of the
model synthetic singly phosphorylated peptides LFTGHPEpSLER (TpSR) and
LFpTGHPESLER (pTSR), using a linear quadrupole ion trap mass spectrometer,
are shown in Figure 3.1 and Figure 3.2, respectively. Consistent with previous
phosphopeptide fragmentation studies, a range of product ions resulting from
both non-sequence and sequence fragmentation pathways were observed in

these spectra, the magnitudes of which were dependant on the proton mobility of

104

the precursor ion.206 For example, the % total product ion abundance for the

non-sequence product ions corresponding to the loss of 98 Da (typically

assigned as -H3PO4 and labeled in Figure 3.1 and Figure 3.2 with a superscript

A) from the singly protonated (i.e., non-mobile) precursor ion of the pTSR peptide

shown in Figure 3.2A, was found to be 72%, while the same loss from the doubly
(i.e., partially mobile) and triply (i.e., mobile) precursor ions (Figure 3.28 and C,

respectively) were found to be 43% and 13%, respectively. In addition, non-

sequence ions corresponding to the loss of 80 Da (-HP03, labeled in Figure 3.1
and Figure 3.2 with a superscript 0) and 18 Da (-H20, labeled in Figure 3.1 and

Figure 3.2 with a superscript 0), as well as various products corresponding to the

sequential losses of these species from the protonated precursor ions were also

observed.

105

Figure 3.1 lon trap ClD—MS/MS product ion spectra of the (A) singly, (B) doubly,
and (C) triply protonated precursor ions of the model synthetic phosphopeptide
LFTGHPEpSLER (TpSR). The spectra are labeled according to the known or
expected Ser phosphorylation site. Ions labeled with bold text are
unambiguously indicative of the expected site of phosphorylation at the Ser
residue. A = -98 Da (-H3PO4 or —(H20+HPO3)); [:1 = —80 Da (—HP03); o = —18

Da (—H20); I = +80 Da (+HPO3).

106

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108

Figure 3.2 Ion trap ClD—MS/MS product ion spectra of the (A) singly, (B) doubly
and (C) triply protonated precursor ions of the model synthetic phosphopeptide
LFpTGHPESLER (pTSR). The spectra are labeled according to the known or
expected Thr phosphorylation site. Ions labeled with bold text are
unambiguously indicative of either the expected or correct site of phosphorylation
at the Thr residue, or the unexpected or incorrect site of phosphorylation at the

Ser residue. A = —98 Da (—H3PO4 or —(H20+HPO3)); [:1 = —80 Da (—HPO3); ° =
—18 Da (-H20); I = +80 Da (+HPO3).

109

Figure 3.2 (cont’d)

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111

Note that the spectra shown in Figure 3.1 and Figure 3.2 are labeled

according to the expected (i.e., known) phosphorylation sites at Serg of the TpSR

peptide sequence and Thr3 of the pTSR peptide sequence. However, the

possibility of phosphorylation occurring at the alternative (i.e., unexpected) site

(i.e., Thr3 of the TpSR peptide sequence and Serg of the peptide sequence

TpSR) must also be considered prior to unambiguously assigning the site of
phosphorylation in this peptide. Figure 3.3 shows the same spectra as those in

Figure 3.2, but where the product ions in each spectra are labeled according to

the alternative (i.e., unexpected) phosphorylation site at Serg. By comparison of

these Figures, it can be seen that the majority of the observed product ions can

be assigned to either of the two possible phosphorylated peptide sequences. For

example, yn—type product ions assigned to the pTSR peptide in Figure 3.2 that

contain the non-phosphorylated serine residue (e.g., y4, y5, W and ya in Figure

3.2A) may also be assigned to the TpSR peptide in Figure 3.3 as corresponding

to ynD-type product ions (e.g., y4D. Yea. WU and y3D in Figure 3.3A) formed via

the sequential loss of 80 Da (-HPO3) from yn-type ions that contain the

phosphorylated serine residue (i.e., sequential sequence and non-sequence

fragmentation pathways). Similarly, the yn-type product ions labeled in Figure
. . O 0 O O O . .

3.2 With a superscript (e.g., y4 , y6 , w and ya In Figure 3.2A),

corresponding to the loss of 18 Da (-H20) from product ions containing the non-

112

phosphorylated serine residue in the expected pTSR sequence may also be

labeled as corresponding to the loss of 98 Da (-H3PO4) from the phosphorylated
. . . A A A A .
senne resrdue In the unexpected TpSR sequence (e.g., y4 , ye , w and ya In
Figure 3.3A). b-type product ions labeled in Figure 3.2 with a superscript A
corresponding to the loss of 98 Da (-H3PO4,), which contain only a single
. . . A A A A. .
potential srte of phosphorylation (e.g., b3 , b4 , b5 and by In Figure 3.2B), may
also be assigned in Figure 3.3 as corresponding to the loss of 18 Da (-H20)
when a non-phosphorylated threonine residue is present (e.g., b3o, b4o, b50 and
b7° in Figure 338). Finally, b—type product ions labeled with a superscript D in
Figure 3.2 (e.g, b5D, bit3 in Figure 3.2A) corresponding to the loss of 80 Da

(HPO3). when a phosphorylated residue is present, may also be assigned in

Figure 3.3 as intact b-type ions where the non-phosphorylated residue is present.

Without definitively knowing the extent to which the losses of H3PO4, HP03 or

H20 occur in these peptides, these ions must all be considered ambiguous, and

therefore may not be used to provide conclusive evidence to unambiguously
assign the site of phosphorylation. It should also be noted that conclusive

evidence to unambiguously localize the site of phosphorylation cannot be

obtained from b- or y-type product ions containing a phosphate group (e.g., b9,
y9 and b1o in Figure 3.2A), or from bnA- or ynA-type product ions formed via the

113

loss of 98 Da (e.g., bgA, b1oA, ygA and y1oA in Figure 3.2A), when multiple

potential phosphorylation sites within these regions of the sequence are present.
These results therefore indicate that the only ions in the ClD-MS/MS spectra that
can provide conclusive (i.e., unambiguous) evidence for the site of
phosphorylation are those in which the phosphate group remains intact and

where the product ion contains only a single potential site of phosphorylation.

114

Figure 3.3 lon trap ClD—MS/MS product ion spectra of the (A) singly, (B) doubly
and (C) triply protonated precursor ions of the model synthetic phosphopeptide
LFpTGHPESLER (pTSR). The spectra are labeled according to the unexpected
(i.e., incorrect) site of phosphorylation at the serine residue. A = —98 Da (—

H3PO4 or —(H20+HPO3)); «:1 = —80 Da (—HPO3); ° = —18 Da (4120); I = +80 Da
(+HPO3).

115

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Inspection of the product ion 'spectrum obtained upon CID-MS/MS of the
singly, doubly and triply protonated precursor ions of the TpSR peptide (Figure
3.1) revealed multiple ions in each case (labeled in the Figures in bold text) that
were unambiguously indicative of the expected site of phosphorylation at the
serine amino acid. In contrast, inspection of the product ion spectrum obtained
upon CID-MS/MS of the singly protonated precursor ion of the pTSR peptide
(Figure 3.2A) did not reveal any ions that were unambiguously indicative of the
expected site of phosphorylation at the threonine amino acid. However, an

unexpected series of product ions were observed, whose mlz values

corresponded to localization of the phosphate moiety (+80 Da, ') at the serine
residue of the peptide (labeled as y4-. ye'. WI, and ya' in Figure 3.2A, and as

y4, ye, y7, and ya in Figure 3.3A). CID-MS3 of the ye. ion (sequence

PESLER+80 Da) from Figure 3.2A (see Figure 3.4A) revealed the presence of an

unmodified y3 ion (LER) as well as a product corresponding to a y4 ion

containing the phosphate group (y4') (mlz 584.4), confirming that the phosphate

group was located on the serine residue. Importantly, this spectrum was

essentially identical (see Figure 3.4B) to that obtained by CID-MS3 of the

phosphorylated y5 product ion from the TpSR peptide in Figure 3.1A. These data

potentially suggest therefore, that the incorrect peptide (i.e., one containing
phosphoserine (TpSR) rather than phosphothreonine (pTSR)) had been

introduced to the mass spectrometer, or indicate that transfer of the phosphate

118

residue had occurred either in the interface of the mass spectrometer during the

electrospray ionization process, or in the gas-phase during ClD-MS/MS.

119

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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HPO3) product ion formed from ClD-MS/MS of the singly protonated pTSR
precursor ion in Figure 3.2A and (B) the ye product ion formed from ClD—MS/MS
of the singly protonated TpSR precursor ion in Figure 3.1A.

120

An unexpected ye. product ion was also observed upon analysis of the

ClD-MS/MS spectrum obtained from the doubly protonated precursor ion of the

pTSR peptide (Figure 3.2B). However, products corresponding to the correct site

of phosphorylation were also observed in this spectrum, (see the b3, a5, b5 and

b7 ions), suggesting that a mixture of the threonine and serine phosphorylated

peptide sequences were present. In contrast, no evidence was observed for
unexpected product ions indicative of the TpSR sequence in the ClD-MS/MS
spectrum obtained from the triply protonated precursor ion of the pTSR peptide
(Figure 3.20), while various products were observed that could be used to

conclusively assign the site of phosphorylation to the expected site of

phosphorylation (see the b3, a5, b5, b52+ and b72+ ions). As the presence of the

TpSR peptide during introduction of the pTSR peptide to the mass spectrometer
would be expected to result in observation of the unexpected product ions in
each of the precursor ion charge states subjected to ClD-MS/MS, this data
provides evidence for formation of these ions via a gas-phase phosphate transfer
process. Further confirmation that the unexpected product ions were not due to
the presence of the isomeric phosphoserine (i.e., TpSR) containing peptide being
present within the pTSR sample that was introduced to the mass spectrometer,
was obtained by repeating the MS/MS analysis following individual RP-HPLC
purification of the pTSR and TpSR peptides. Using the conditions outlined in the
Materials and Methods section above, the retention times of these peptides were

found to be 22.8 min and 20.1 min, respectively. Reinjection of each peptide

121

following their purification did not result in any absorbance at the retention time of
the other peptide, indicating that the purification process did not result in
rearrangement of the phosphate group.

The effects of the ionization method employed (ESI versus MALDI), the
type of ion trap mass spectrometer in which the dissociation experiments were
carried out (linear versus 30), as well as the time scale for ion activation (ion trap
versus quadanole) on the origin of the phosphate group transfer products were
also examined. Acquisition of the ClD-MS/MS product ion spectrum from the
singly protonated precursor ion of the pTSR peptide following ionization by
MALDl on a linear quadrupole ion trap mass spectrometer yielded an identical
spectrum to that shown in Figure 3.2A, suggesting that the rearrangement
process did not occur during the ESI process. Analysis of the product ion
spectra obtained from the singly, doubly, and triply protonated precursor ions of
the pTSR peptide following ESI-ClD—MS/MS in a conventional 30 ion trap
resulted in essentially identical product ion spectra to those observed using the
linear ion trap, including observation of the rearrangement product ions from the
singly and doubly protonated precursor ions (data not shown), indicating that the
rearrangement process was not limited to a particular ion trap platform. The
abundance of the product ions resulting from gas-phase rearrangement reactions
upon ClD-MS/MS of the singly protonated precursor ion of the pTSR peptide in
the linear ion trap mass spectrometer was found to vary significantly as a
function of the activation time (2-2000 msec). In particular, a decrease in the

abundance of the phosphate group rearrangement product ions was observed

122

with decreasing activation time (Figure 3.5). However, even at the shortest
activation time that could be employed while still observing reasonable activation
efficiencies (2 msec); the rearrangement product ions were still present. In
contrast, upon analysis of the singly, doubly and triply protonated peptide ions by
ESI-ClD-MS/MS in a triple quadrupole mass spectrometer (psec activation
timescale), no product ions resulting from the gas-phase phosphate group
rearrangement reactions were observed, while the overall appearance of the
spectra were relatively unchanged compared to that obtained using the ion trap
(Figure 3.6 and Figure 3.7). These results indicate the phosphate group
rearrangement reaction occurs in the gas-phase during ClD-MS/MS, in either
linear or 3D ion trap mass spectrometers, due to the relatively long (millisecond)

timescales associated with ion activation in these instrument platforms,

consistent with that previously hypothesized by DeGnore and Qin.89

123

Figure 3.5 lon trap ClD—MS/MS product ion spectra of the singly protonated
precursor ion of the model synthetic phosphopeptide LFpTGHPESLER (pTSR)
using (A) 10 msec and (B) 2000 msec ion activation times at an activation q
value of 0.25. The spectra are labeled according to the known (i.e., expected)
site of phosphorylation (i.e, Thr). Ions labeled with bold text are unambiguously
indicative of the unexpected site of phosphorylation (i.e, Ser), resulting from the
gas-phase rearrangement reaction. A = —98 Da (—H3PO4 or -(H20+HPO3)); El =

—80 Da (—HPO3); ° = -18 Da (—H20); I = +80 Da (+HP03); RA = relative
abundance.

124

Figure 3.5 (cont’d)

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125

Figure 3.6 Triple quadrupole ClD-MS/MS product ion spectra of the (A) singly,
(B) doubly, and (C) triply protonated precursor ions of the model synthetic
phosphopeptide LFpTGHPESLER (pTSR). The spectra are labeled according to
the known (i.e., expected) site of phosphorylation (i.e, Thr). Ions labeled with
bold text are unambiguously indicative of the expected site of phosphorylation at

the threonine residue. A = —98 Da (—H3PO4 or —(H20+HPO3)); D = —80 Da (—
HPO3); ° = —18 Da (—H20).

126

Figure 3.6 (cont’d)

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127

Figure 3.6 (cont’d)

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128

Figure 3.7 Expanded regions of the product ion spectra obtained by (A) ion trap
(Figure 3.2A) and (B) triple quadrupole (Figure 3.6A) ClD-MS/MS of the singly
protonated precursor ion of the model synthetic phosphopeptide
LFpTGHPESLER (pTSR). The spectra are labeled according to the known (i.e.,
expected) site of phosphorylation (i.e, Thr). Ions labeled with bold text are
unambiguously indicative of the unexpected site of phosphorylation (i.e, Ser),

resulting from the gas-phase rearrangement reaction. E] = —80 Da (—HPO3); O =
-18 Da (—H20); I = +80 Da (+HPO3).

129

Figure 3.7 (cont’d)

 

 

 

 

 

 

 

 

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to
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130

950

910

870

830

790

750

710

m/z

To determine the generality of these gas-phase phosphate group transfer
reactions, 33 singly or doubly phosphorylated peptides, each containing multiple
potential phosphorylation sites, were independently synthesized then examined
by ESI- and MALDI- ClD-MS/MS using linear quadrupole ion trap mass
spectrometers (Table 3.1). Of these peptides, 70% (23 of 33) were singly
phosphorylated (12 containing phosphoserine, 7 containing phosphothreonine
and 4 containing phosphotyrosine) while the remaining 30% (10 of 33) were
doubly phosphorylated (4 containing two phosphoserine, 1 containing two
phosphothreonine, 2 containing one phosphoserine and one phosphothreonine,
2 containing one phosphoserine and one phosphotyrosine, and 1 containing one

phosphothreonine and one phosphotyrosine). These sequences were taken

from a previously published dataset of identified phosphopeptide sequences,24

and were selected in order to provide a reasonable representation of the overall
characteristics of the peptides contained within the dataset (i.e., the type and
number of phosphorylation sites, peptide mass, complete versus incomplete
tryptic cleavages, arginine versus lysine terminated, etc.). All of the precursor ion
charge states that were observed by either ESI (primarily doubly and triply
protonated precursor ions) or MALDI (singly protonated precursor ions) using the
experimental conditions described in Chapter Four and were subjected to CID-
MS/MS. Product ion assignment was achieved by manual interrogation of each
of the MS/MS spectra. For all of the precursor ion charge states from each of the
peptides listed in Table 3.1, the number of observed amide bond cleavages that

were used to unambiguously assign either the correct or incorrect site(s) of

131

phosphorylation, as well as the total number of possible amide bond cleavages
that could be used to unambiguously assign the site(s) of phosphorylation, have
been determined (Table 3.1). Amide bond cleavages that enable unambiguous
assignment of the site of phosphorylation to the correct (i.e., expected) residue
result from product ion(s) that contain the intact phosphate modification at the
expected residue, and (i) where no other potentially phosphorylated residues
(i.e., no unmodified serine, threonine or tyrosine) are present within the product
ion, and (ii) when the m/z of the product ion is higher than the low mass cutoff
and lower than the high mass limit of the instrument, and (iii) where the nominal
mlz of the product ion does not overlap with any other product ion. Similarly,
amide bond cleavages that enable unambiguous assignment of the
phosphorylation site to an incorrect (i.e., unexpected) residue result from product
ion(s) that contain a phosphate modification (+80 Da) at an unexpected serine,
threonine or tyrosine (i.e., incorrect) residue within the product ion, and (i) where
the formerly phosphorylated (i.e., correct) residue is not present within the
product ion, and (ii) when the m/z of the product ion is higher than the low mass
cutoff and lower than the high mass limit of the instrument, and (iii) where the

nominal mlz of the product ion does not overlap with any other product ion.

132

Table 3.1 Summary of the gas-phase phosphate group transfer product ions
observed following ESl- and/or MALDI-ClD-MS/MS of independently synthesized
tryptic phosphopeptides. a Proton mobility classifications defined by Kapp, et
al: Non-mobile: number of ionizing protons 5 number of arginine residues;
Partially-mobile: number of arginine residues < number of ionizing protons 5
combined number of arginine, lysine and histidine residues; Mobile: number of
ionizing protons > combined number of arginine, lysine and histidine residues. b

I indicates +80 Da (i.e., +HP03), II indicates +160 Da (i.e., +2HPO3). c
Singly protonated precursor ions observed only by MALDI.

133

 

Table 3.1 (cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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134

Table 3.1 (cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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135

Table 3.1 (cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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136

Table 3.1 (cont'd)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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137

Table 3.1 (cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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138

Table 3.1 (cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

v F.N 0.0 000.. .0... 0.50:. 2.00.00 N
0.03005505050513
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139

Table 3.1 (cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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0N... 0.0 - 0.50:.:0: 0F
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140

Table 3.1 (cont'd)

 

 

 

 

 

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141

From the data in Table 3.1, it was determined that 15 of the 33 peptides
(45%) exhibited product ions resulting from gas-phase phosphate group transfer
reactions. Importantly, the site of phosphorylation could be unambiguously
correctly assigned in only 12 of the 33 (36.4%) peptides, while 10 of the 33
(30.3%) peptides gave rise to product ion spectra from which the site of
phosphorylation was unambiguously incorrectly assigned. For the remaining 11
peptides (33.3%), the site of phosphorylation could not be definitively assigned to
either the correct or incorrect site(s), as either both or neither of the required
product ions were observed. When divided according to the ionization method
employed, only 10 out of the 33 peptides ionized by MALDI (30.3%) and 12 out
of the 33 peptides ionized by ESI (36.4%) resulted in unambiguously correct
phosphorylation site assignment.

Further analysis of the data in Table 3.1 revealed that 28 of the 94
observed precursor ion charge states (29.8%) gave rise to product ions resulting
from gas-phase phosphate group transfer reactions (14 of the 33 precursor ions
observed by MALDI (42.4%) and 19 of the 78 precursor ions observed by ESI
(24.0%)). From these data, the number of unambiguously correct,
unambiguously incorrect and ambiguous phosphorylation site assignments were
31 of 94 (33.0%), 21 of 94 (22.3%) and 42 of 94 (44.7%), respectively.

Although this is a relatively limited data set, such that a comprehensive
evaluation of the effect of peptide sequence, length, overall basicity/acidity and
the relative location of the donor/acceptor residues was not possible, a clear

trend was observed based on the effect of the precursor ion proton mobility on

142

the observation of the rearrangement products. The majority of the precursor
ions that exhibited evidence for the phosphate group transfer were classified as
“non-mobile” (number of ionizing protons 5 number of arginine residues) or

“partially-mobile” (number of arginine residues < number of ionizing protons 5

combined number of arginine, lysine and histidine residues).193 Note that the

percentage of the precursor ion charge states that could be unambiguously
correctly assigned following ClD-MS/MS under “non-mobile”, “partially-mobile” or
“mobile” protonation conditions was 23.8% (5 of 21 ), 30.3% (10 of 33) and 42.5%
(17 or 40), respectively.

Based on this observation, a potential mechanism for the observed
rearrangement reaction is shown in Scheme 3.3. This mechanism involves
nucleophilic attack by the hydroxyl oxygen of a non-phosphorylated serine or
threonine residue to the phosphorous atom of a phosphoserine or
phosphothreonine residue, with a concerted proton transfer. Due to the
observed effect of proton mobility on the formation of these rearrangement
products, it is proposed that this mechanism is facilitated by the initial formation
of strong hydrogen-bonding interactions between the phosphate group of the
phosphoamino acid and the side-chain of a protonated arginine residue. This is

consistent with our previous report indicating the role of such intramolecular

interactions in the loss of H3PO4 from protonated phosphoserine and

phosphothreonine containing peptides.206 This proposal is also consistent with

previous reports with a report describing gas-phase phosphorylation within non-

143

covalent complexes of serine or threonine containing peptide cations and

triphosphate anions.221 Moreover, this rationale is in agreement with previously

observed intermolecular gas-phase phosphate group transfer within noncovalent
heterodimeric peptide complexes. In particular, Woods and coworkers showed
that due to the “covalent-like” stability of the phosphate-arginine (and phosphate-
quatemary amine) electrostatic interactions, ClD-MS/MS of such non-covalent
complexes causes removal of the phosphate group from the serine, threonine, or
tyrosine residues by scission of the P-O phosphoester bond, while the phosphate

remains noncovalently bound to arginine or quaternary amines of the formerly

106, 107, 109, 110

nonphosphorylated peptide in the complex. Moreover, Jackson

et al. have shown that the propensity for noncovalent intermolecular gas-phase

phosphate group transfer decreases with increasing charge state.110

 

‘71 H N + H
.N + N .
Ho ————— H I HQ"'H \Nr
Ho-eio ----- H' ‘H ClD-MS/M§ HO'P=0'"H’ H OH
0 OWH'O NH OH 0 0H 0 NH 0
”'JLN N 0 FAN N
H o H o H o H 0

Scheme 3.3 Proposed mechanism for the intramolecular gas-phase phosphate
group transfer facilitated by hydrogen bonding to a protonated basic residue.

Several of the phosphopeptides listed in Table 3.1 were also subjected to
MS/MS analysis via electron transfer dissociation (ETD), following esterification

of the peptides to increase the relative basicity and promote the formation of

144

multiply protonated ions, and performed with or without the aid of supplemental

activation. As expected, no evidence was observed in any of the product ion

spectra for the loss of H3PO4. Furthermore, no evidence was observed for the

presence of products formed via gas-phase rearrangement reactions, thereby
readily allowing the unambiguous determination of the location of the correct site
of phosphorylation in these peptides in each case (data not shown). These
findings therefore clearly support the increased use of electron transfer

dissociation (ETD) (or electron capture dissociation (ECD)). under which the

102, 160, 161, 16

phosphate group remains intact, 4 for large scale or high

throughput phosphoproteome analysis studies.

3.2.2 CID-MS3 of [M+nH-98]'1+ Ions May Not Be Used for Unambiguous

Phosphorylation Site Localization.

The loss of H3PO4 (98 Da) from a phosphoserine or phosphothreonine

residue upon ClD-MS/MS would result in the formation of a dehydrated amino
acid at the formerly phosphorylated residue (Scheme 3.1), while any non-

phosphorylated tyrosine, serine or threonine residues that are present within the

peptide would remain intact. Further M83 dissociation of the [M+nH-98]n+

product ions could therefore be use to obtain evidence to assign the site of

phosphorylation. However, as mentioned in the Introduction section above (3.1),

if the loss of 98 Da occurs via the loss of HP03 from a phosphotyrosine,

145

phosphoserine or phosphothreonine residue combined with the loss of H20 from

a non-phosphorylated serine or threonine residue (Scheme 3.2) an isomeric
product ion containing an unmodified tyrosine, serine or threonine residue at the
formerly phosphorylated amino acid, and a dehydrated amino acid at the formerly

non-phosphorylated amino acid would be produced. Analysis of the product ions

formed by MS3 of this population would therefore result in assignment of the site

of phosphorylation to the incorrect position. Under conditions where gas-phase
phosphate group transfer occurs prior to dissociation, an isomen’c mixture of
protonated precursor ions with respect to the site of phosphorylation may also be
present. For example, the pTSR peptide discussed above was shown to

undergo gas-phase phosphate group rearrangement to yield a mixture of itself

and the phosphoserine isomer, TpSR. Subsequent MS3 of the product ion

corresponding to the neutral loss of H3PO4 from this isomeric mixture would

therefore result in the formation of product ions from which the site of
phosphorylation could be assigned to either the expected (i.e., correct) or

unexpected (i.e., incorrect) phosphorylated residue. Note that the structure of

the product ions formed by the combined losses of HP03 and H20, as described

above, would be indistinguishable from those formed via the loss of H3PO4 from

a precursor ion population that had undergone a gas-phase phosphate group

transfer reaction prior to its dissociation.

146

Definitive evidence for the combined losses of HPO3 and H20 was
obtained here, however, by analysis of the spectra produced by CID-MS3 of the

[M+nH-98]ln+ ions from the singly, doubly and triply protonated precursor ions of

the TpSR peptide from Figure 3.1 (see Figure 3.8A-C, respectivelY). where no

evidence for the gas-phase phosphate group rearrangement was observed upon

MS/MS. In these spectra, a series of product ions (labeled as yn')

corresponding to the presence of an unmodified serine residue rather than the
expected dehydrated residue at the site of the formerly phosphorylated serine

residue, were observed (Figure 3.8). It was also noted that the abundance of the

-80 Da (-HPO3) neutral loss product ion in the MS/MS spectra of these ions were

increased relative to that of the -98 Da neutral loss product with decreasing

activation times in the linear ion trap (Figure 3.5), consistent with the loss of 98

Da being formed, at least to some extent, via the initial loss of HPO3 from the

precursor ion followed by the further loss of H20. Further evidence for the

presence of a population of ions formed via the combined losses in the -98 Da

ClD-MS/MS product ion spectra was also obtained by inspection of the neutral

loss product ions observed in the MS3 spectra of the singly and doubly

protonated TpSR peptide (Figure 3.8A and B, respectively). As mentioned in the

Introduction section above (3.1), a recent study to determine the mechanisms

and other factors that influence the formation and abundance of H3PO4 neutral

147

loss product ions resulted in the observation of characteristic product ions formed

via the losses of 30 Da (CH20) and 44 Da (CH3CHO) upon CID-M83
dissociation of the initial [M+H-98]+ products formed from phosphoserine- and
phosphothreonine-comaining model peptides, respectively.206 The potential

utility of these MS3 aldehyde neutral loss product ions were noted in this prior

study as diagnostic indicators of the site of phosphorylation (i.e, pSer or pThr) in
peptides containing multiple possible sites of phosphorylation. For the TpSR

peptide, therefore, if the ClD-MS/MS neutral loss of 98 Da occurred exclusively

via loss of H3PO4 from the phosphoserine residue, the loss of CH20 should be
observed upon M83, rather than the loss of CH3CHO. It can be seen however,

that an abundant ion corresponding to the loss of CH3CHO (44 Da) was

observed in these spectra, indicating that the loss of 98 Da had occurred via the

combined loss of HPO3 from the phosphoserine residue and the loss of H20

from the threonine residue. This result is consistent with other studies that have

shown that the loss of H20 from serine or threonine residues198 can result in the

formation of product ions with identical structures to those determined for the loss

of H3PO4 from phosphoserine or phosphothreonine residues,117 and can

therefore undergo similar subsequent fragmentation reactions.

148

I." 1.4
- .

..“fl 5
'1‘ -!',

II.

Figure 3.8 lon trap CID—M83 product ion spectra of the (A) singly, (B) doubly,

and (C) triply protonated ([M+nH—98]n+) neutral loss product ions from the TpSR
peptide in Figure 3.1A—C, respectively. Ions labeled with bold text correspond to
the presence of an unmodified serine residue rather than the expected
dehydrated residue. The inset in panel B shows the expanded m/z region from

600-630. ° = -18 Da (—H20); ' = +18 Da (+H20).

149

Figure 3.8 (cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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150

Figure 3.8 (cont’d)

 

 

 

 

 

 

 

 

_ l
(I)
>.
N ........ II
> t
o D
F l
x
‘0
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é?__—
o ......... W
(D
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m .......
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1' Ln ..........
N >.
54-0 ............. _..,
F ooooooo
+ o >__~3 ................ b.
N S ........ Lo.
93 > .o
+ + _
m0 + N00
N NN >5 ............... .
I. > ...................................
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<0
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N—
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(0/0) eouepUHQV GARBIGH

151

300 400 500 600 700 800 900 1 000

200

m/z

Finally, a series of product ions were observed in the M83 spectra from

the TpSR peptide in Figure 3.8 (as well as an analogous product ion series in

Figure 3.9), that could be assigned as being due to the expected presence of a

dehydrated residue at the site of the formerly phosphorylated serine (e.g., y4, y5,
ye, w and ya) as well as for the presence of the unmodified threonine residue

(b3, b5 and b7), following the loss of H3PO4 from the phosphoserine residue.

However, these ions could also be assigned as being due to the loss of water

from an unmodified serine residue and the loss of HPO3 from a

phosphothreonine residue following the combined loss of H20 and HPO3 from a

phosphothreonine containing peptide (i.e., pTSR). Indeed, all of the product ions

observed in the MS3 spectra could be assigned as being formed from either of

the phosphorylated peptide isomers.

152

Figure 3.9 lon trap CID-M83 product ion spectra of the (A) singly, (B) doubly,

and (C) triply protonated ([M+nH—98]n+) neutral loss product ions from the pTSR
peptide in Figure 3.2A—C, respectively. lons labeled with bold text correspond to
the presence of an unmodified threonine residue rather than the expected
dehydrated residue. The inset in panel B shows the expanded m/z region from

600-630. ° = —18 Da (—H20); ' = +18 Da (+H20).

153

Figure 3.9 (cont’d)

oov _.

omo

0N0

com—-

on. ...o; 9...... «.3 3..

omo

ooor

9.

 

 

«00-.

$6303..-

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3.0103310-

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... 3......- _ 3..
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(%) eouepunqv eAueIeu

 

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(%) eouepunqv eAneIeu

 

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154

Figure 3.9 (cont’d)

 

10x

 

 

 

 

 

 

 

 

 

 

 

1001-(3

(%) eouepunqv eAneIea

155

300 400 500 600 700 800 900 1000

200

an

 

Similar results to those described above for the TpSR peptide were also

obtained upon CID-MS3 of the [M+nH-98]n+ product ions from the pTSR peptide

(Figure 3.9), where a5., b5. and b7. ions indicative of the presence of an

unmodified threonine residue rather than the expected dehydrated residue were

observed, as well as the loss of CH20.

Taken together, these data all indicate that without definitively knowing a

priori the extent to which gas-phase phosphate group transfer occurs in a given

phosphopeptide, or the extent to which the combined losses of HP03 and H20

contribute to the loss of 98 Da, the product ions formed by CID-MS3 must all be

considered ambiguous (note the similarity between the product ions, but not the

product ion assignments, in the MS3 spectra of the TpSR peptide in Figure 3.8A

and the pTSR peptide in Figure 3.9A), and therefore may not be used to provide
conclusive evidence to unambiguously assign the site of phosphorylation within

peptides containing multiple potential phosphorylation sites.

3.3 Conclusions

Although tandem mass spectrometry based analysis strategies have been
widely employed for phosphoproteome analysis, the potential for gas-phase
phosphate group transfer rearrangement reactions under typical ion trap CID-
MS/MS conditions can significantly impede the ability to unambiguously assign

the correct site of phosphorylation in protonated phosphopeptides containing

156

 

multiple potential sites of phosphorylation. Furthermore, given that competing

fragmentation reactions corresponding to the loss of H3PO4 or the combined
losses of HPO3 and H20 may be responsible for the neutral loss of 98 Da, the

spectra acquired by CID-MS3 of [M+nH-98]n+ ions may not be used for

unambiguous phosphorylation site localization. Thus, great care should be taken
when exclusively using ion trap ClD-MS/MS methods for phosphorylation site
determination. Indeed, the validity of the protein phosphorylation site

assignments that have previously been reported using ion trap ClD-MS/MS or

MS3 analysis strategies should be re-evaluated prior to making any conclusions

regarding their biological significance. It is recommended that this is achieved by
either (i) taking into consideration the presence of sequences with known kinase
motifs, (ii) by using alternative data acquisition methods such as ETD or ECD, or
(iii) by comparison of the experimentally observed CID-MS/MS spectra with those
obtained from the dissociation of independently synthesized phosphopeptide

standards.

3.4 Other Recent Studies Demonstrating Intramolecular Gas-phase
Phosphate Group Transfer

Since the publication of this work on the intramolecular gas-phase

phosphate group transfer of protonated peptides by CID,222 an analogous

phosphate migration has been reported for deprotonated phosphopeptides by

Lehmann and coworkers (Scheme 3.4).223 In that study, the phosphate group

157

transfer from phosphotyrosine residues to other functional groups such as

hydroxyl and carboxyl followed by H3PO4 neutral loss was investigated.

Although no sequence product ions containing the transferred phosphate were
observed, ClD-MS/MS of differentially labeled peptides suggested phosphate
group transfer from phosphotyrosine to carboxyl groups. In particular,
fragmentation of the deprotonated phosphopeptides studied commonly gave rise

to the neutral loss of 140 Da, which was attributed to the concurrent losses of 98

Da (H3PO4) from the transferred phosphate and 42 Da (HN=C=NH, 42 Da) from

the C-tenninal arginine residue by the mechanism shown in Scheme 3.4.223

Note that this proposed mechanism involves the removal of one of the oxygen

atoms from the C-terminal carboxyl as part of the neutral loss of H3PO4.
Therefore, upon CID-MS/MS of an 180 C-terrninal carboxyl labeled peptide, the

neutral loss would be expected to be 142 Da or H3P1603180 (100 Da) and

HN=C=NH (42 Da). In fact, the neutral loss of 142 Da was observed for a

deprotonated phosphotyrosine-containing peptide that was 180 labeled on the C-

temninal carboxyl upon ClD-MS/MS, providing confirmation of the migration of the

phosphate group.223

158

 

 

 

 

OH
_ |+/-\. NH —
°Ei"°“ 0 ca - 7
° )4 ”3 0 A n
O NH HO. 1,0 H’VVNH
CID-MS/MS ’P\O
P > HO L ,NH
hosphate CH
0 CH2 Transfer 0 CH2 0&( 23’
H'JL N NH
N
H 0 H O
L— _
- H3PO4(98 Da)

- HN=C=NH (42 Da)

HN
0 CH2 0
A WW

N

H 0
Scheme 3.4 Proposed mechanism for intramolecular phosphate group transfer
for deprotonated peptide ions.
3.5 Additional Commentary

Certainly the results reported in this chapter, which demonstrate that

intramolecular gas-phase phosphate group transfer occurs, imposes limitations
on the effective use of ClD-MS/MS spectra for accurate and unambiguous
assignment of phosphorylation sites in ion trap instruments. However, these
results do not imply that CID is not capable of accurate phosphorylation site
assignments under these conditions. Moreover, these results do not imply that
CID should be replaced by alternative dissociation methods such as electron

capture dissociation (ECD) and electron transfer dissociation (ETD), which

159

 

maintain the phosphorylation site. In fact, the information derived from CID and
ECD/ETD spectra may largely overlap, despite the complications originating from
the Iability of the phosphate group and the potential for phosphate group transfer
by CID. This is because multiply protonated ions have a lower propensity to

undergo phosphate group neutral losses and intramolecular phosphate group

87, 89, 111, 206, 222

transfer upon CID, and ECD/ETD are only amenable to the

analysis of multiply protonated ions. In brief, ECD/ETD may result in redundant
information to that achieved by CID in the analysis of multiply protonated
peptides. In light of this discussion, however, it is clear that the analysis of singly
protonated peptide ions and peptide ions with low proton mobility by these
established mechanisms are limited and the results from such analyses may
require validation using alternative dissociation methods of those ions (e.g., using
ultraviolet photodissociation, femtosecond laser-induced ionization/dissociation,
metastable atom-activated dissociation; discussed in detail Chapter One), the
consideration of those suggestions listed in the Conclusions section of Chapter

Four, or previously published data on related multiply protonated species.

160

 

CHAPTER FOUR

Experimental Methods for Gas-Phase Fragmentation Studies of

Phosphopeptides (Chapters Two and Three)

4.1 Materials

All aqueous solutions were prepared using deionized water purified by a
Bamstead nanopure diamond purification system (Dubuque, IA). Sequenal
grade trifluoroacetic acid (TFA) was purchased from Pierce (Rockford, IL).
Peptides were prepared using manual stepwise Fmoc-based solid-phase peptide
synthesis (SPPS) on Wang resins pre-loaded with N-q-Fmoc protected amino
acids (100-200 mesh) (EMD Biosciences, San Diego, CA). N-a-Fmoc protected
acid building blocks with acid-labile orthogonal side-chain protection, where
appropriate, were purchased from EMD Biosciences (San Diego, CA) and are
listed in Table 4.1. Boc-glycine-OH and dibenzyI-N,N-diisopropylphosphoamidite
were also purchased from EMD Biosciences. Anhydrous dimethylforrnamide
(DMF) was purchased from Spectrum Chemicals (Gardena, CA) and further
dried with a 4 A molecular sieve. Reagent grade piperidine, O-(Benzotriazol-1-
yI)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU), 1-
hydroxybenzotriazole hydrate (HOBt), diisopropylethylamine (DIPEA), ninhydrin,

phenol, potassium cyanide, pyridine, ethanedithiol (EDT), triisopropylsilane (TIS),

and monodeuterated acetic acid (CH3COzD) were purchased from Sigma Aldrich

(St. Louis, MO). Deuterium oxide (D20) and deuterated methanol (CD30D) were

161

obtained from Cambridge Isotope Laboratories (Andover, MA). D,L-Serine-2,3,3-
d3 (99.4 atom % D) was purchased from CDN Isotopes Inc. (Quebec, Canada).
Recrystallized 2,5-dihydroxybenzoic acid (2,5-DHB) was purchased from Laser
BioLabs (SOphia-Antipolis Cedex, France). HPLC grade methanol (MeOH) was
purchased from Mallinckrodt (Hazelwood, MO) and HPLC grade acetonitrile
(ACN) was purchased from EMD (Gibbstown, NJ). All other reagents were

commercially available and used without further purification.

162

 

 

Table 4.1 Common amino acid building blocks used for solid-phase peptide

 

 

 

 

synthesis.
Abbreviation Solid Phase Peptide Synthesis

Amino Acid 3 Letter 1 Letter Building Block

Alanine Ala A Fmoc-AIa-OH

Arginine Arg R Fmoc-Arg(Pmc)-OH
Asparagine Asn N Fmoc-Asn(Trt)-OH

Aspartic Acid Asp D Fmoc-Asp(OtBu)-OH

Cysteine Cys C Fmoc-Cys(Trt)—OH

Glutamic Acid Glu E Fmoc-Glu(OtBu)—OH
Glutamine Gln Q Fmoc-Gln(Trt)-OH

Glycine Gly G Fmoc-Gly-OH

Histidine His H Fmoc-His(Trt)-OH

Isoleucine lle | Fmoc-IIe-OH

Leucine Leu L Fmoc-Leu-OH

Lysine Lys K Fmoc-Lys(Boc)—OH
Methionine Met M Fmoc-Met-OH

Phenylalanine Phe F Fmoc-Phe—OH

Phosphoserine pSer pS Fmoc-Ser(PO(Obzl)OH)—OH
Phosphothreonine pThr pT Fmoc-Thr(PO(Obzl)OH)-OH
Phosphotyrosine pTyr pY Fmoc-Tyr(P03H2)-OH

Proline Pro P Fmoc-Pro-OH

Serine Ser 8 Fmoc-Ser(tBu)-OH

Threonine Thr T Fmoc-Thr(tBu)-OH
Tryptophan Trp W Fmoc-Trp(Boc)«OH

Tyrosine Tyr Y Fmoc-Tyr(tBu)-OH

Valine Val V Fmoc-VaI-OH
4.2 Phosphopeptides

The phosphoserine- and phosphothreonine—containing peptides

GAILpSGAILK, GAILpTGAILK. GAILpSGAILR, GAILpTGAILR.

LFTGHPEpSLEK, LFTGHPEpTLEK, LRRApSLG, LRRApTLG, LFpTGHPESLER

 

(pTSR), and LFTGHPEpSLER (TpSR) were prepared by manual stepwise Fmoc-
based solid-phase peptide synthesis, as described in detail below. Detailed
experimental methods for the synthesis of the regioselectively deuterated peptide
GAIL(d3-D,L)pSGAILK also follow.

All other phosphopeptides mentioned in Chapter 3 were synthesized by

Sigma-Genosys (The Woodlands, TX) and used without further purification.

4.2.1 Fmoc Solid-Phase Peptide Synthesis (SPPS)
4.2.1.1 Apparatuses

Manual solid-phase peptide synthesis was completed in disposable 5 mL
Toriq syringes with frits (Fisher Scientific, Pittsburgh, PA). Multiple peptides were
synthesized simultaneously using Kontes three-way stopcocks with HDPE plugs
(VWR, West Chester, PA) on a Vac-Man laboratory vacuum manifold (Promega,
Madison, WI). All steps requiring continuous mixing were completed using a

Labquake tube rotator (Thenno Fisher Scientific Bamstead, San Diego, CA).

4.2.1.2 Synthesis

Generally, peptide synthesis was completed on a 0.05 mmol scale. The
N-d-Fmoc protected C-terminal residue bound to a Wang-resin was swelled in
DMF (5 mL) for 15 min with continuous mixing and washed with 2 x 5 mL DMF.
C-terminal elongation of the peptide was completed via repetitive Fmoc

deprotection and amino acid coupling until the target peptide sequence was

164

achieved. Cleavage of the peptide from the resin and acid-labile orthogonal

protecting groups followed.

4.2.1.2.1 Fmoc Deprotection

Deprotection of the resin-bound peptide N-terminal Fmoc group to form a
free amine N-terminus was achieved by washing with 3 mL 30% piperidine in
DMF followed by continuous mixing with 3 mL 30% piperidine in DMF for 25 min.
The peptidyl resin was then washed with DMF until pH < 8 was achieved

(approximately 8 x 5 mL).

4.2.1.2.2 Amino Acid Coupling

N-d-Fmoc amino acids (0.25 mmol) were pre-activated by thorough mixing
with DIPEA (65 pl) and a DMF solution consisting of 0.5 M TBTU, 0.5 M HOBt
(0.5 mL). To increase coupling efficiencies, monobenzyl-protected N-d-Fmoc-
phosphoamino acids were pre-activated using a 3—fold excess of DIPEA (195 pl).
Coupling of the pre-activated building blocks to the peptidyl resin was completed
by continuous mixing for 15 min. The peptidyl resin was then washed 5 x 5 mL
DMF.

Directly following coupling and washing, the Kaiser test was completed to
determine the coupling efficiency. This color test determines the presence of
primary amines, such as the N-terminus of a peptide, by reaction with ninhydrin.

A dark blue color is generated upon reaction of ninhydrin with a primary amine

. . . . . 224 .
and a yellow color IS maintained If no reaction occurs. Therefore, here It was

165

used to indicate the completeness of the coupling of an N-d-Fmoc amino acid to
the free amine N—tenninus of the peptidyl resin. Briefly, approximately 2 mg of
peptidyl resin was placed in a small glass test tube and washed with 2 x 0.5 mL
ethanol. Equal volumes (125 pL) of 0.28 M ninhydrin in ethanol, 42.5 M phenol
in ethanol and 20 (M potassium cyanide in water/pyridine (2 mL 0.001 M KCN
aq. in 100 mL pyridine) were added to the washed resin and mixed thoroughly.
After incubation at 100 °C for 10 min, the color was visually inspected. Upon the
formation of any blue resin, the same N-d-Fmoc amino acid was re-coupled to

the resin, using the coupling procedure described above.

4.2.1.2.3 Resin Cleavage and Deprotection of Acid-Labile Orthogonal
Protecting Groups

Upon completion of the target peptide sequence, a final Fmoc
deprotection was done. The peptidyl resin was dried by washing with 5 x 5 mL
ethanol. Then the peptide was cleaved from the resin and any acid-labile
orthogonal side chain protecting groups simultaneously by continuous mixing
with a cleavage solution (5 mL) for 3 hr. The composition of the cleavage
solution was dependent upon the composition of the peptidyl resin. A flow chart
for its determination is summarized in Figure 4.1. Product peptides were
precipitated in cold diethyl ether (20 mL), filtered, reconstituted in a minimum
volume of 25% acetic acid (aq) solution and Iyophilized. Lyophilized crude

peptides were stored at room temperature.

166

 

 

 

Does the peptide ® Does the peptide
contain Arg(Mtr)or contain Cys(Trt) or

Met?

unprotected Trp?

 

 

 

 

 

 

 

 

Does the peptide Cleave with:
contain > 2 95% TFA, 2.5% H20,
Arg(Mtr)? 2.5% TIS

 

 

 

 

Cleave with:

94.5% TFA, 2.5% H20, ‘—
V 0 0
Cleave with: 2.5 /o EDT, 1 /o TIS
1 M TMSBr/thioanisole in

TFA with m-cresol/EDT

 

 

 

 

 

 

 

Cleave with:
81.5% TFA, 2.5% EDT,
5% thioanisole, 5%
phenol, 5% H20, 1% TIS

 

 

 

 

figure 4.1 Flowchart to determine cleavage solution composition. Adapted from

4.2.2 Synthesis of GAIL(d3-D,L)pSGAILK

The regioselectively deuterated peptide GAIL(d3-D,L)pSGAILK was
prepared by the synthesis and incorporation of (d3-D,L)-Fmoc-Ser(O-tert-
butyldimethylsilyl (TBDMS))-OH into the protected peptide Boc-GAIL(d3-
D,L)S(O-TBDMS)GAILK(Boc). On-resin deprotection of the serine side chain
and functionalization of the resin bound hydroxyl group using dibenzyl-N,N-
diisopropylphosphoramidite followed. Oxidation, peptide cleavage, and
extraction from the resin afforded the product phosphopeptide. Detailed

experimental methods for this procedure are below.

167

4.2.2.1 Synthesis of (d3-D,L)-Fmoc-Serine-OH (4.a)
A solution of (d3-D,L)-serine (150 mg, 1.39 mmol) in 10% Na2CO3 (aq)

(7.5 mL) and 1,4-dioxane (3.75 mL) was treated dropwise with 9-fluorenylmethyl
chlorofon'nate (395 mg, 1.53 mmol) in 1,4-dioxane (7.5 mL) at 0 °C. After 1 hr at
0 °C, the mixture was warmed to room temperature, and then stirred overnight.
The resulting mixture was diluted with cold water (15 mL) and extracted with
ether (2 x 20 mL). The aqueous layer was acidified with 5% HCI (aq) solution

followed by extraction with ethyl acetate (2 x 20 mL). The combined organic

phases were dried with MgSO4, filtered, and concentrated in vacuo resulting in

an off-white solid (388 mg, 85%). The resulting Fmoc-protected amino acid 4.a
was used without further purification. 1H NMR (300 MHz), (CD3)2CO: 5 1.29 (s,
1H), 4.25 (t, J = 7.1 Hz, 1H), 4.30-4.39 (m, 2H), 6.57 (s, 1H), 7.32 (ddd, J = 7.5,
1.1 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.74 (dd, J = 7.3, 4.0 Hz, 2H), 7.85 (d, J =
7.4 Hz, 2H), 11.01 (s, 1H); 130 NMR (75 MHz), (003)2co: 6 47.93, 56.79,

62.31, 67.32, 120.74, 126.13, 126.16, 127.89, 127.91, 128.48, 142.04, 144.97,

145.04, 157.02, 172.21. Chemical shifts are reported relative to the residue

peaks of the solvent (CD3)2CO (2.05 ppm for 1H and 29.84 ppm for 13C).

4.2.2.2 Synthesis of (d3-D,L)-Fmoc-Serine(O-TBDMS)-OH (4.b)
A solution of 4.a (250 mg, 0.76 mmol) in 2.5 mL dry DMF was treated with

ted-butyldimethylsilyl chloride (148 mg, 0.98 mmol) and imidazole (2.27 mmol) at

0 °C while stirring under N2 (9) atmosphere. After 1 hr at 0 °C, the solution was

168

 

warmed to room temperature and then stirred overnight. The solution was
diluted with 1 N HCI (aq) (5 mL) and extracted with ether (2 x 8 mL). The
combined organic layers were concentrated in vacuo and the resulting residue

was partitioned between 10% LiBr (aq) solution (5 mL) and ether (2 x 8 mL). The

combined organics were dried with MgSO4, filtered, and concentrated in vacuo.

The silyl protected product 4.b (210 mg, 62%) was recovered as a yellow oil after
silica gel chromatography (EM Science Silica Gel 60 (230-400 mesh)). An initial
mobile phase composition of 5% ethyl acetate, 95% hexanes was used to elute

impurities followed by elution of the product with 80% ethyl acetate, 20%

hexanes. 1H NMR (300 MHZ), (CD3)2CO: 6 0.08 (s, 6H), 0.90 (s, 9H), 4.25 (t, J

= 7.0 Hz, 1H), 4.34 (d, J = 7.0 Hz, 2H), 6.45 (s, 1H), 7.32 (t, J = 7.3 Hz, 2H), 7.41

(t, J = 7.4 Hz, 2H), 7.72 (t, J = 7.1 Hz, 2H), 7.85 (d, J = 7.4 Hz, 2H), 11.03 (s,
1H); 13c NMR (75 MHz), (003)2co: 6 -5.34, -5.27, 18.83, 26.18, 47.94, 56.46,

63.54, 67.39, 120.79, 126.12, 126.14, 127.92, 128.52, 142.08, 144.94,

145.07,156.80, 171.90.

4.2.2.3 On-resin SPPS of Boc-GAIL(d3-D,L)pSGAILK(Boc) (4.c)

The side chain protected peptide Boc-GAIL(d3-D,L)S(O-
TBDMS)GAILK(Boc) was synthesized as described above in section 4.2.1 using
4.b for serine incorporation and Boc-eg-OH as the N-terminal amino acid. The
peptidyl resin (0.05 mmol) was dried by washing with ethanol (2 x 5 mL) and

dichloromethane (2 x 5 mL). Selective deprotection of the serine silyl protecing

169

group was completed by incubation of the dried peptidyl resin with a solution of

tetrabutylammonium fluoride trihydrate (TBAF-3H20, 31.6 mg, 0.10 mmol) in

dimethylacetamide (DMA) (2 mL) while rotating overnight. The peptidyl resin
was washed with DMA (3 x 5 mL) and dichloromethane (3 x 5 mL) and dried
overnight in vacuo. A 0.45 M tetrazole solution in acetonitrile (8.9 mL, 4.0 mmol)
was mixed with dry DMF (5 mL) and dibenzyI-N,N-diisopropylphosphoramidite

(1.0 g, 2.9 mmol). The solution was then added to the peptidyl resin and the

mixture was stirred for 2.5 hr at room temperature under a N2 (9) atmosphere.

The resin was washed with DMF followed by oxidation with 6 M t—butyl
hydroperoxide (0.36 mL, 2 mmol) for 0.5 hr, resulting in 4.c. The resin was then
washed with DMF and cleaved and extracted as described in section 4.2.1.2.3

affording GAIL(d3-D,L)pSGAILK.

4.2.3 Peptide Purification

The product peptides GAILpSGAILK, GAILpTGAILK, GAILpSGAILR,
GAILpTGAILR. LFTGHPEpSLEK, LFTGHPEpTLEK, LRRApSLG, LRRApTLG,
and GAIL(d3-D,L)pSGAILK were purified by reversed phase-high performance
liquid chromatography (RP-HPLC) using an Aquapore RP-300 column (4.6 mm;
Perkin Elmer, Wellesley, MA). Peptides were eluted at 1 mL/min with monitoring
at 215 nm using a linear gradient elution from 0-100% B. Solvent A was 0.1%
TFA in water and solvent B was 0.089% TFA, 60% acetonitrile in water.

The product peptides LFpTGHPESLER (pTSR), and LFTGHPEpSLER

(TpSR) were purified by RP-HPLC (Shimadzu, model LC-20AB, Columbia, MD)

170

using a Vydac Protein and Peptide C18 column (150 mm x 10 mm) (Grace,
Hesperia, CA). Peptides were eluted at 5 mL/min with monitoring at 215 nm
using the following gradient elution conditions: 045 min, 0-25% B; 15-35 min, 25-
35% B. Solvent A was 0.1% TFA in water and solvent B was 0.089% TFA, 60%
acetonitrile in water. This method afforded separation of LFpTGHPESLER and
LFTGHPEpSLER and their retention times were 22.8 min and 20.1 min,

respectively.

4.3 Multistage Tandem Mass Spectrometry Analysis of Phosphopeptides
4.3.1 Phosphopeptide Sample Preparation

All peptide samples were prepared in 50% methanol, 49% water, 1%
acetic acid (v/v/v) for subsequent mass spectrometry analyses and stored at -20

°C. For H/D exchange experiments, peptides were prepared in 50% deuterated

methanol (CD3OD), 49% deuterated water (D20), and 1% deuterated acetic acid

(CH3002D) (v/v/v), Iyophilized, and then redissolved in the same deuterated

solvent system to maximize exchange. Mass spectrometry analysis of the

deuterated samples immediately followed sample preparation, purging of the

source with N2 (9), and equilibration of the sample injection lines and syringe

with deuterated methanol. For matrix-assisted laser desorption/ionization
(MALDI) analysis, 5 pmol of each sample was mixed on-plate with 0.25 pL of a

2,5-DHB matrix solution (10 mg/mL in 50% acetonitrile, 50% water (v/v)).

171

Samples subjected to triple quadrupole mass spectrometry analysis were

dissolved in 50% methanol, 50% water (v/v).

4.3.2 Mass Spectrometry

Mass spectrometry analysis was performed using (i) an LCQ DECA 3D
quadrupole ion trap mass spectrometer or (ii) an LTQ linear quadrupole ion trap
mass spectrometer, each equipped with electrospray (ESI) or nano electrospray
ionization (nESI) sources, (iii) an LTQ XL linear quadrupole ion trap mass
spectrometer equipped with a vMALDl source, or (iv) a TSQ Quantum Ultra triple
quadrupole mass spectrometer equipped with a chip-based nESI source (Advion
NanoMate, Ithaca, NY). All mass spectrometers used were manufactured by
Therrno Fisher Scientific (San Jose, CA).

Samples subjected to ion trap ESI analysis (100 11M) (Chapter Two) were
introduced by infusion at a flow rate of 5 pL/min. The spray voltage was
maintained at 4.5 kV. Nitrogen sheath gas and auxiliary gas (H/D exchange
only) were supplied at 25 (arbitrary unit) and 10 (arbitrary unit), respectively. The
heated capillary temperature was set to 180 °C.

Samples subjected to ion trap nESI analysis (20 pM for purified peptides
and 50 pM for the crude peptides) (Chapter Three) were introduced by infusion at
a flow rate of 0.5 pL/min. GAIL(d3-D,L)pSGAlLK (Chapter Two), however, was
infused at a flow rate of 1.0 pL/min. The spray voltage was maintained at 2.1 kV

and the heated capillary temperature was set at 200 °C.

172

' r "."-".!'_'i

 

Ion trap collision-induced dissociation (ClD)—MS/MS and -MS3

experiments were performed on mass selected precursor ions. Typical isolation
widths were 5.0 m/z for singly protonated ions, 3.5 m/z for doubly protonated ions

and 1.5 m/z for triply or higher protonated charge states. The automatic gain

control (AGC) was set to 3.0 x 104 for full MS and 1.0 x 104 for MS". The

activation time was maintained at 30 msec using an activation q value of 0.25,
unless otherwise stated.

For triple quadrupole mass spectrometry analysis, 13 uL of sample (20
pM) was loaded into a Whatman Multichem 96-well plate (Fisher Scientific,
Pittsburgh, PA) and sealed with Teflon Ultra-Thin Sealing Tape (Analytical Sales
and Services, Pompton Plains, NJ). Infusion mode operation was achieved
using an ESI HD-A chip, a spray voltage of 1.4 W, a gas pressure of 0.3 psi and
an air gap of 2 11L. The ion transfer tube of the mass spectrometer was

maintained at 150 °C. MS and MS/MS spectra were acquired using scan rates of

500 mlz-second'1, while maintaining Q1 and QS peak widths at 1.0 Th. The Q2

argon collision gas pressure was set at 0.7 mTorr.

All MS/MS and MS3 experiments were performed by collision induced

dissociation (CID) on mass selected ions using standard isolation and excitation
procedures. The spectra shown were typically the average of 20—200 scans (3
microscans / scan) and were collected in profile mode. Repeated analysis of
individual samples resulted in less than 5% variation in relative product ion

abundances.

173

4.3 Quantitative Analysis of Percent Product Ion Abundances

Quantitative analysis of non-sequence percent product ion abundances
was performed by initially filtering the spectra to remove any ions present below
the empirically determined level of chemical noise (5-200 counts). Then, the %

total product ion abundances were determined by expressing the summed

abundances of product ions formed by the loss of H3PO4 (98 Da) or HPO3 and

H20 (98 Da), CH20 (30 Da), CD20 (32 Da), or CH3CHO (44 Da) as a fraction of

the total product ion abundance.

174

 

CHAPTER FIVE

Application of Phosphoproteomic Analysis Strategy to Elucidate p65-
Associated Phosphoproteins in the NF-KB Signaling Pathway From Treated

and Untreated THP-1 Cell Nuclear Extracts

5.1 Introduction

Since its first discovery in 1986,226 and more than 30,000 scientific

publications later,227 the protein nuclear factor (NF)—KB has proven central in the

coordination of innate and adaptive immunity and inflammatory responses in
nearly all cell types. NF-KB exerts its activity by functioning as a transcription
factor, regulating over 150 genes that are involved in the development of immune

cell function, promotion of inflammatory mediators, inhibition of apoptosis, and

regulation of cellular growth.(rew'med m 228-230) These genes include those that

produce pro-inflammatory cytokines and chemokines (e.g., lL-6, TNF-a, and IL-

8)231'234, cell adhesion molecules (e.g., lCAM-1)235, growth factors (e.g., FGF-

236

8) : and apoptosis regulators (e.g., Bcl-2 and caspase11)237' 238

. Due to its

biological importance, it is not surprising that aberrant regulation of NF-KB and
the associated biological pathways that control its activity have been implicated

in many different diseases including arthritis, asthma, diabetes, and

AIDSWViewed in 239’ 240) In addition, the NF-KB pathway has been implicated in
a variety of processes leading to the development and progression of cancer,

175

including tumor proliferation, prevention of apoptosis, metastasis, and

. . ' d' 231, 241-24 .
angrogenesrs.(remewe m 3) For these reasons, the mechanisms by

which NF-KB exerts its influence, specifically pertaining to its activation and

modulation of its transcriptional activity, have been of great interest for the

- . 44- 4
treatment and prevention of cancer and other diseases.2 2 6

NF-kB consists of different homo— and hetero-dimers comprised of five
proteins: NF-kB1 (p50 and its precursor p105), NF-KB2 (p52 and its precursor
p100), RelA (p65), RelB, and c-Rel. Each of these proteins has an N-terminal
Rel homology domain (RHD), which contains a nuclear localization signal (NLS)

and is involved in dimerization, sequence-specific DNA binding, and interaction

(structures reviewed in 247)

with inhibitory proteins. In addition to the RHD, p65,

RelB, and c-Rel each contain non-homologous C-terminal transactivation

domains, which are responsible for the recruitment of co-activators and basal

(structures reviewed in 247)

transcriptional machinery. In mammalian cells, the

p50/p65 heterodimer is ubiquitous and is largely responsible for regulating

inflammation as well as the life cycle of lymphoid cells during the immune

response.248 For these reasons, the remainder of this introduction and the

research described in Chapter Five will focus on the p50/p65 isoform, specifically
highlighting the p65 subunit. (From this point on the discussion of “NF-KB” will
refer to the p50/p65 isoforrn.)

In unstimulated cells, NF-KB is negatively regulated in the cytoplasm by its

interactions with inhibitory (I) KB proteins (illustrated in Figure 5.1). Activation of

176

the NF-KB pathway may be initiated by a host of cellular stimuli, including pro-

inflammatory cytokines, such as tumor necrosis factor (TNF)-d (as shown in

Figure 5.1)249; microbial components, such as Iipopolysaccharide (LPS)250; and

' . 2 1 . . ' d' 252
cytotoxrc agents, such as chemotherapeutics, 5 ox1dative stress,(rev'ewe '" )

and UV light.253 In response to such inducers, the upstream IKB kinase (IKK)

complex is activated by phosphorylation, which in turn phosphorylates IKB,
subsequently targeting it for ubiquitination and proteasomal degradation (Figure
5.1). This liberates the p50/p65 dimer, allowing nuclear translocation and
transcriptional regulation of target genes, resulting in variety of cellular

responses.

177

 

Extracellular matrix

mm mm writ? :ff “ ‘ j"

 

Cytoplasm

i_

Serine

‘ kinases

IKK kinases
%® L» 8%"
Inhibited NF-KB
J‘—

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6 9°
.36

_ f—“K IKB degradation

     
 

Gene
transcription

Cell survival
> Inflammation
Immunity

 

\
\
\
\
F \
|
I

Figure 5.1 The NF-KB pathway. Adapted from 254.

178

Given the host of cellular stimuli that may induce the NF-KB pathway and
the variety of NF-kB gene targets, many regulatory mechanisms are likely
involved that relate a specific stimulus to a specific NF-KB-induced cellular
response (i.e., NF-KB gene specificity and the resulting translational profile) in a
specific cell line. Regulatory mechanisms of the NF-KB pathway may be divided
into two distinct parts: (i) those involving translocation of NF-KB into the nucleus
and (ii) those involving mediation/promotion of transcriptional activity. It is
generally accepted that regulation of NF-KB nuclear transport, regardless of
stimulus, originates from activation of the IKK complex by phosphorylation, which

subsequently phosphorylates IkB, leading to dissociation of the IKB-NF-KB

complex in the cytosol (as shown in Figure 5.1).(mwev‘led in 227’ 255) Although it

is known that phosphorylation is the key regulatory mechanism for IKK activation

(reviewed in 255)

and subsequent NF-KB nuclear localization, the kinases that are

directly responsible are largely unknown. Additionally, the mechanisms that
ultimately converge to enable phosphorylation/activation of IKK by such diverse
cellular stimuli also remains to be answered.

Numerous studies have demonstrated that NF-KB transcriptional activity,
including DNA binding, gene specificity, and resulting translational profile, is

complex, and depends on phosphorylation of NF-KB and involves the interactions

KB (reviewed in 255-258;259)

of numerous proteins with NF- In fact, for the p65

subunit alone, ten phosphorylation sites have been elucidated (summarized in

Figure 5.2, including the responsible kinases and phosphatases involved), many

179

of which mediate NF-kB transcriptional activity.(reVIeWGd m 256’ 257) For example,

phosphorylation of p65 at Ser536 has been shown to enhance transcription of

interleukin-8 (IL-8), which is responsible for production of pro-inflammatory

chemokines.260 Another example includes phosphorylation of Ser468, which,

interestingly, has been described to both enhance and suppress p65

transcriptional activity under different conditions.261'263 The differential function

of Ser468 phosphorylation may arise from a difference in phosphorylation status
of other p65 sites (e.g., phosphorylation of both Ser468 and Ser536 under one
condition and phosphorylation of only Ser468 in another condition). Or, perhaps,
the difference in p65 activity despite constant phosphorylation of Ser468
corresponds to a difference in the transcriptional co-activators or other p65-
interacting proteins involved in p65—transcriptional regulation. In fact, greater

than 130 proteins have shown to directly interact with the p65 subunit alone and

have demonstrated involvement in NF-KB-mediated transcriptional activity.257

For example, increased lL-4 gene expression, has been directly linked to the

interaction between p65 and STAT6 (signal-transducer and activator of

transcription 6) in cells stimulated with lL-4.264 Another example includes

increased NF-kB transcriptional activity as a result of the nuclear association

between p65 and the protein UXT (ubiquitously expressed transcript) in

comparison with systems that have reduced UXT expression.265

180

[IKKa]

[IKKB]
[lKKs]

[PKAc] [NAK]
[MSK1/2] [2] [Chk2][RSK1]
S276 8281 3529 S536

['KKB]
[?l [?l w lPKCfJ [Fig] [$515313] [Chk1] w
5205 T254 S311 T435 S468 T505
9 9 \/ 9 9 9 9 \/
| | RHD I l TAD I
1 19 301 428 551

 

Figure 5.2 Summary of the known phosphorylation sites of p65 and the
corresponding [kinases] and [phosphatases*]. The kinases responsible for
Ser205, T254, and Thr435 phosphorylation are25a6t Egg/sent unknown, indicated

with [?]. This information was compiled from ; and adapted from 2 6
RHD is the Rel homology domain and TAD is the transactivation domain

Given that phosphorylation of p65 is highly involved in the regulation NF-
KB transcriptional activity, it is expected that the phosphorylation status of p65
influences the recruitment of p65 transcriptional co-activators and other NF-KB
regulatory proteins. Additionally, the post-translational modifications of the p65-
interacting proteins may also play a role in p65-interactions and subsequent
regulation of p65-mediated transcriptional activity. In fact, a recent study has

shown that tyrosine phosphorylation of STAT6 is required for both p65-binding to

STAT6 and the resulting p65-STAT6-mediated IL-4 gene expression.264 Despite

the significance of this result and the results of other individual studies described
above that associate a specific phosphorylation event or a specific NF-KB-
interacting protein with particular NF-KB cellular responses, many of these
studies may not be comparable or may not be generally applicable. This is

because some of these studies have involved overexpression of either p65, the
181

interacting protein, or both and, therefore, may not be truly representative of
“real” systems. Moreover, each study varies in the stimulating agents,
concentrations of the stimulating agent, incubation times, and cell lines used,
resulting in different phosphorylation events and different p65 interactions with
different proteins. Accordingly, a true representation of the NF-KB regulatory
mechanisms and the relationship between stimulus and NF-KB activity would
require global examination of the stimulus-specific interacting proteins, their post-
translational modifications, and the stimulus-specific NF-KB transcriptional
response. Such a global study would involve a variety of stimuli, treatment
regimens, and cell types. Additionally, the analysis technique used in such a
study should not be limited by the post-translational modifications that may be
examined and should be able to unambiguously and accurately localize those
modifications. To date, however, such a comprehensive study that would
elucidate context-specific regulatory mechanisms and NF-KB activity has yet to
be completed.

To aid in this achievement, here, a phosphoproteome analytical approach
was developed and applied to determine the p65-associated phosphoproteins
from the nuclear fractions of untreated and TNF-d-treated THP-1 acute
monocytic leukemia cells. A schematic of the analytical strategy employed is
shown in Figure 5.3. Briefly, p65-associated proteins were co-
immunoprecipitated with nuclear p65 and the resulting protein mixture was
proteolytically digested with trypsin. The phosphorylated peptides were then

enriched using a titanium-based method followed by collision-induced

182

 

dissociation tandem mass spectrometry analysis. The resultant product ion
spectra were interpreted by database searching and manual analysis for protein
identification and phosphorylation site localization. Importantly, for
phosphorylation site assignment of the identified phosphorylated peptides, the
effects of gas-phase ion chemistry of phosphorylated peptides (as discussed in
Chapters Two and Three) on the formation of analytically relevant information
was considered during the downstream data interpretation. Using this process,
five p65-associated proteins were identified, two of which have not been

previously reported to interact with p65.

183

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Figure 5.3 Schematic of the phosphoproteome analytical strategy employed for
identification and phosphorylation site determination of p65-interacting proteins

from the nuclear fractions of untreated and TNF-d-treated THP-1 cells.

184

5.2 Results and Discussion
5.2.1 Development of the Phosphoproteome Analysis Strategy Employed
for Identification of p65-Associated Phosphoproteins From

Differentially Treated THP-1 Cells

To determine the identity and phosphorylation sites of the p65-associated
phosphoproteins from the nuclear fractions of TNF-d-treated and untreated THP-
1 cells, immunoprecipitation of these samples was completed using a p65
polyclonal antibody (p65 C-terminal epitope) that had been covalently cross-
linked to agarose beads. The co-immunoprecipitated, p65-associated proteins
were then analyzed using the phosphoproteome approach described in detail

below.

5.2.1.1 Proteolytic DigestiOn

The immunoprecipitated proteins were digested directly on the
immunoprecipitation resin, using trypsin. On-resin digestion was used here
because it enables decreased risk of sample loss as compared to in-gel digestion
or in-solution digestion post protein elution, which may incur losses from
inefficient peptide extraction from the gel or inefficient protein elution,
respectively. Additionally, utilizing the covalently cross-linked IP resin decreases
the presence of extraneous p65 antibody peptides, by decreasing the number of
tryptic sites available through modification of the lysine residues on the p65

anfibody.

185

 

5.2.1.2 Phosphopeptide Enrichment

Phosphopeptide enrichment was completed by Mr. Weihan Wang
(Professor Merlin Bruening’s group, Department of Chemistry, Michigan State
University) on a matrix-assisted laser desorption/ionization (MALDI) sample
plate, utilizing a titanium-based metal-oxide affinity chromatography (MOAC)
method developed by Mr. Weihan Wang and Professor Merlin Bruening. On-

plate phosphopeptide enrichment methods have exhibited high recovery of

phosphopeptides (70%) from mixtures containing a few proteins.(reViewed in 50)

Additionally, these on-plate methods enable decreased sample handling and,

therefore, decreased sample loss, and are convenient for samples with small

volumes (“Lionewewe‘d in 50) As a result, on-plate phosphopeptide enrichment

strategies are optimal for immunoprecipitation samples and hence were used

here.

5.2.1.3 Mass Spectrometry Analysis and Database Searching

MALDI-MS and ClD-MS/MS analyses of the enriched phosphopeptides
were performed using a linear ion trap mass spectrometer. MALDI-MS allows for
decreased complexity in the MS spectrum because only singly protonated ions
are formed, as opposed to electrospray ionization (ESI), where multiple
protonated charge states for each peptide would be present. Also, MALDI is
compatible with the upstream on-plate enrichment strategy.

The work described in Chapter Two demonstrate that the precursor ions

targeted here (singly protonated phosphorylated tryptic peptides i.e., precursor

186

ions with low proton mobility) under ClD—MS/MS conditions acquired on the
MALDI-ion trap instrument platform promote predominant formation of the
nonsequence product ion resulting from the neutral loss of 98 Da. As a result,
these nonsequence product ions were used for this work as diagnostic markers
for the presence of phosphorylated peptides, enabling targeted mass
spectrometry analyses. However, as discussed in Chapter Two, when the
product ion resulting from the neutral loss of 98 Da is dominant, the formation of
sequence ions, which may be used for peptide identification and localization of

the phosphorylation site, may be limited. To overcome this potential limitation,

strategies involving CID-MS3 of “the initial 98 Da neutral loss precursor ion has

been used in other studies to obtain the sequence information and

24. 98-101

phosphorylation site localization. However, the work described in

Chapter Three, demonstrate that the sites of phosphorylation should not be

determined from product ion spectra acquired using such CID-MS3 strategies,

since the initial neutral loss product ion may represent a mixture of ions. For

these reasons, CID-MS3 activation was not used here for that purpose. Instead,

high quality ClD-MS/MS product ion spectra were achieved by averaging
between 500-3000 scans (with 3 microscans), such that sequence ions could be
clearly observed above the level of noise for peptide identification and
phosphorylation site determination. Additionally, isolation and activation of the

protonated precursor ion was limited to either the monoisotopic ion or the

monoisotopic ion and the 130 isotope (the first heavy isotope), to decrease the

187

amount of signal present in the product ion spectrum resulting from activation of
extraneous ions with similar m/z.

The work described in Chapter Three also demonstrate that the propensity
for intramolecular gas-phase phosphate group migration is highest for precursor
peptide ions with low proton mobility (i.e., singly protonated tryptic peptides), and
using ClD-MS/MS in ion trap instruments, which are the conditions used in this
study. As a result, unambiguous phosphorylation site analysis may be hindered.
This may be particularly limiting when using database search algorithms for
phosphorylation site determination, since they do not account for the possibility of
such rearrangement reactions. However, this potentially limiting case does not
apply for peptides that contain equivalent numbers of phosphate groups and
potential phosphorylation sites (serine, threonine, and tyrosine residues). This
was true for two of the phosphopeptides identified in this study. In cases where
the number of phosphate groups was greater than the number of potential
phosphorylation sites, the ambiguities of phosphorylation site assignment were
decreased by considering phosphorylation sites that have been previously
identified from product ion spectra obtained using alternative dissociation
techniques to CID. Additionally, all ClD-MS/MS product ion spectra were
manually interpreted to obtain partial sequence information, information
pertaining to the amino acid composition, and other modifications (e.g,
methionine oxidation) to enhance the specificity of the database search as well
as enable validation of those search results. In many cases, manual

interpretation also enabled elucidation of the phosphate-intact sequence ions.

188

Importantly, manual interpretation was completed considering the potential for

the formation of sequence product ions resulting from (i) the neutral losses 98 Da

(H3PO4 or HPO3+H20), 80 Da (HPO3), 18 Da (H20) and combinations thereof;

and (ii) intramolecular gas-phase phosphate group transfer.

For this study, peptide identities and the proteins from which they are
derived were determined using the Mascot database search engine. The
definable search parameters for each Mascot query include: taxonomy;
proteolytic enzyme; number of proteolytic missed cleavages; constant and
variable modifications (either post-translational or process-induced); and
precursor and product ion mass tolerances. To determine an initial set of search
parameters as well as to empirically determine the tolerances of those
parameters for accurate identification of phosphorylated peptides, Mascot was
used to examine the ClD-MS/MS spectra of singly protonated synthetic “tryptic”
phosphorylated peptides (data not shown). From this study it was found that
trypsin (cleavage C-terminal to KR, but not when P is the subsequent residue)
could be chosen as the proteolytic enzyme allowing for up to three missed
cleavages. However, for the real samples, if the search results did not return a
manually validated peptide, then “semi-trypsin” was chosen as the proteolytic
enzyme to account for peptides that have tryptic specificity at one terminus, but
where the other terminus may be a non-tryptic cleavage. No constant
modifications were chosen. From the standard peptides, it was determined that if
the modifications that were present in the peptide were not chosen as a variable

modification for the search, then the correct peptide could not be identified. For

189

 

 

 

this reason, an initial set of variable modifications were chosen including:
oxidation of methionine; S-carboxyamidomethylation of cysteine; and
phosphorylation of serine, threonine, and tyrosine residues. Additionally, if no
result could be manually verified, then other modifications such as lysine
acetylation were also included in the search. Based on the analysis of the
synthetic peptides, mass tolerances of 10.5 Da for both the precursor and
product ions were used to optimize the search specificity, while decreasing the

resulting number of false positives.

5.2.2 Differentially Treated THP-1 Cells and Resultant Differential Levels in

Nuclear p65

THP-1 cells, which activate the NF-KB pathway upon stimulation with a

variety of known stimulating agents including TNF-a,266'268 were used in this

study. The nuclear fraction from cells treated with TNF-o (10 ng/mL for 0.5 hr)
and untreated cells were extracted, and the amount of nuclear p65 was

determined by Western blot (Figure 5.4). Similar to the results from previously

published studies,268-270 the TNF-o-treated cells exhibited significantly higher

levels of nuclear p65 than the untreated cells.

190

 

 

Untreated TN Fa Treated

I j I l

1 2 1 _ 2 _ReplicatelD

 

 

 

 

    

M ”M ”r Actin

Figure 5.4 Western blot detection of p65 (total, C-terrninal epitope) and actin
from replicate untreated and TNF-a-treated THP-1 nuclear extracts. Western
blot of the actin loading control indicates that the untreated and TNF-o-treated
THP-1 nuclear extracts contain comparable amounts of protein; and therefore,
that the amount of nuclear p65 is greater for the TNF-a-treated nuclear extract
than the untreated nuclear extract. This gel was edited to enable facile
comparison between the untreated and treated samples. The unaltered image is
shown in Figure 5.15. '

Since the TNF-o stimulated cell nuclear extracts contained significantly
more p65 than their untreated counterparts, immunoprecipitation of p65 and its
interacting proteins was initially completed for the treated cell samples only. At a
later time (~2 months later), the untreated cell samples (prepared on the same
day as the treated cellular extracts) were also immunoprecipitated. Western blot
analysis of p65 from these immunoprecipitated samples is shown in Figure 5.5.
As expected, the amount of p65 immunoprecipitated from the nuclear fraction of
the TNF-o-treated cells resulted in significantly higher nuclear p65 than that of
the untreated cells. Identification of the p65-associated proteins was completed

for both the trypsin digested, phosphopeptide-enriched nuclear fractions of the

treated and untreated THP-1 cells. Discussion involving the protein identification

191

 

 

process, comparison of the results from the treated and untreated samples, and

the relevance of the identified proteins is given in detail below.

IPT |P*

 

* - f + TNFd

*1" . _.. . 'l’i'. .' . “1-3131. ....

 

 

Figure 5.5 Western blot detection of p65 (total, N-terminal epitope) from p65-
immunoprecipated from untreated (IPT) and TNF-o-treated (IP ) THP-1 nuclear

extracts. The immunoprecipitation of p65 from the untreated and treated
samples were completed on separate occasions. For this reason the
experiments are differentiated here. Control experiments involving
immunoprecipitation of p65 from phosphate-buffered saline are also shown (*).
Higher amounts of nuclear p65 is present in the treated than the untreated
samples after p65-immunoprecipitation, consistent with the results shown in
Figure 5.4 for the samples prior to p65-immunoprecipitation. The unaltered
image is shown in Figure 5.16.
5.2.3 Identification of p65-Associated Phosphoproteins From TNF-q-

Treated THP-1 Cells

A representative mass spectrum of the phosphopeptide-enriched tryptic
digests of the nuclear extracts from TNF-d-treated THP-1 cells is shown in Figure
5.6. Nearly all of the ions present in the mass spectrum were determined to be
peptides, as indicated by the appearance of their ClD-MS/MS product ion
spectra. The only exceptions included those ions corresponding to matrix
clusters (labeled as “m” in Figure 5.6), which were also present in the product ion

spectra of standard peptides (data not shown). The observed predominant

neutral loss of 98 Da in nine ClD-MS/MS product ion spectra indicated that those

192

precursor ions were phosphorylated peptides (labeled as “*” in Figure 5.6). This
diagnostic ion was, however, not present in the product ion spectra of other
peptide ions. Upon further investigation of these ions, three were identified as
nonphosphorylated peptides (labeled as “o” in Figure 5.6) and some others were
unfortunately not identified from their ClD-MS/MS product ion spectra (labeled as
“7" in Figure 5.6). However, the unidentified peptides did not give rise to
predominant 98 Da neutral product ions in their spectra, and therefore, may not

be phosphorylated peptides.

193

 

 

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3000

 

 

 

 

 

 

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Figure 5.6 Representative MALDI-mass spectrum of the samples resulting from
phosphopeptide enrichment of the trypsin digested p65-immunoprecipitated

nuclear extracts from TNF-o-treated THP-1 cells, where * = identified

phosphorylated peptides; O = identified nonphosphorylated peptides; ? =
unidentified peptides; and m = matrix ions. '

194

 

 

The ClD-MS/MS product ion spectra were used to identify the peptides,
and, when present, their phosphorylation sites. The ClD-MS/MS product ion
spectrum of the most abundant ion in the mass spectrum, m/z 2061, is shown in
Figure 5.7. The spectrum contains product ions resulting from single and double
neutral losses of 98 Da from both the precursor (labeled as “A” and AA,
respectively, in Figure 5.7) and some sequence product ions (i.e., sequence ions
that have differences in 98 Da), demonstrating that the peptide is multiply
phosphorylated. Despite the significant amount of sequence product ions
present, and the quality of the product ion spectrum, the identity of the peptide
was initially not determined by either manual interpretation or by Mascot
searching (while allowing for carboxyamidomethylation of cysteine; oxidation of
methionine; and phosphorylation of serine, threonine, and tyrosine residues).
However, upon further investigation of the ClD-MS/MS product ion spectra from
other peptide ions in the mass spectrum (Figure 5.6), the identity of m/z 2061
was elucidated and the limitations that initially interfered with its identification

were made clear, vide infra.

195

Figure 5.7 ClD-MS/MS product ion spectrum of m/z 2061 (from the MS
spectrum shown in Figure 5.6). This peptide was identified as
KEEpSEEpSDDDM(cam-105)GFGLFD from the P1 or P2 isoforms of the 608
acidic ribosomal protein, and resulting from the gas-phase neutral loss of 2-
(methylthio)acetamide (105 Da) from the S-carbamidomethylmethionine
(M(cam))-containing species (m/z 2166). A = —98 Da (—H3PO4 or -

(H20+HPO3)); [:1 = —80 Da (-HP03); ° = -18 Da (—H20).

196

Figure 5.7 (cont'd)

 

 

  

 

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197

The ClD-MS/MS product ion spectra of m/z 2109 and 2125, also gave rise
to single and double neutral losses of 98 Da from both the precursor and some
product ions (Figure 5.8A and 5.63, respectively), indicating that those ions were
also multiply phosphorylated. Comparison of the product ion spectra of m/z 2109
and 2125 show that they have similar fragmentation patterns as well as many of
the same product ions, indicating that the two ions are related (Figure 5.8). And,
the difference in precursor ion mass (16 Da) suggest that mlz 2125 is an oxidized
form of m/z 2109. Further manual interpretation of these product ion spectra
resulted in identification of the partial sequence -DDM— for mlz 2109 (as shown
in Figure 5.8A) and the oxidized methionine (M(ox)) —DDM(ox)—- for m/z 2125 (as
shown in Figure 5.88). Additionally, the product ion resulting from the neutral

loss of 64 Da (methane sulfenic acid; labeled as i), which is a diagnostic

115, 271, 272

indicator of the presence of M(ox) (methionine sulfoxide), was also

observed in the product ion spectrum of mlz 2125 (Figure 5.83). This partial
sequence and the product ion masses enabled identification of these peptides
using Mascot as the doubly phosphorylated unmodified methionine-containing
and oxidized methionine-containing species of (K)KEESEESDDDMGFGLFD(C—
terminus) from the human P1 or P2 isoforms of the 608 acidic ribosomal protein.
Unambiguous phosphorylation site assignment was readily determined on the
two serine residues for this peptide (KEEpSEpSDDDMGFGLFD), since there are

only two phosphate groups and only two possible phosphorylation sites.

198

 

 

Figure 5.8 ClD-MS/MS product ion spectra of m/z 2109 and 2125 (from the MS
spectrum shown in Figure 5.6). These peptides were identified as the
unmodified methionine- and oxidized methionine-containing species of
KEEpSEEpSDDDMGFGLFD from the P1 or P2 isoforms of the 608 acidic

ribosomal protein, respectively. A = -98 Da (—H3PO4 or —(H20+HPO3)); El = —

so Da (—HP03); ° = —18 Da (—H20); 1 = —64 Da (—CH3$OH or methane sulfenic
acid).

199

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

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200

CID-MS/MS of mlz 2166, resulted in the predominant formation of a

product ion at m/z 2061 (shown in Figure 5.9), which is 105 Da less than the

precursor ion. Subsequent ClD-MS/MS activation (CID-M83) of the 2061

product ion (data not shown), resulted in an identical product ion spectrum as
that shown in Figure 5.7, indicating that the precursor ions m/z 2061 and 2276
were related. Additionally, comparison of the ClD-MS/MS product ion spectra of
m/z 2061 (Figure 5.7) and that of 2109 and 2125 from the identified peptide
KEEpSEEpSDDDMGFGLFD (Figure 5.8) showed that some of the observed
product ions were the same. Considering all of this information, it was
determined that m/z 2166 was a modified form (+57 Da) of m/z 2109
(KEEpSEEpSDDDMGFGLFD). Typically, a shift in mass of 57 Da is attributed to
an S-carbamidomethylcysteine residue (C(cam)) resulting from alkylation of
cysteine using iodoacetamide, which was employed in the treatment of this
sample. However, since no cysteine residues are present in the identified
peptide, the mass shift was attributed'to an alkylated methionine residue, 8-
carbamidomethylmethionine (M(cam)) (structure shown in Figure 5.10).
Therefore, mlz 2166 was identified as KEEpSEEpSDDDM(cam)GFGLFD.
Importantly, alkylation of methionine residues has been previously observed for

human lens crystallins proteins upon treatment with iodoacetamide under

commonly used conditions (similar to what was used for this study).273

Moreover, upon ClD-MS/MS of peptides containing 8-
carbamidomethylmethionine, the product ion resulting from the neutral loss of

105 Da (S-(methylthio)acetamide) has been previously predominantly

201

 

 

 

observedzn'275 These observations, therefore, justify the appearance of the

ClD—MS/MS product ion spectrum of mlz 2166 (Figure 5.9). In light of this, the
presence of the precursor ion m/z 2061 in the MS spectrum (Figure 5.6),
therefore, likely results from in-source fragmentation of m/z 2166 (shown in
Figure 5.10). The gas-phase ion chemistry of the S-carbamidomethylmethionine-
containing peptide provides a rationale for the initial inability to identify m/z 2061,
since it was not considered while completing the identification. However, upon
inclusion of “dethiomethyl methionine” as a variable modification, which mimics
the structure of the gas-phase product ion that corresponds to the neutral loss of
S-(methylthio)acetamide from the modified methionine, identification of m/z 2061
as KEEpSEEpSDDDM(cam-105)GFGLFD using Mascot was achieved.
However, identification of the alkylated methionine-containing peptide (m/z 2166)
was not possible using Mascot, since this variable modification was not an
available parameter in the search. Importantly, these results highlight how gas-
phase peptide ion chemistry can critically affect the utility of proteomic analysis

strategies.

202

 

 

 

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spectrum shown in Figure 5.6). This peptide was identified as the S-
carbamidomethylmethionine-containing species of KEEpSEEpSDDDM(cam)-
GFGLFD from the P1 or P2 isoforms of the 608 acidic ribosomal protein.

203

(105 Da)
+

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K:
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S-carbamidomethylmethionine (M(cam)).

A similar grouping of ions was also present in a lower mass region of the
MS spectrum of the phosphopeptide-enriched nuclear extracts from the TNF-d-

treated THP-1 cells (Figure 5.6), corresponding to a peptide containing an

204

unmodified methionine (m/z 1677), oxidized methionine (m/z 1693), S-
carbamidomethylmethionine (mlz 1734), and the product ion resulting from the
gas-phase neutral loss of S-(methylthio)acetamide (m/z 1629). The ClD—MS/MS
product ion spectra for these four ions are shown in Figure 5.11. As expected,
the appearance of these product ion spectra exhibited similar relationships to one
another as had the product ion spectra for the isoforms of the
KEEpSEEpSDDDMGFGLFD peptide. Briefly, the product ion spectra
corresponding to the methionine-containing ion (mlz 1677; Figure 5.11A),
oxidized methionine-containing ion (m/z 1693; Figure 5.118), and the product ion
resulting from the gas-phase neutral loss of 2-(methylthio)acetamide (m/z 1629;
Figure 5.110) from the alkylated methionine-containing ion (mlz 1677; Figure
5.11D), each contained many of the same product ions. The product ion
resulting from the neutral loss of 64 Da (methane sulfenic acid) was also present
in the product ion spectrum of the M(ox)-containing species (m/z 1693; Figure
5.11B labeled as 1:). Additionally, the S-carbamidomethylmethionine-containing
peptide (mlz 1677; Figure 5.11D) gave rise to the product ion resulting from the
gas-phase neutral loss of 2-(methylthio)acetamide (105 Da) (mlz 1629).
Moreover, the ClD-MS/MS product ion spectra from these peptide isoforms
(Figure 5.11) demonstrated some identical product ions as those resulting from
the previously identified KEEpSEEpSDDDMGFGLFD peptide (Figure 5.7, Figure
5.8, Figure 5.9), suggesting that these lower mass peptide ions represented a
truncated version of that peptide. Indeed, these lower mass peptide ions were

identified as modified forms of the (K)KEEpSEEpSDDDMGF(G) peptide. Again,

205

unambiguous phosphorylation site assignment was achieved since the peptide is
doubly phosphorylated and there are only two possible phosphorylation sites.
The origin of this truncated peptide could be rationalized by chymotrypsin
proteolysis occurring during sample preparation, since chymotrypsin cleaves C-
terrninal to F, W, Y, and L. Alternatively, this truncated peptide could result from
incomplete translation of the C-terminus of the protein or proteolytic processing

occurring in the cells prior to extraction.

206

Figure 5.11 ClD—MS/MS product ion spectra of m/z (A) 1677, (B) 1693, (C)
1629, and (D) 1734 (from the MS spectrum shown in Figure 5.6). These
peptides were identified as various isoforms of KEEpSEEpSDDDMGF, including
the unmodified methionine (mlz 1677), oxidized methionine (mlz 1693), product
ion resulting from the gas-phase neutral loss of 2—(methylthio)acetamide (105 Da)
from the S-carbamidomethylmethionine (mlz 2166), and S-
carbamidomethylmethionine-containing species (m/z 1734) from the P1 or P2

isoforms of the 608 acidic ribosomal protein. A = —98 Da (—H3PO4 or —

(H20+HPO3)); [3 = —80 Da (—HPO3); ° = —18 Da (—H20); 1: = -64 Da (—CH3SOH
or methane sulfenic acid).

207

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The last phosphopeptideidentified from the TNF-o-treated sample was the
doubly phosphorylated peptide (R)SRSFDYNYR(R) (m/z 1367) from the human
translocation liposarcoma (TLS)—associated serine-arginine protein (TASR). The
ClD-MS/MS product ion spectrum of m/z 1367 is shown in Figure 5.12, where the
labeling represents the pSRpSFDYNYR isofon'n. The product ion corresponding
to the 98 Da neutral loss is the most abundant ion in the spectrum, indicating that
this species is phosphorylated. Other neutral loss product ions observed include
the 196 Da (labeled as “AA") and 178 Da (labeled as “150”) indicating that the ion
is multiply phosphorylated. Since there are two phosphate groups and four
potential phosphorylation sites (two serine residues and two tyrosine residues),
phosphorylation site assignment required consideration of many factors. The
Mascot results indicated that the two serine residues were the phosphorylation

sites and that assignment is reflected in Figure 5.12. Inspection of the assigned

product ion spectrum demonstrate that there exists a y5/y7 ion pair with a

difference of 163 03, indicating Ser3 phosphorylation. Additionally, no

phosphate-intact sequence ions unambiguously corresponding to tyrosine
phosphorylation were present upon manual interpretation. However, the
potential for intramolecular gas-phase phosphate group transfer remains under
these conditions, and gives rise to some ambiguity to the origin of any

phosphate-intact sequence ions. Previous mass spectrometry-based studies

have shown that Sen and Ser3 are phosphorylation sites, further justifying these

164, 276-279

assignments. Notably, electron transfer dissociation tandem mass

210

spectrometry (ETD-MS/MS) has been used to unambiguously assign these
phosphorylation sites of the identified quadruply protonated

RRpSRpSRpSFDYNYR and triply protonated pSRpSRpSFDYNYR peptides,

which were derived from trypsin digestion and Ti02 phosphopeptide enrichment

of human embryonic 293T whole cell lysates.164 Note that under ETD-MS/MS

conditions, the phosphate group remains intact and therefore the potential

limitations associated with CID (i.e., phosphate Iability and phosphate migration)

do not apply. Additionally, phosphorylation of Wm and Wm of this peptide has

not been described previously. Considering all of this information, it is likely that
m/z 1367 predominantly represents the doubly serine-phosphorylated peptide
pSRpSFDYNYR. However, using an alternative tandem mass spectrometry
method for activation of this ion from this sample would further validate this

hypothesis.

211

Figure 5.12 ClD-MS/MS product ion spectrum of m/z 1367 (from the MS
spectrum shown in Figure 5.6). This peptide was identified as pSRpSFDYNYR
from the translocation liposarcoma (TLS)—associated serine-arginine protein

(TASR). A = —98 Da (-H3PO4 or —(H20+HPO3)); E] = —80 Da (—HPO3); O = —18
Da (-H20).

212

 

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213

Since the proteins identified in this sample have other known
phosphorylation sites, the corresponding precursor ion masses for those
modified peptides were searched for in the MS spectrum. Additionally,
phosphorylation of p65 may also occur, so the presence of those
phosphopeptides was also examined for the TNF-o-treated sample. This was
completed by allowing for tryptic cleavages only, with up to three missed
cleavage sites for the 608 acidic ribosomal protein (P1 and P2 isoforms) and p65
and up to five missed cleavage sites for the TASR protein; as well as variable
modifications including the known phosphorylation sites, 8-
carbamidomethylcysteine, S-carbamidomethylmethionine, and other known post-
translational modifications of those proteins (e.g., lysine acetylation). However,
using these parameters, no other peptides from 608 acidic ribosomal protein (P1
and P2 isoforms), TASR, or p65 were identified from this sample. One plausible
explanation for the lack of their presence includes that the peptides were not
phosphorylated in this sample. Alternatively, if these phosphorylated peptides
were present in the sample, they may not have been effectively enriched; they
may not have been eluted from the enrichment material; they may not have
ionized above the noise; or their masses were above or below the user defined
mass range (m/z 800-4000).

Nonphosphorylated peptides were also identified from the TNF-d-treated
sample. Those peptides included HMYHSLYLK from ribosomal protein L19 (m/z
1191; product ion spectrum shown in Supplemental of Chapter Five Figure 5.12),

AAIDWFDGK from the translocation liposarcoma (TLS) protein (mlz 1022;

214

pi

product ion spectrum shown in Supplemental of Chapter Five Figure 5.18), and
IWHHTFYNELR from actin (B isoforrn) (m/z 1515; product ion spectrum shown in

Supplemental of Chapter Five Figure 5.19). Since TLS and actin are known to

interact with p65 (as discussed in detail below),280'283 these nonphosphorylated

peptides were likely non-specifically enriched during MOAC, but are likely

specifically associated with p65.

5.2.4 Identification of p65-Associated Phosphoproteins From Untreated

THP-1 Cells

For comparison to the TNF-d-treated cell sample, immunoprecipitation,
trypsin digestion, and phosphopeptide enrichment of p65 and its associated
proteins was also performed for untreated THP-1 cells. Since p65 was present in
significantly lower abundance in the untreated sample as compared to the TNF-
a-treated sample (Figure 5.4 and Figure 5.5), it was expected that any protein
that has specific interactions with p65 (rather than nonspecific interactions with
the p65 antibody) would be present in lower abundance in the untreated sample
than in the treated sample, assuming similar enrichment efficiencies. A
representative MS spectrum of the untreated sample is shown in Figure 5.13.
Similar to the treated sample, the KEEpSEEpSDDDMGFGLFD peptide (mlz
2061, 2109, 2125, and 2166) from the P1 and P2 isoforms of the 608 acidic
ribosomal protein, the pSRpSFDYNYR peptide (mlz 1367) from TASR, and the
IWHHTFYNELR peptide (m/z 1515) from actin were present. The most

predominant ions in the spectrum, however, were those relating to the matrix

215

(labeled as “m” in Figure 5.13). In fact, many more matrix ions were present in
the untreated sample (Figure 5.13) than in the treated sample (Figure 5.6),

potentially due to a lower abundance of peptides in the untreated sample.

 

216

 

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from phosphopeptide enrichment of the trypsin digested p65-immunoprecipitated

nuclear extracts from untreated THP-1 cells, where * = identified phosphorylated
peptides; O = identified nonphosphorylated peptides; ? = unidentified peptides;
and m = matrix ions.

217

To compare the relative quantities of the peptides in the treated and
untreated samples, the phosphopeptides from these samples were enriched in

parallel and after elution, 100 amol of an internal standard phosphorylated

peptide CD3CD2CO-LFTGHPEpSLEK (“053284 was added followed by addition

of matrix. The resulting MS spectra for these samples is shown in Figure 5.14.
By comparison, it is clear that the abundance of the ions identified from the TNF-
d-treated sample are significantly lower than that in the untreated sample. These
results rationalize the high abundance of matrix ions in the MS spectrum of the
untreated sample (Figure 5.13). As a result, the corresponding proteins from
which these peptides are derived are likely to be specifically associated with p65

in the cell.

218

4000

 

 

   

 

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Figure 5.14 MALDI-mass spectra of the (A) TNF-d-treated THP-1 cells and (B)
untreated THP-1 samples resulting from p65 immunoprecipitation, trypsin
digestion, phosphopeptide enrichment, and addition of 100 amol of an internal
standard CD3CD200-LFTGHPEpSLEK (“D5", m/z 1398), where * = identified
phosphorylated peptides; O = identified nonphosphorylated peptides; ? =
unidentified peptides; and m = matrix ions.

219

5.2.5 Relevance of Identified Proteins Co-lmmunoprecipitated With p65
5.2.5.1 Ribosomal Proteins: 60$ Acidic Ribosomal Protein from the P1 or
P2 Isoforms and Ribosomal Protein L19
Ribosomal proteins are largely thought of as pertaining to cytoplasmic
translational processes, however, many studies have demonstrated that a
number of ribosomal proteins have “extra-ribosomal” functions, including

regulation of gene transcription, mRNA translation outside of the ribosome, and

(reviewed in 285, 286)

DNA repair. In fact, a study that aimed to determine a

physical and functional map of the NF-KB pathway using tandem affinity

purification (TAP) and key NF-KB proteins demonstrated that a variety of

ribosomal proteins are involved in its regulation. 59 In fact, nine ribosomal

proteins and four other proteins involved in ribosomal function were co-purified

with p65.259 Additionally, a recent study has showed that the ribosomal protein

S3 (RPS3) is a DNA-binding partner for both the p65 homodimer and the
p50/p65 heterodimer, and enhances DNA-binding of those dimers to specific
genes.287 These results are consistent with the identification of ribosomal
proteins originating from co-immunoprecipitation with p65 demonstrated in this
study. A brief description of these identified ribosomal proteins as well as the
implications regarding their associations with p65 are discussed below.

The large subunit (608) of eukaryotic ribosomes contain the P0, P1, and

P2 isoforms of the 608 acidic ribosomal protein (collectively known as the “P-

proteins”), whose sequences are highly conserved across species.288 Both the

220

 

P1 and P2 proteins have demonstrated involvement in peptide synthesis and

interact with elongation factors (EF) 1 and 2, which facilitate translational

elongation.289'291 A recent study has also demonstrated that the P1 and P2

proteins may modulate cytoplasmic translation by influencing the interaction

between the two ribosomal subunits.292 Importantly, the P2 isoform of the 608

acidic ribosomal protein has been previously co-immunoprecipitated with key
players of the NF-KB pathway, including IKK (y subunit), p52, NF—kB-inducing

kinase (NIK), and RelB, indicating that it too plays a potentially important role in

this pathway.259 However, no reports have been described that associate the P-

proteins directly with p65. Consequently, the association between the P1 or P2
isoform of the 608 acidic ribosomal protein and p65 demonstrated here
substantiates a function for these proteins in the NF-KB pathway. Since, at
present, there is no evidence for a non-cytoplasmic role for the P-proteins, this
identified association may result from a cytosolic contaminant or may indicate a
potentially novel nuclear function for these proteins.

Ribosomal protein L19 (RPL19) is part of the eukaryotic 60$ ribosomal
subunit and its exact function has yet to be elucidated. Interestingly, over-

expression of RPL19 has been associated with malignant prostate cells and

. . . . . . 294 .
tissues,293 IS differentially expressed in colorectal cancer tissue, and is up-

regulated in human breast tumors.295 No known interactions between RPL19

and p65 have been reported to date. As a result, the demonstrated association

221

here may be novel. Similar to the association between the ribosomal P-protein

and p65, this association could be cytoplasmic or the nuclear.

5.2.5.2 TLS and TLS-Associated Serine-Arginine Protein (TASR)
Translocation liposarcoma (TLS), is a protein that has shown cellular
involvement with the formation of the transcription factor (TF)IID complex and

association with RNA polymerase II, implicating TLS in transcriptional

activation.296'299 Recently, it has been shown that TLS enhances NF-KB

mediated transactivation induced by TNF-d, interleukin-1B, and overexpression

of NIK by its interaction with the p65 subunit.280 These results suggest that TLS

acts as a co-activator of NF-KB and are consistent with its identification in this
study resulting from its co-immunoprecipitation with nuclear p65.

TLS-associated serine-arginine protein (TASR) serves as a splicing factor,

processing pre-mRNA, and as the name implies, interacts with TLS.298' 300

Specifically, it has been shown that the C-temiinal region (residues 266 to the C-

tenninal residue 526) of TLS links gene transcription and RNA splicing by

298, 300

recruitment of TASR to RNA polymerase II. However, when TLS

constitutes part of an oncogenic fusion protein, where its C-tenninal region is

replaced with another protein, it is unable to recruit TASR to RNA-polymerase

298, 301

II In the study discussed here, the TLS peptide identified constitutes

residues 349-357, indicating that TLS is likely present in its wild-type form.

Collectively these results suggest that the TASR protein co-immunoprecipitated

222

with p65 here may result from a higher order complex of p65, TLS, TASR, and

RNA polymerase II.

5.2.5.3 Actin
Actin is a highly conserved major component of the cytoskeleton and
plays a critical role in a variety of essential cellular processes. These include

traditional mechanical functions, such as cell motility, contractility, mitosis,

cytokinesis, intracellular transport, endocytosis, and secretion.(rev'ewed m 302’

303) And, within the last five years, it has been generally accepted that actin is

also involved in the regulation of gene transcription, through sub—cellular

localization of transcription factors and in the assembly of transcriptional

' ' 4- 7 . .
(rev'ewed m 30 30 ) Importantly, interaction

regulatory complexes in the nucleus.

between p65 and actin has been previously demonstratedzm'283 In particular, it

has been shown that the state of the actin cytoskeleton (e.g., filamentous)

dictates the cytoplasmic localization of p65 via the p65-actin interaction in rat

embryo fibroblastas.281 In addition, recent evidence has suggested that the

cytoplasmic p65-actin interaction regulates p65 nuclear transport, and is

sensitive to actin filament stabilization/destabilization in endothelial cells treated

282. 283

with thrombin. These studies, therefore, are consistent with the co-

immunoprecipitation of actin with p65 demonstrated here. However, since actin

is also a nuclear protein that is known to interact with transcriptional machinery

223

 

(reviewed in 304-307)

(e.g., all three RNA polymerases), and the samples prepared

here represent a nuclear fraction, the p65-actin interaction proposed in this study

may suggest a nuclear role for p65 regulation.

5.3 Conclusions and Future Directions

The results from this initial investigation indicate that the
phosphoproteomic approach involving co-immunoprecipitation, phosphopeptide
enrichment, and collision-induced dissociation tandem mass spectrometry-based
methods holds promise for protein interaction discovery and signaling
mechanism elucidation. Using this technique five p65-associated proteins were
determined, two of which have not been previously reported. This study also
confirmed previously published results that implicate a role for three identified
proteins in the NF-KB pathway. Additionally, use of internal standards allowed for
the comparative analysis of the extent of p65-protein association in treated and
untreated cells. Collectively, these results broaden the perspective on the
processes involved in the NF-KB pathway.

Due to the success of this initial phosphoproteomic strategy, it is clear that
a more global study involving a variety of cell types and treatment regimens is
well within reach and would enable a greater understanding of the role of protein
interactions and signaling mechanisms in biochemical pathways. Such a study
would undoubtedly benefit from the maturation of the phosphoproteomic analysis
strategy used here. Initial adaptations to this approach should include

explorations of alternative phosphopeptide enrichment strategies. This is

224

particularly important considering that only two of the five proteins identified here
were derived from their associated phosphorylated peptides. This likely resulted
from limitations regarding the capacity of the phosphopeptide enrichment
material and the enhanced enrichment efficiency (and adequate elution) of the
peptides identified. In fact, all three distinct phosphorylated peptides (and their
modified isoforms resulting from sample preparation) were doubly
phosphorylated and two of those phosphopeptides were highly acidic, which may
suggest that these are optimal peptide conditions for this titanium-based
enrichment material. As a result, it is likely that other phosphopeptides were
present within these samples, in particular singly phosphorylated peptides, but

were either not enriched or not eluted from the enrichment material.

225

5.4 Supplemental Figures

Untreated TN Fa Treated

 

* 1 2 * 1 2 Replicate ID
250

150
100

75 p65

50
Actin

37

25

20

 

Figure 5.15 Unedited Western blot of p65 (total, C-terminal epitope) and actin
from replicate untreated and TNF-d-treated THP-1 nuclear extracts. Replicate
samples labeled with * were prepared previously (1 year prior to the preparation
of replicates 1 and 2) by another laboratory member and had been stored at -80
°C. Due to their similar amount of p65 in the untreated and treated previously
prepared samples, they were not used for any of the experiments described in
Chapter Five nor were they included in Figure 5.4.

226

THP-1 NE IPT IPi

 

- + * - * + TNFd
250

150

100

75 p65

50

37
IP3

25

20

 

Figure 5.16 Unedited Western blot of p65 (total, N-terminal epitope) from
untreated and TNF-d-treated THP-1 nuclear extracts (THP—1 NE); and p65-

immunoprecipated untreated (IP?) and TNF-d-treated (lPt) THP-1 nuclear
extracts. The immunoprecipitation of p65 from the untreated and treated
samples were completed on separate occasions. For this reason they are
differentiated here. Control experiments including immunoprecipitation of p65
from phosphate-buffered saline are also shown (*).. An additional protein band

(IPa) is present in the samples resulting from immunoprecipitation of the treated
sample (lPi). However, because this band is also present in the PBS control
sample, it IPa results from the immunoprecipitation process.

227

Figure 5.17 ClD-MS/MS product ion spectrum of m/z 1191 (from the MS
spectrum shown in Figure 5.6). This peptide was identified as HMYHSLYLK

from ribosomal protein L19. 0 = —18 Da (—H20).

228

 

 

 

 

OONM. oo _. —. coo _. com com CON com com Dov
JMIM . m .30 ....om m33m .....m 3.33 0033
3 m ...... ....
+—I+_>_M 00.... w> ... N> of omn >
. Nooi own 0&3 0> 002
333
033
3300:-

v_._>._mI>S_I

5:. 9252-90

 

(%) eouepunqv aAiielea

229

Figure 5.18 ClD-MS/MS product ion spectrum of mlz 1022 (from the MS
spectrum shown in Figure 5.6). This peptide was identified as AAIDWFDGK

from translocation liposarcoma (TLS) protein. 0 = -18 Da (—H20); * = —17 Da (—
NH3).

230

 

3300:-

 

x00m>>9<<

 

 

coo

Nmow 92392-90

 

o

o

_.

(%) eouepunqv eAiiEieu

231

Figure 5.19 ClD—MS/MS product ion spectrum of m/z 1515 (from the MS
spectrum shown in Figure 5.6). This peptide was identified as IWHHTFYNELR

from actin (B-isoform). o = -18 Da (-H20).

 

232

 

 

N33:
com? 00.3. com? com? 09. _. Door com com com com com

 

 

 

 

 

 

 

0.33 .4 30.0.30
03333.
0.33
000. 0090333
3300:-
m4m2>uFII>>_ mwmr 92392-90

 

 

%) eouepunqv eAiieiea

003..

233

CHAPTER SIX

Experimental Methods for Applications of Mass Spectrometry Methods for

Phosphoproteomic Analysis (Chapter Five)

6.1 Materials

Primary p65 polyclonal antibodies (C-20, C-20 agarose conjugate, F6),
protein A/G PLUS agarose, and normal goat lgG were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). B—actin primary antibody and E2 Blue
Coomassie stain were purchased from Sigma-Aldrich (St. Louis, MO). HyGLO
HRP Western blotting detection reagents and XAR film were purchased from
Denville Scientific (Metuchen, NJ) and secondary antibodies were obtained from
Amersham Biosciences (Piscataway, NJ). Bradford reagent dye, materials used
to prepare mini gels, and blocking grade non-fat dairy milk (NFDM) were from
BioRad (Hercules, CA). 10% SDS-polyacrylamide gels were prepared according
to the manufacturer’s protocol and were 0.75 mm in thickness. PBS, cellular
growth media, cellular growth media supplements, and TNF-d were purchased
from lnvitrogen (Carlsbad, CA).

All aqueous solutions were prepared using deionized water purified by a
Bamstead nanopure diamond purification system (Dubuque, IA). HPLC grade
methanol (MeOH) was purchased from Mallinckrodt (Hazelwood, MO) and HPLC
grade acetonitrile (ACN) was purchased from EMD (Gibbstown, NJ). Sequenal
grade trifluoroacetic acid (TFA) was purchased from Pierce (Rockford, IL).

Dimethyl pimelimidate (DMP) was purchased from Sigma Aldrich (St. Louis, MO).

234

Sequencing grade modified trypsin was purchased from Promega (Madison, WI).
Recrystallized 2,5-Dihydroxybenzoic acid (DHB) was purchased from Laser

Biolabs (Sophia-Antipolis, Cedex, France). Information regarding the reagents

used for synthesis of the “D5 internal standard may be found in Chapter Four.

D1o-propionic anhydride was obtained from CDN Isotopes (Pointe-Claire,

Quebec, Canada). All other reagents were commercially available of biological

grade and used without further purification.

6.2 Cells
THP-1 cell line (ATCC, Manassas, VA) was cultured in RPMI-1640
medium, complemented with 10% fetal bovine serum, 2 mM L-glutamine, 100

units/ml penicillin, 100 pg/ml streptomycin, and 10 mM HEPES buffer without

phenol red. Cells were propagated at 37 °C with 5% 002 and ambient oxygen.

6.3 Nuclear Extraction of THP-1 Cells

Cells were seeded at 1.0 x 106 cell/mL in 150 mm x 15 mm plates (30 mL)

the night before treatment. On the day of treatment, cells (20 replicate plates)

were treated with TNF-o (final concentration 10 ng/mL) for 30 min at 37 °C with

5% C02 and ambient oxygen. Untreated cells (2 replicate plates) were

maintained at 37 °C with 5% C02 and ambient oxygen.

235

Unless otherwise stated, all centrifugation steps were completed at 5000
xg for 5 min at 4 °C. Cells were harvested by centrifugation and the cellular
pellet was resuspended in ice cold PBS (1 mL/10 mL initial cell volume). After
centrifugation, the supernatant was aspirated and the cellular pellet was washed
with ice cold PBS (1 mL/10 mL initial cell volume). The cellular pellet was lysed
with cytoplasmic extraction buffer (50 pL/10 mL cells) (10 mM HEPES buffer, pH
7.9, 10 mM potassium chloride, 1.5 mM magnesium chloride, 1.0% Nonidet p-40,
0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) supplemented with
protease inhibitors (1 pg/mL leupeptin and 1 ug/mL aprotinin) and phosphatase
inhibitors (1 mM sodium orthovanadate for phosphotyrosine phosphatases and
alkaline phosphatase; 10 mM sodium fluoride for acid phosphatases; and 1 mM
B-glycerophosphate for serine/threonine phosphatases) for 10 min on ice. The
nuclei were pelleted by centrifugation and the cytosolic supernatant was
aspirated and stored at -80 °C. Nuclei were lysed with nuclear extraction buffer
(60 uL/ 10 mL initial cell volume) (20 pM HEPES buffer, pH 7.9, 0.42 M sodium
chloride, 1.5 mM magnesium chloride, 25% glycerol, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) supplemented with protease
inhibitors (1 pg/mL leupeptin and 1 pg/mL aprotinin) and phosphatase inhibitors
(1 mM sodium orthovanadate for phosphotyrosine phosphatases and alkaline
phosphatase; 10 mM sodium fluoride for acid phosphatases; and 1 mM (3-
glycerophosphate for serine/threonine phosphatases) at 4 °C for 20 minutes

while shaking at 2000 rpm. Samples were centrifuged at 5000 xg for 15 min at 4

236

°C, and the supernatant of similar treatment was collected, combined, and

transferred into chilled centrifuge tubes, and stored at —80 °C until further use.

6.3.1 Bradford Protein Assay: Determination of Protein Concentration in
Cellular Extracts

The Bradford protein assay utilizes the color change that Coomassie stain

undergoes upon binding to protein.308 Here, this change was measured

spectroscopically to quantify the concentration of protein in the cellular extract
samples. Duplicate IgG standard solutions were made in 40 pL in water (0.000,
0.088, 0.175, 0.350, 0.700, and 1.050 ug/uL). Cellular extract samples to be
quantified were prepared in duplicate by diluting 2 pL cellular extract in 38 pL
water. Bradford reagent dye (500 pL) was added to each standard and sample

and allowed to react for 5 min. Absorbance was measured at 595 nm on a

SpectraMax M5e plate reader (Molecular Devices, Sunnyvale, CA). Protein

concentration of each sample was then determined by fit to the lgG calibration

curve.

6.3.2 Western Blot Analysis of p65 from Nuclear Extracts

Nuclear extracts (30 pg total protein) were boiled for 3 min in 6x reducing
Laemmli buffer (0.35 M tris, 600 mM dithiothreitol, 0.36 M SDS, 0.18 mM
bromophenol blue in 30% glycerol) and were loaded onto 10% polyacrylamide
gels, and electrophoresed for 50 min at 200 V in running buffer (2.5 mM tris

base, 2 M glycine, and 10% or 35 mM SDS). The gel was wet transferred to a

237

PVDF membrane for 75 min at 60 V in Towbin buffer (25 mM tn's base, 19 mM
glycine, 5% methanol). Membranes were blocked with 4% NFDM in tris-buffered
saline-tween-20 (TBS-T: 50 mM tris base, 154 mM NaCl, 0.1% tween-20) for 1 hr
at room temperature, followed by washing with TBS-T while rocking for 10 min at
room temperature. The membranes were then probed with primary p65 antibody
(C-20 rabbit polyclonal 1:1000 in 4% NFDM in TBS-T) while rocking overnight at
4 °C. The membranes were then washed 3xTBS-T while rocking for 10 min at
room temperature, followed by incubation with horseradish peroxidase
conjugated to secondary antibody anti-rabbit (1:3000 in 4% NFDM in TBS-T)
while rocking for 1 hr at room temperature. The blots were washed 3 times with
TBS-T while rocking for 10 min at room temperature and developed with HyGLO
chemiluminescent detection for 1 min at room temperature and exposed on XAR

film.

6.4 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-
PAGE) of lmmunoprecipitated Proteins from Nuclear Extracts
6.4.1 Preparation of Immunoprecipitation (IP) Matrix: Covalently Cross-
Linked p65 Primary Antibody to Protein AIG-Agarose
The p65 antibody was covalently cross-linked at its fragment crystallizable
(Fc) domain to protein A/G-agarose using the diimidoester lysine cross-linking
reagent dimethylpimelimidate. For the cross-linking process, all centrifugation
steps were completed at 5000 xg for 3 min at 4 °C, unless otherwise stated. p65

goat polyclonal primary antibody agarose conjugate (up to 1 mL of resin) was

238

 

washed with 10 mL with ice-cold PBS and centrifuged. The pelleted matrix was
washed with 3x10 mL 0.2 M borate-NaOH solution, pH 9 and centrifuged.
Covalent cross-linking was achieved from incubation of dimethylpimelimidate
(DMP, 15.5 mg, 0.06 mmol) dissolved in 2 mL 0.2 M triethanolamine-HCI, pH 9
(final concentration of DMP 30 mM) with the drained matrix while rotating for 30
min at room temperature. The suspension was centrifuged and the reaction was
quenched by washing the resin with 10 mL 0.2 M ethanolamine-HCI, pH 9 while
rotating for 5 min at room temperature. The suspension was centrifuged and the
supernatant was aspirated. The resin was then incubated with 10 mL 0.2 M
ethanolamine-HCI, pH 9 while rotating for 1 hr at room temperature. The
suspension was centrifuged, the supernatant was aspirated, and the resin was
washed with 2x10 mL PBS, 2x10 mL 100 mM glycine, pH 3, and 2x10 mL PBS
each for 2 min while rotating at room temperature. The resin was resuspended
in 0.02% sodium azide (w/v) in PBS to make a 25% slurry (v/v) and stored at 4
°C until further use.

Efficiency of the cross-linking reaction was tested each time the reaction
was carried out by running samples before and after the cross-linking reaction by
SDS-PAGE and Coomassie staining. Equivalent samples (approximately 2 pg
p65 antibody) were aliquoted before cross-linking (after the last 0.2 M borate-
NaOH washes), after cross-linking (after the second PBS wash), and after cross-
linking and stringent 100 mM glycine, pH 3 washes. These samples were
microcentrifuged at maximum speed for 30 sec at 4 °C and suspended in 20 pL

2X SDS-PAGE buffer (10 mM tris pH 6.8, 0.2 M sodium dodecyl sulfate, 20%

239

glycerol, 0.01% bromophenol blue, 2% 2-mercaptoethanol). The samples were
incubated in boiling water for 3 min and loaded onto a 10% polyacrylamide gel
and electrophoresed for 50 min at 200 V in running buffer (25 mM tris base, 19
mM glycine, 3.5 mM SDS pH 8.3). The gel was washed three times with water
while rocking for 5 min at room temperature to remove residual SDS. The
protein bands of the gel were fixed with 50% methanol, 10% acetic acid (v/v)
while rocking for 15 min at room temperature. Removal of the fixing solution was
completed by washing the gel 2x5 min with water at room temperature. Protein
bands were visualized with Coomassie stain while rocking for 2 hrs at room
temperature. Residual Coomassie stain was removed by washing with water for
1-2 hr, changing water as necessary until the background of the gel was clear.

Cross-linking was typically achieved with greater than 95% efficiency.

6.4.2 Immunoprecipitation of Cellular Extracts Using Covalently Cross-

Linked p65 Primary Antibody to Protein AIG-Agarose

All centrifugation steps used for immunoprecipitation methods were
completed at maximum speed in a microcentrifuge for 1 min at 4 °C.

Samples for immunoprecipitation (IP) were prepared with 1000 pg of total
nuclear protein (quantified by Bradford assay; 20 replicates of TNF-d-treated
nuclear extracts and 2 replicates of untreated nuclear extracts) and diluted with
ice cold PBS to 800 pL. Blank samples (800 pL PBS) were also

“immunoprecipitated". IP samples were precleared with 1 pg normal goat lgG

240

and 20 pL Protein A/G PLUS agarose and incubated while rotating for 30 min at
4 °C to ensure complete removal of endogenous lgGs.

Samples were centrifuged and the supernatant was collected into chilled
microcentrifuge tubes containing 200 pL cytoplasmic extraction buffer
supplemented with protease and phosphatase inhibitors (see 6.3 Nuclear
Extraction of THP-1 Cells) and covalently cross-linked p65 antibody to agarose
(30 pL, 25% v/v). IP samples were incubated overnight while rotating at 4 °C.
Samples were centrifuged and the supernatant was aspirated. The pelleted
matrix was washed 2x500 pL cytoplasmic extraction buffer and 2x500 pL PBS.

Drained samples were stored at -20 °C until further use.

6.4.3 Western Blot Analysis of p65 From p65-lmmunoprecipitated Nuclear

Extracts of THP-1 Cells

For Western blot analysis, IP samples were resuspended in 20 pL 6x
reducing Laemmli buffer (0.35 M tris, 600 mM dithiothreitol, 0.36 M SDS, 0.18
mM bromophenol blue in 30% glycerol) and 5 pL aliquots (or 250 pg of initial total
protein) were loaded onto 10% polyacrylamide gels in running buffer (2.5 mM tris
base, 2 M glycine, and 10% or 35 mM SDS). For comparison, 30 pg of nuclear
extract samples (without immunoprecipitation) were also loaded onto the gels.
The gels were electrophoresed for 50 min at 200 V. The gel was wet transferred
to a PVDF membrane for 75 min at 60 V in Towbin buffer (25 mM tris base, 19

mM glycine, 5% methanol). Following transfer, membranes were treated as

241

indicated above (6.3.2 Western Blot Analysis of p65 from Nuclear Extracts). The

p65 F6 primary antibody was used to probe the samples post-IP.

6.5 ln-Solution Tryptic Digestion and Subsequent Phosphopeptide
Enrichment of lmmunoprecipitated Proteins

The immunoprecipitated samples were concentrated to dryness by Speed
Vacuum. The sample pellets were denatured and reduced by incubation with 20
pL 6 M urea in 50 mM Tris-HCI and 5 pL of 10 mM dithiothreitol at 65 °C for 1 hr.
After cooling, 160 pL 50 mM ammonium bicarbonate and 10 pL of 100 mM
iodoacetamide were added, followed by incubation at room temperature in the
dark for 1 hr. Sequencing grade modified trypsin (10 pL of 0.5 pg/pL solution)
was added and protein digestion was performed overnight at 37 °C. The
digestion was quenched by addition of 11 pL glacial acetic acid to achieve a 5%
solution. The digested samples were then centrifuged for 1 min at maximum
speed and the supernatant was transferred to 300 pL PCR tubes. The residual
resin was washed with 20 pL water, centrifuged for 1 min at maximum speed,
and the wash solution was collected and combined with the initial supernatant.
The combined supernatant was concentrated and stored at -20 °C.

Phosphopeptide enrichment of the tryptic digest samples was completed
on polymer-oxotitanium hybrid materials on gold wafers by Mr. Weihan Wang
(Professor Bruening’s group, Michigan State University). Briefly, lP samples
were redissolved in 0.1% trifluoroacetic acid (final volume 20 pL) and 2 pL (10%

of the total sample) aliquots were enriched using the oxotitanium enrichment

242

 

wafers prepared by Mr.. Weihan Wang. After enrichment and washing,

phosphopeptides were eluted with 1 pL of 1% H3PO4, followed by addition of
0.25 pL of 40 mg/mL DHB (prepared in 1% H3PO4J 50% ACN), and co-

crystallization. For incorporation of the 05 internal standard, enriched

phosphorylated peptides were first eluted, followed by application of 0.5 pL of a

200 amol/pL solution of D5 and the matrix solution.

6.6 Synthesis of the lntemal Standard “D5” CD3CD2CO-LFTGHPEpSLEK

Fmoc-solid phase peptide synthesis of the D5 internal standard was

completed as described in Dunn, et al. and using the detailed methods in

Chapter Four.284 Briefly, after C-terrninal elongation and Fmoc-deprotection of

the N-terrninal leucine residue, acetylation was performed on-resin by addition of

d1o-propionic anhydride (3 mmol eq.) and DIPEA (5 mmol eq.) in DMF, while

shaking for 15 min. Cleavage of the resin and orthogonal protecting groups was
achieved as described by Dunn, et al. (with consideration of Figure 4.1). Product
peptides were purified by reversed-phase-high performance liquid
chromatography (RP-HPLC) using an Aquapore RP-300 column (4.6 mm; Perkin
Elmer, Wellesley, MA) and a linear gradient elution at a flow rate of 1 mL/min

from 0-100 % B, where solvent A was 0.1 % TFA in water, and solvent B was

0.089 % TFA/60 % acetonitrile in water. The concentrations of aqueous D5

243

solutions were determined by amino acid analysis completed by the Genomics

Technology Support Facility at Michigan State University.

6.7 Matrix Assisted Laser Desorption/Ionization and Multistage Tandem
Mass Spectrometry

Mass spectrometry analysis was performed using an LTQ XL linear

quadrupole ion trap mass spectrometer equipped with a matrix assisted laser

desorption/ionization source (vMALDl) (Therrno Fisher Scientific, San Jose, CA).

Ion trap ClD-MS/MS and M83 experiments were performed on mass selected

precursor ions. For precursor ions with m/z less than 2000, isolation and
activation was completed on the monoisotopic precursor ion using an isolation
width of approximately 1.1. For those precursor ions with m/z greater than 2000,
isolation and activation was achieved using an isolation width of approximately

2.5 centered at the m/z of the monoisotopic precursor ion. The automatic gain

control (AGC) was set to 3.0 x 104 for full MS and 1.0 x 104 for MS". The

activation time was maintained at 30' ms using an activation q value of 0.25,
unless otherwise stated. All spectra shown were typically the average of 500-
3000 scans (3 microscans / scan), were collected in profile mode, and are shown
with a 5 point Gaussian smooth. MALDI-MS analysis of technical replicates
resulting from individually immunoprecipitated samples and phosphopeptide
enrichment of those samples gave rise to reproducible and consistent results
similar to what is shown in Figure 5.6 and Figure 5.13 for the TNF-d-treated and

untreated samples, respectively.

244

6.8 Peptide Identification by Database Searching

The Mascot algorithm (http://www.matrixscience.com/; Matrix Sciences,
UK) was used for interpretation of both uninterpreted MS/MS data (using MS/MS
Ion Search) and MS/MS data that was manually interpreted to obtain sequence,
composition and fragment ion data (using MS Query). DTA files were generated
for MS/MS Ion Search from the CID data (RAW files) using Bioworks 3.3
(Therrno Fisher Scientific, San Jose, CA) (parameters: absolute intensity
threshold of 1; minimum ion count of 10). All data was searched against the
NCBI non-redundant database, compiled from GenBank CDS translations,
Protein Information Resource (PIR), Swiss-Prot, Protein Research Foundation,
and Protein Data Bank (National Center for Biotechnology Information, National
Library of Medicine, National Institutes of Health, Bethesda, MD; 9,680,073
entries). The search parameters used were as follows: human; semi-tryptic;
three missed cleavages allowed; carbamidomethylation (C); oxidation (M);
phosphorylation (STY) as variable modifications; peptide tolerance of 0.5 Da;
MS/MS product ion tolerance 0.5 Da; peptide charge state 1+; experimental
mass values monoisotopic; instrument ESI-Trap. The taxonomy filter resulted in
223,846 protein sequences being considered in the database search. All

identifications and determined phosphorylation sites were manually validated.

245

10.

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