{a . figkfi gauehwnmx . v.73. i : ”wag“ H i 4C...“ u.u..ui..u.«.norv .luqifiztpl. ‘34.; 77 III . n.- . . . t. 3... .u n is .3 4: t‘. I. 161:5? THESIS J 00? MLil‘UHAgY MIC Ulfig'suy l taTe This is to certify that the dissertation entitled MULTISTAGE TANDEM MASS SPECTROMETRY STRATEGIES FOR THE TARGETED ANALYSIS OF OXIDATIVE PROTEIN MODIFICATIONS presented by JENNIFER M FROELICH has been accepted towards fulfillment of the requirements for the PhD degree in Chemistry &7;/ Major Professor’s Signature DEC. 84‘ 200?? I Date MSU is an Affirmative Action/Equal Opportunity Employer PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:IProj/Acc&PrelelRC/DateDue.indd MULTISTAGE TANDEM MASS SPECTROMETRY STRATEGIES FOR THE TARGETED ANALYSIS OF OXIDATIVE PROTEIN MODIFICATIONS By Jennifer M. Froelich A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chemistry 2008 ABSTRACT MULTISTAGE TANDEM MASS SPECTROMETRY STRATEGIES FOR THE TARGETED ANALYSIS OF OXIDATIVE PROTEIN MODIFICATIONS By Jennifer M. Froelich The origin and control of ex vivo sample handling related oxidative modifications of methionine-, S-alkyl cysteine- and tryptophan-containing peptides obtained fi'om typical in-solution or in-gel proteolytic digestion strategies, have been examined by capillary HPLC and MS/MS. The origin of increased oxidation levels was found to be predominately associated with the extensive ex vivo sample handling steps required for gel electrophoresis and/or in-gel proteolytic digestion of proteins prior to analysis by MS. Conditions for deliberately controlling the oxidation state (both oxidation and reduction) of these peptides, as well as for those containing cysteine, have also been evaluated using a series of model synthetic peptides and standard tryptic protein digests. Optimal conditions for oxidation and reduction were achieved via reaction with 30% hydrogen peroxide/5% acetic acid and reaction with 1 M dimethylsulfide/ 10 M hydrochloric acid, respectively. The mechanisms for the gas-phase fragmentation reactions of singly and multiply protonated precursor ions of a series of model S-alkyl cysteine sulfoxide-containing peptides prepared by reaction with iodomethane, iodoacetamide, iodoacetic acid, acrylamide or 4-vinylpyridine, followed by oxidation with hydrogen peroxide have also been examined using multistage tandem mass spectrometry (MS/MS, MS3 and M84), hydrogen/deuterium exchange and molecular orbital calculations (at the B3LYP/6-31 + G(d,p) level of theory). Dissociation of uniformly deuterated precursor ions of these model peptides confirmed that the non-sequence neutral loss of alkyl sulfenic acid (XSOH) in each case occurred via a charge-remote five-centered cis-1,2 elimination reaction. Similarly, the charge state dependence to the mechanisms and product ion structures for the losses of C02, C02 + H20 and C02 + CHZO from S-carboxymethyl cysteine sulfoxide-containing peptides, and for the losses of CHZCHCONHZ and CHZCHC5H4N, respectively from S-amidoethyl and S-pyridylethyl cysteine sulfoxide- containing peptide ions have also been determined. A strategy involving the fixed-charge sulfonium ion derivatization, stable isotope labeling, capillary HPLC and automated neutral loss MS/MS and data dependent “pseudo MS3” scans in a triple quadrupole mass spectrometer has also been developed for the targeted gas-phase identification, characterization and quantitative analysis of low abundance methionine-containing peptides present within complex protein digests. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method was able to achieve accurate quantification for individual peptides at levels as low as 100 fmol and at abundance ratios ranging from 0.1 to 10, present within a complex protein digest. Using a similar fixed—charge sulfonium ion derivatization and tandem mass spectrometry-based analysis strategy, methionine oxidation was successfully quantified following hydrogen peroxide treatment of the Ca2+/calmodulin dependent serine/threonine phosphatase calcineurin. Cepyn'ght by JENNIFER M. FROELICH 2008 ACKNOWLEGEMENTS The road leading to the completion of my dissertation was not always an easy one for me and I know that I would not have made it to the end without the help of many people along the way. First and foremost, I would like to thank my advisor, Dr. Gavin Reid, for his constant support, dedication, creativity and unwillingness to let me give up. I would also like to thank the members of my committee, Dr. Merlin Bruening, Dr. Greg Swain and Dr. J etze Tepe. I would like to thank the members of the Reid Research Group, both past and present, for all of their knowledge, friendship and support over the years and for helping me to enjoy my time here at MSU. I would especially like to thank Amanda “Pocahontas” Palumbo for always being there to listen and for helping me through all of those difficult times. I could never thank my family enough, especially my Mom, Ryan, Grandpa and Grandma Fritz and Aunt Debbie and Uncle Bob, for all of their support and encouragement, not only through graduate school, but through my entire life. My Mom, in particular, has been a constant source of strength for me and without her I would not be where I am today. Last, but certainly not least, I want to thank my husband Brad Cox for always believing in me, for making me a better person and for always being my biggest fan. I could not have done this without him. I also wish to acknowledge the award of a US. Department of Homeland Security Fellowship, which is administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement with the Department of Energy (DOE). TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... ix LIST OF FIGURES ......................................................................................................... x LIST OF SCHEMES ........................................................................................................ xvi 1. CHAPTER ONE: Introduction .................................................................................. 1 1.1 An Overview of Mass Spectrometry-based Approaches for Proteome Analysis .......................................................................................................... 1 1.2 Analytical Challenges Associated with Mass Spectrometry-based Approaches for Proteome Analysis ................................................................ 3 1.2.1 Mixture Complexity and Dynamic Range ........................................... 3 1.2.2 Quantitative Analysis .......................................................................... 5 1.3 Tandem Mass Spectrometry and Gas-phase Chemistry of Protonated Peptide Ions ..................................................................................................... 14 1.3.1 Concepts and Terminologies Associated with the Gas-phase Fragmentation Reactions of Protonated Peptide Ions ......................... 14 1.3.2 Overview of the Mechanisms Responsible for the Formation of “Sequence” Ions from the Gas-phase Fragmentation of Protonated Peptides ............................................................................. 16 1.3.3 Overview of the Mechanisms for the Formation of “Sequence” and “Non-Sequence” Ions from the Gas—Phase Fragmentation of Protonated Peptides containing Post-Translational or Process- Induced Modifications ......................................................................... 20 1.3.4 Alternative Dissociation Methods and Chemical Derivatization Strategies for Controlling the Formation of “Sequence” versus “Non-Sequence” Product Ions ............................................................. 22 1.3.4.1 Alternative Dissociation Techniques ..................................... 23 1.3.4.2 Chemical Derivatization Strategies for Protein Identification and Characterization ........................................ 23 1.3.4.3 Chemical Derivatization Strategies for Quantitative Analysis of Protein Expression .............................................. 29 1.4 Aims of this Dissertation ................................................................................ 33 2. CHAPTER TWO: Instrumentation ............................................................................ 34 2.1 Mass Spectrometry ......................................................................................... 34 2.1.1 Ionization ............................................................................................. 35 2.1.1.1 Electrospray Ionization (ESI) ................................................ 35 2.1.1.2 Matrix-Assisted Laser Desorption Ionization (MALDI) ....... 37 2.1.2 Mass Analyzers ................................................................................... 38 vi 2.1.2.1 The Quadrupole Mass Analyzer ............................................ 38 2.1.2.2 The Quadrupole Ion Trap Mass Analyzer ............................. 43 2.1.3 Detectors .............................................................................................. 49 3. CHAPTER THREE: Experimental ............................................................................ 51 3.1 Materials ......................................................................................................... 51 3.2 Synthesis of 13C6 Phenacylbromide ................................................................ 53 3.3 S-alkylation of Cysteine-containing Peptides ................................................. 53 3.4 Oxidation of Synthetic Peptides ..................................................................... 54 3.5 Oxidation of Calcineurin ................................................................................ 54 3.6 Reduction of Synthetic Peptides ..................................................................... 54 3.7 One-dimensional SDS-PAGE Separation of Standard Proteins ..................... 55 3.8 In-gel Tryptic Digestion of Standard Proteins ................................................ 55 3.9 Solution-phase Tryptic Digestion of Standard Proteins ................................. 56 3.10 Solution-phase LysC Digestion of Calcineurin .............................................. 57 3.11 Side Chain Fixed-Charge Derivatization of Methionine-Containing Peptides ........................................................................................................... 57 3.12 Mass Spectrometry of Model Peptides ........................................................... 58 3.13 Liquid Chromatography/Mass Spectrometry .................................................. 59 3.13.1 Model Peptides and Standard Protein Digests .................................... 59 3.13.2 Fixed-Charge Methionine-containing Peptides within Standard Protein Digests .................................................................................... 60 3.13.2.1 Data Dependent Identification and Characterization ............. 61 3.13.2.2 Quantitative Analysis ............................................................. 61 3.13.3 LysC Digests of Native and Oxidized Calcineurin Prior to and Following Side Chain F ixed-Charge Derivatization ........................... 62 3.14 Molecular Orbital Calculations ....................................................................... 63 3.15 Data Analysis .................................................................................................. 63 3.15.1 Calculation of Percent Oxidation and Reduction ................................ 63 3.15.2 MS/MS Database Analysis .................................................................. 64 3.15.3 Quantitative Analysis of Product Ion Abundances ............................. 64 3.15.4 Quantitative Analysis of Calcineurin Methionine Oxidation .............. 65 4. CHAPTER FOUR: The Origin and Control of Ex Viva Oxidative Peptide Modifications Prior to Mass Spectrometry Analysis ................................................. 67 4.1 Introduction ..................................................................................................... 67 4.2 Determining the Origin of Ex Viva Sample Handling Related Oxidative Peptide Modifications under In-solution versus In-gel Proteolytic Digestion Conditions ...................................................................................... 70 4.3 Determining Optimized Conditions for Peptide Oxidation Prior to MS/MS Analysis ............................................................................................. 82 4.4 Determining Optimized Conditions for Peptide Reduction Prior to MS/MS Analysis ............................................................................................. 93 4.5 Conclusions ..................................................................................................... 97 vii 5. CHAPTER FIVE: Mechanisms for the Proton Mobility Dependent Gas-Phase Fragmentation Reactions of S-alkyl Cysteine Sulfoxide-Containing Peptide Ions ............................................................................................................................ 98 5. 1 Introduction ..................................................................................................... 98 5.2 Multistage Tandem Mass Spectrometry and H/D Exchange Reactions 5.3 5.4 5.5 for the Fragmentation of S-Methyl and S-Carboxyarnidomethyl Cysteine Sulfoxide-Containing Peptide Ions .................................................. 102 Multistage Tandem Mass Spectrometry and H/D Exchange Reactions for the Fragmentation of S—Carboxymethyl Cysteine Sulfoxide- Containing Peptide Ions .................................................................................. 118 Multistage Tandem Mass Spectrometry, H/D Exchange Reactions and Molecular Orbital Calculations for the Fragmentation of S-Amidoethyl and S-Pyridylethyl Cysteine Sulfoxide-Containing Peptide Ions ................... 123 Conclusions ..................................................................................................... 141 6. CHAPTER SIX: Automated Neutral Loss and Data Dependent Energy Resolved “Pseudo M83” for the Targeted Identification, Characterization and Quantitative Analysis of Methionine-containing Peptides ........................................ 142 6.1 Introduction ..................................................................................................... 142 6.2 Optimization of “Low” and “High” Collision Energies Employed for Neutral Loss MS/MS and “Pseudo M83” Analysis ........................................ 144 6.3 Selective Identification and Characterization of Fixed-charge 6.4 6.5 Methionine-containing Peptides by Neutral Loss MS/MS and Data Dependent Energy Resolved “Pseudo M83” .................................................. 145 Differential Quantitative Analysis of Fixed-charge Methionine- containing Peptides by Neutral Loss MS/MS ................................................. 152 Conclusions ..................................................................................................... l 5 8 7. CHAPTER SEVEN: Quantitative Analysis of Calcineurin Methionine Oxidation via Fixed-Charge Chemical Derivatization and Tandem Mass Spectrometry .............................................................................................................. 160 7.1 Introduction ..................................................................................................... 160 7.2 Results Obtained for the Quantitative Analysis of Calcineurin Methionine Oxidation Using Different Measurement Strategies ................... 163 7.3 Comparison of the Results Obtained for the Quantitative Analysis of Calcineurin Methionine Oxidation Using Different Measurement Strategies ......................................................................................................... 172 7.4 Conclusions and Future Directions ................................................................. 173 APPENDIX ...................................................................................................................... 175 REFERENCES ................................................................................................................ 185 viii TABLE 4.1 4.2 4.3 4.4 4.5 4.6 5.1 5.2 5.3 LIST OF TABLES Triplicate analysis of the ex viva sample handling related oxidation of methionine, tryptophan and S-cam cysteine residues of the standard protein bovine apo-transferrin following in-solution or in-gel tryptic digestion ................. 72 Ex viva sample handling related oxidation and deliberate reduction of methionine residues of standard proteins following in-solution or in-gel tryptic digestion ....................................................................................................... 73 Ex viva sample handling related oxidation and deliberate reduction of tryptophan residues of standard proteins following in-solution or in-gel tryptic digestion ....................................................................................................... 74 Ex viva sample handling related oxidation and deliberate reduction of S- carboxyamidomethyl (S-cam) cysteine residues of standard proteins following in-solution or in-gel tryptic digestion ..................................................... 75 Oxidation of individual amino acid residues following the reaction of model tryptic peptides with 30% H202/5% CH3COOH at room temperature ................... 84 Reduction of individual amino acid residues following the reaction of oxidized model tryptic peptides with 1 M DMS/lO M HCl at room temperature .............................................................................................................. 95 Cleavage intensity ratio (CIR) values for the “non-sequence” side chain fragmentation reactions of S-alkyl cysteine sulfoxide- and methionine sulfoxide-containing peptide ions ......................................................................... l 10 Percent total product ion abundances for the “non-sequence” side chain neutral loss of HzNCOCHZSOH from S-carboxyamidomethyl (S-carn) cysteine sulfoxide—containing peptide ions of an oxidized tryptic digest of bovine serum albumin ........................................................................................... 117 Total energies (Emmi), zero point vibrational energies (ZPVE) and relative energies (Erel) computed for the precursor ions, transition states and product ion structures associated with each reaction pathway at the B3 LYP/6-31 + G (d,p) level of theory ............................................................................................... 129 ix PAGE FIGURE 1.1 2.1 2.2 2.3 2.4 2.5 4.1 LIST OF FIGURES Generic summary of the stable isotope labeling strategies currently employed for mass spectrometry-based relative protein quantitation. All reactions shown to occur on the amino terminus also apply to the e-amino group of lysine residues. The numbers in parentheses indicate the respective references cited in the review article by Julka et al. J. Proteome Res. 2004, 3, 350-363 .................................................................................................................... 10 Components of a mass spectrometer ....................................................................... 34 Stability regions as a function of U and V for positively charged ions with different masses (m. < m2 < m3 < m). Each ion can be observed successively by changing U linearly as a function of V while maintaining a constant ratio of UN ...................................................................................................................... 42 Cross-section of a three-dimensional quadrupole ion trap ...................................... 44 Typical Mathieu stability diagram for the quadrupole ion trap. The larger balls represent high mass ions whereas the smaller balls represent low mass ions .......................................................................................................................... 46 Diagram of a two-dimensional linear quadrupole ion trap ...................................... 49 CID-MS/MS product ion spectra of reduced and oxidized forms of methionine- and tryptophan-containing peptides derived from in-gel tryptic digests of bovine serum albumin (Figures 4.1A-4.1D) and bovine apo- transferrin (Figures 4.1E and 4.1F). (A) the [M+2H]2+ precursor ion of reduced MPC(S-cam)TEDYLSLILNR, (B) the [M+2H]2+ precursor ion of singly oxidized M(ox)PC(S-cam)TEDYLSLILNR, (C) the [M+2H]2+ precursor ion of reduced ALKAWSVAR, (D) the [M+2H]2+ precursor ion of singly oxidized ALKAW(ox)SVAR, (E) the [M+3H]3+ precursor ion of reduced WCTISTHEANK and (F) the [M+3H]3+ precursor ion of singly oxidized W(ox)CTISTHEANK ............................................................................... 79 PAGE 4.2 4.3 4.4 5.1 CID-MS/MS product ion spectra of the [M+2H]2+ precursor ions of (A) reduced VTMAHFWNFGK (MWK), (B) singly oxidized VTM(ox)AHFWNFGK (M(ox)WK) formed by reaction with 30% H202/S% CH3COOH at room temperature for 5 minutes, (C) doubly oxidized VTM(ox)AHFW(ox)NFGK (M(ox)W(ox)K) formed by reaction with 30% H202/5% CH3COOH at room temperature for 45 minutes and (D) singly oxidized VTMAHFW(ox)NFGK (MW(ox)K) formed by reaction with 1 M DMS/ 10 M HCl at room temperature for 45 minutes ............................................. 86 CID-MS/MS product ion spectra of the [M+2H]2+ precursor ions of (A) reduced VTMGHFCNFGK (MCK), (B) quadruply oxidized VTM(ox)GHFC(OX3)NFGK (M(ox)C(OX3)K) formed by reaction with 30% H202/5% CH3COOH at room temperature for 5 minutes and (C) triply oxidized VTMGHFC(ox3)NFGK (MC(0X3)K) formed by reaction with 1 M DMS/ 10 M HCl at room temperature for 10 minutes ............................................. 89 CID-MS/MS product ion spectra of the [M+2H]2+ precursor ions of (A) reduced VTMGHFC(S-cam)NFGK (MC(S-cam)K), (B) singly oxidized VTM(ox)GHFC(S-cam)NFGK formed by reaction with 30% H202/5% CH3COOH at room temperature for 5 minutes, (C) doubly oxidized VTM(ox)GHFC(S-cam)(ox)NFGK (M(ox)C(S-cam)(ox)K) formed by reaction with 30% H202/5% CH3COOH at room temperature for 5 minutes and (D) singly oxidized VTMGHFC(S-cam)(ox)NFGK (MC(S-cam)(ox)K) formed by reaction with 1 M DMS/ 10 M HCl at room temperature for 10 minutes .................................................................................................................... 91 Multistage tandem mass spectrometry of the S-methyl cysteine sulfoxide- containing peptide GAILCGAILK (C(S-me)(ox)K). (A) CID-MS/MS product ion spectrum of the [M+H]+ ion. (B) CID-MS/MS product ion spectrum of the [M+2H]2+ ion. (C) CID MS3 product ion spectrum of the [M+H-CH3,SOH]+ neutral loss product ion from panel A. (D) CID MS3 product ion spectrum of the [M+2H-CH3SOH]2+ neutral loss product ion from panel B. The insets to panels A and B, showing expanded regions of the product ion spectra obtained by CID-MS/MS of the unifome deuterated [M+D]+ and [M+2D]2+ precursor ions, confirm that the loss of CH3SOH occurs via the charge-remote cis-1,2 elimination pathway. Key: A = -CH3SOH; o = -H20; * = -NH3 ............................................................. 105 xi 5.2 5.3 5.4 5.5 Capillary HPLC-mass spectrometry analysis of S-carboxyamidomethyl cysteine sulfoxide- (S-cam(ox)) containing peptides from an oxidized tryptic digest of bovine serum albumin. Mass spectra obtained from region 1 (28.2- 29.2 minutes) and region 2 (22.5-23.5 minutes) of the LC-MS chromatogram (shown in the inset to panel A) are shown in panels A and B, respectively. The CID-MS/MS product ion spectra obtained from dissociation of the doubly (m/z 797.5) and triply (m/z 532.3) protonated precursor ions of the singly oxidized peptide LKPDPNTLC(S-cam(ox))DEFK in panel A are shown in panels C and D, respectively. The CID-MS/MS product ion spectra obtained fi'om dissociation of the doubly (m/z 989.1) and triply (m/z 660.2) protonated precursor ions of the triply oxidized peptide C(S-cam(ox))C(S- carn(ox))AADDKEAC(S-cam(ox))FAVEGPK in panel B are shown in panels E and F, respectively. Key: A = -H2NCOCHZSOH; o = -H20; *=-NH3 ................................................................................................................ 116 Multistage tandem mass spectrometry of the S-carboxymethyl cysteine sulfoxide-containing peptide GAILCGAILK (C(S-cm)(ox)K). (A) CID- MS/MS product ion spectrum of the [M+H]+ ion. (B) CID-MS/MS product ion spectrum of the [M+2H]2+ ion. (C) CID MS3 product ion spectrum of the [M+H-C02]+ neutral loss product ion from panel A. (D) CID MS4 product ion spectrum of the [M+H-CH20]Jr neutral loss product ion from panel C. Key: A = -H02CCHZSOH; D = -C02; :1: = —(COz+CH20); 0 = - CHzO; o = -H‘zO; * = -NH3 ................................................................................... 120 Multistage tandem mass spectrometry of the S-amidoethyl cysteine sulfoxide-containing peptide GAILCGAILK (C(S-ae)(ox)K). (A) CID- MS/MS product ion spectrum of the [M+H]+ ion. (B) CID-MS/MS product ion spectrum of the [M+2H]2+ ion. (C) CID MS3 product ion spectrum of the [M+H-CH2CHCONH2]+ neutral loss product ion from panel A. (D) CID MS4 product ion spectrum of the [M+H-H20]+ neutral loss product ion from panel C. Key: A = -H2NCOCH2CH2SOH; T = -(CH2CHCONH2+H20); o = - H20; * = -NH3 ...................................................................................................... 125 Optimized precursor, transition state and product ion structures (at the B3LYP/6-31+G(d,p) + ZPVE level of theory) for (A) the loss of CH3SOH from the neutral model system CH3CONHCH(CH2S(O)CH3)CONHCH3 (equivalent to the loss of XSOH from the S-methyl cysteine sulfoxide- containing peptides) and (B) the loss of CHZCHCONHZ from the neutral model system HZNCOCH2CH2S(O)CH3 (equivalent to the loss of CHZCHX’ from the S-amidoethyl cysteine sulfoxide-containing peptides), via 5- membered cis-1,2 elimination pathways ............................................................... 127 xii 5.6 5.7 6.1 6.2 CID-MS/MS product ion spectra of the methionine sulfoxide- and S- pyridylethyl cysteine sulfoxide-containing peptide VTMGHFCNFGK (M(ox)C(S-pe)(ox)K). (A) [M+H]+ ion. (B) [M+2H]2+ ion. (C) [M+3H]3+ ion .......................................................................................................................... 135 Optimized precursor, transition state and product ion structures (at the B3LYP/6-31+G(d,p) + ZPVE level of theory) for (A) the loss of CHZCHC5H4N from the neutral model system NC5H4CH2CH2S(O)CH3 and (B) the loss of CHZCHC5H4NH+ from the protonated model system HNC5H4CH2CH2S(O)CH3+ (equivalent to the loss of CHzCHX’ fi'om the S- pyridylethyl cysteine sulfoxide-containing peptides) ............................................ 138 Neutral loss MS/MS and data dependent energy resolved MS3 for the selective identification and characterization of a single methionine- containing phenacylsulfonium ion derivative of GAILMGAILK (500 final) spiked into a complex tryptic digest mixture of seven proteins (1 pmol each). (A) Total ion current trace obtained by capillary HPLC-mass spectrometry analysis. The inset to panel A shows the mass spectrum obtained from the region of the chromatogram spanning 19.5-20.5 min. (B) Total ion current trace obtained by neutral loss scan mode MS/MS analysis (neutral loss of 83.0 m/z). (C) Summed neutral loss scan mode CID-MS/MS spectrum from the 19.5-20.5 min region indicated in panel B. (D) Date dependent energy resolved “pseudo” MS3 product ion spectrum of the doubly charged precursor ion at m/z 553.1 identified from the neutral loss scan in panel C ......... 150 Selective identification and differential quantitative analysis of a 1:1 mixture of methionine-containing phenacylsulfonium and I3C6 phenacylsulfonium ion derivatives of GAILMGAILK (100 frnol each) spiked into a complex tryptic digest mixture of seven proteins (1 pmol each). (A) Total ion current trace following capillary HPLC-mass spectrometry analysis. The inset to panel A show the mass spectrum obtained from the region of the chromatogram spanning 19.5-20.5 min. (B) Expanded region of the mass spectrum containing the triply charged “light” (m/z 369.0) and “heavy” (m/z 371.0) labeled peptide ions. (C) Expanded region of the mass spectrum containing the doubly charged “light” (m/z 552.9) and “heavy” (m/z 555.9) labeled peptide ions. (D) Neutral loss scan mode MS/MS spectrum for the neutral loss of 55.3 m/z from 12C6 containing triply charged ions. (E) Neutral loss scan mode MS/MS spectrum for the neutral loss of 57.3 m/z from 13C6 containing triply charged ions. (F) Neutral loss scan mode MS/MS spectrum for the neutral loss of 83 m/z from ”CG containing doubly charged ions. (G) Neutral loss scan mode MS/MS spectrum for the neutral loss of 86 m/z from 13C6 containing doubly charged ions .................................................................... 157 xiii 6.3 7.1 7.2 7.3 7.4 Observed versus theoretical abundance ratios for 0.1:], 0.5:], 1:1, 1:0.5 and 1:0.1 pmol mixtures of phenacylsulfonium and 13C6 phenacylsulfonium ion derivatives of GAILMGAILK in a tryptic digest mixture of seven proteins (1 pmol each) obtained by MS analysis of the doubly and triply charged precursor ions (panels A and B, respectively) and by neutral loss scan mode MS/MS analysis (83.0 and 86.0 m/z for doubly charged ions and 55.3 and 57.3 m/z for triply charged ions) (panels C and D, respectively) ......................... 158 Extracted ion chromatograms of the quadruply charged ([M++3H]4+) precursor ion (m/z 579.2) of the fixed-charge methionine-containing peptide M406ARVFSVLREESESVLTLK (Met406) following treatment of calcineurin with 12 mM H202 for 0 (panel A), 1 (panel B) or 4 (panel C) h, respectively. Mass spectra obtained from the 27.0-27.3 minute region of the extracted ion chromatograms in panels A-C are shown in panels D-F ....................................... 167 CID-MS/MS product ion spectra of the [M‘L+3H]4+ precursor ion of the fixed-charge methionine containing peptide M406ARVFSVLREESESVLTLK (Met406) following treatment of calcineurin with 12 mM H202 for 0 (panel A), 1 (panel B) or 4 (panel C) h .......................... 168 Fraction of the calcineurin-A peptide M406ARVFSVLREESESVLTLK remaining in the reduced form following treatment of calcineurin with 12 mM H202 for 0, 1, 2, 4 or 10 h calculated using either the peptide mapping procedure described in Carruthers et al. (A), the fixed-charge chemical derivatization-MS (O) or the fixed-charge chemical derivatization-MS/MS (I) approach. All data are the mean of three experiments each analyzed in triplicate. Error bars are i the standard deviation ................................................. 169 Fraction of the calcineurin-A peptide GLTPTGM431LPSGVLSGGK remaining in the reduced form following treatment of calcineurin with 12 mM H202 for 0, 1, 2, 4 or 10 h calculated using either the peptide mapping procedure described in Carruthers et al. (A), the fixed-charge chemical derivatization-MS (O) or the fixed-charge chemical derivatization-MS/MS (I) approach. All data are the mean of three experiments each analyzed in triplicate. Error bars are :t the standard deviation. ................................................ 170 xiv Fraction of the calcineurin-A peptide EPPAYGPM227CDILWSD- PLEDFGNEK remaining in the reduced form following treatment of calcineurin with 12 mM H202 for 0, 1, 2, 4or 10hcalculated using either the peptide mapping procedure described in Carruthers et al. (A), the fixed- charge chemical derivatization-MS (O) or the fixed-charge chemical derivatization-MS/MS (I) approach. All data are the mean of three experiments each analyzed in triplicate. Error bars are i: the standard deviation ................................................................................................................ 170 Fraction of the calcineurin-A peptide VTEM364LVNVLNICSDDELGSE- EDGFDGATAAARK remaining in the reduced form following treatment of calcineurin with 12 mM H202 for 0, 1, 2, 4 or 10 h calculated using either the peptide mapping procedure described in Carruthers et al. (A), the fixed- charge chemical derivatization-MS (O) or fixed-charge chemical derivatization-MS/MS (I) approach. All data are the mean of three experiments each analyzed in triplicate. Error bars are i the standard deviation ................................................................................................................ 171 Fraction of the calcineurin-B peptide LRFAFRIYDMIOIDK remaining in the reduced form following treatment of calcineurin with 12 mM H202 for 0, 1, 2, 4 or 10 h calculated using either the peptide mapping procedure described in Carruthers et al. (A), the fixed-charge chemical derivatization-MS (O) or the fixed-charge chemical derivatization-MS/MS (I) approach. All data are the mean of three experiments each analyzed in triplicate. Error bars are i the standard deviation ............................................................................................ 171 XV LIST OF SCHEMES SCHEME PAGE 1.1 1.2 1.3 5.1 5.2 5.3 5.4 6.1 6.2 7.1 7.2 Nomenclature for peptide fragment ions ................................................................. 17 Generally accepted mechanism for the formation of b- and y-type sequence product ions following CID-MS/MS ....................................................................... 19 Gas-phase fragmentation reactions of phenacylsulfonium ion derivatized methionine- and cysteine-containing peptides ........................................................ 28 Potential mechanisms for the loss of XSOH or CHZCHX’ from the side chains of S-alkyl cysteine sulfoxide-containing peptide ions ............................... 101 Proposed mechanisms for the side chain fragmentation reactions of S- carboxymethyl (S-cm) cysteine sulfoxide-containing peptide ions ...................... 121 Proposed mechanisms for the loss of H20 from cysteine sulfenic acid- containing peptide ions .......................................................................................... 132 Proposed mechanism for the loss of vinylpyridine from the protonated side chain of S-pyridylethyl (S-pe) cysteine sulfoxide-containing peptide ions .......... 140 Automated neutral loss MS/MS and data dependent energy resolved “pseudo MS3” for the targeted identification and characterization of phenacylsulfonium ion derivatives of methionine—containing peptides in a triple quadrupole mass spectrometer ..................................................................... 147 Automated neutral loss MS/MS for the quantitative analysis of phenacylsulfonium ion derivatives of methionine-containing peptides in a triple quadrupole mass spectrometer ..................................................................... 153 Amino acid sequence of the calcineurin A subunit. The amino acid residues comprising the regulatory region are underlined. The predicted calmodulin binding domain is indicated by the double underline. Methionine residues identified both in this study and the study by Carruthers et al. are in bold, while the methionine residues identified exclusively in the study by Carruthers et al. are in italics ................................................................................. 161 Amino acid sequence of the calcineurin B subunit. Methionine residues identified both in this study and the study by Carruthers et al. are in bold, while the methionine residues identified exclusively in the study by Carruthers et al. are in italics ................................................................................. 161 xvi CHAPTER ONE Introduction* 1.1 An Overview of Mass Spectrometry-based Approaches for Proteome Analysis The completion of several genome sequencing initiatives, particularly the Human Genome Project in 2003, has led to the identification of thousands of genes involved in cellular regulation. It has been demonstrated, however that mRNA transcript levels do not accurately reflect protein expression,l which is likely due to the vast number of transcriptional, translational and post-translational modifications associated with the proteome. Consequently, research efforts in the post-genomic era have shifted towards determining the unknown functional roles of proteins encoded by these genes, which has led to the field of proteomics. A major goal within the field of proteomics is to identify, characterize, and quantify changes in protein expression either at a particular time throughout the cell cycle or in response to a particular type of stimulation (e.g., disease). The outcome of this research should enable a more complete understanding of the processes which control normal cellular function and the changes in cell regulation that lead to the onset and progression of disease. Due to its speed, sensitivity and specificity, mass spectrometry (MS) has emerged as an integral technique in the field of proteomics.2 * The concepts discussed in this Chapter will be published in: Froelich, J.M.; Lu, Y.; Reid, G.E. Chemical Derivatization and Multistage Tandem Mass Spectrometry for Protein Structural Characterization. In: Practical Aspects of Ion Trap Mass Spectrometry. Vol. 5: Applications. CRC Press 2008, Submitted. (invited chapter) and Froelich, J .M.; Reid, G.E. The Effect of Post-translational and Process-induced Modifications on the Multistage Gas-Phase Fragmentation Reactions of Protontated Peptide Ions. Comb. Chem. High Throughput Screening 2009, In Press. (invited review article) The “bottom-up” or “shotgun” tandem mass spectrometry (MS/MS) approach has emerged as one of the dominant methods employed for protein identification, characterization and quantitative analysis. In a typical bottom-up approach, unresolved protein mixtures, or individual proteins resolved by one- or two-dimensional electrophoretic or chromatographic methods, are subjected to proteolytic digestion, typically using trypsin. The resultant peptide mixture is then separated using one- or two- dimensional capillary liquid chromatography and introduced to the mass spectrometer by electrospray ionization (ESI), or by matrix-assisted laser desorption ionization (MALDI).3'6 Following their mass analysis, individual protonated precursor ions are then automatically isolated and subjected to dissociation by tandem mass spectrometry, most commonly by using collision induced dissociation (CID).7’ 8 The identity of each peptide, and its protein of origin, is then subsequently achieved by de nava sequencing7 or by database search algorithms which correlate the uninterpreted product ion spectrum with theoretically generated product ion spectra determined from peptides of the same mass . . . . 9-12 contained wrthrn a known protein sequence database. Although the bottom-up approach has proven successful, there are several analytical challenges which limit its comprehensive application toward protein identification, characterization and quantitative analysis. Each of these challenges, as well as the strategies which have been developed to address them, will be described in more detail below. 1.2 Analytical Challenges Associated with Mass Spectrometry-based Approaches for Proteome Analysis 1.2.1 Mixture Complexity and Dynamic Range The increase in sample mixture complexity resulting from proteolytic digestion, and the dynamic range associated with the proteome, present a formidable challenge for protein identification and characterization. To address these challenges, off- or on-line multidimensional chromatography (i.e., strong cation exchange followed by reversed- phase) is routinely employed to extensively fractionate proteolytically derived peptide mixtures prior to their analysis by mass spectrometry.13 Numerous other strategies have also been used in conjunction with chromatographic separation to increase the number of unique peptide ions selected for analysis by MS/MS, particularly for those present at low abundance. Dynamic exclusion is one such approach, whereby the m/z values of precursor ions previously selected for fragmentation are automatically placed into an exclusion list for a defined period of time to prevent their reselection.l4'l6 An iterative survey scan approach has also been described, which subjects peptide mixtures to multiple replicate LC-MS/MS analysesm’m. In each individual analysis, precursor ions are selected for fragmentation from a narrow m/z window rather than from the entire m/z range. In addition, Wang et a1. recently described a strategy by which the m/z of peptide ions that are positively identified in an initial LC-MS/MS run are placed into an exclusion list in subsequent runs of the same peptide mixture to prevent these peptides from being re-selected for fragmentation. l 7 The identification of low abundance proteins present in complex biological samples (e. g., human serum or cerebrospinal fluid) can also be improved by removing 3 proteins which are present at high abundance. Traditionally, this has been achieved using dye-ligand affinity chromatography18 or antibody-based methods such as immunoaffinity 19,20 chromatography. In an alternative approach, combinatorial ligand library beads have recently been employed to increase the concentration of low abundance proteins while 21-23 effectively reducing the concentration of high abundance proteins. In this approach, a protein mixture is exposed to beads which have been synthesized with a library of diverse ligands. Each individual protein or peptide present within the sample will bind to the ligand exhibiting the strongest intermolecular interaction. Those proteins which are present at high abundance will continue to bind to the beads until a saturation limit is reached. However, low abundance proteins, which are present at concentrations below the saturation limits of the beads, will be extensively bound. Using this approach, the dynamic range of protein concentrations present within a complex biological sample can be significantly reduced. Also in an effort to decrease sample mixture complexity and improve dynamic range, numerous “targeted” approaches have been described, which analyze only a subset of the peptides contained within a proteolytically derived peptide mixture. For example, affinity capture methods have been extensively employed to enrich for peptides containing specific post-translational modifications or selected amino acid residues. Immobilized metal ion affinity chromatography (IMAC) incorporating Fe3+, Ga3+ or Al3+ has been used to isolate phosphorylated peptides,24’ 25 while the enrichment of histidine- 2+-26 In containing peptides has been achieved using IMAC columns loaded with Cu . an analogous approach, organomercurial agarose beads have been employed to isolate cysteine-containing peptides from a tryptic digest of yeast cell lysates.27 Metal oxide affinity chromatography (MOAC) methods utilizing titanium dioxide (TiOz), zirconium dioxide (ZrOz) and aluminum oxide (A1203) have also been used for the highly selective enrichment of phosphopeptides as an alternative strategy to IMAC.”-3O The enrichment of glycosylated peptides prior to mass spectrometry analysis has also been achieved using lectin affinity chromatography.“ As an alternative “targeted” approach, peptide subsets may be enriched via the chemical derivatization of specific functional groups within a peptide (i.e., amino acid side chains) followed by isolation using affinity capture, covalent capture or 14, 32-48 chromatographic strategies. Specific examples include the biotinylation of cysteine residues within a peptide and. subsequent enrichment using streptavidin affinity 14’ 32’ 33 or the introduction of a quaternary amine tag to the side chain 0f chromatography, cysteine residues followed by their isolation using strong cation exchange (SCX) chromatography.38 Thiol-specific covalent resins have also been employed to enrich for cysteine-containing peptidesM'44 For example, Wang et al. have used a cysteine covalent capture strategy to characterize the mouse brain proteome.44 1.2.2 Quantitative Analysis In addition to protein identification and characterization, another major goal of proteomics research is to quantify protein expression levels. However, mass spectrometry is not inherently quantitative. Thus, the intensity of a peptide ion introduced to the mass spectrometer via ESI or MALDI does not necessarily reflect the amount of peptide 5 present in the sample, due to the strong dependence of ionization on the physical and chemical nature of the analyte. To overcome this challenge, numerous quantitative analysis strategies have been developed which measure the differences in protein abundances between two different cellular states of a biological system (e. g., normal and diseased cells). Differential quantitative analysis has previously been performed at the protein 49-51 In this level using two-dimensional differential gel electrophoresis (2D DIGE). approach, individual protein populations are covalently labeled with structurally similar, but spectrally distinct fluorophores. The protein populations are then combined and separated by 2D polyacrylamide gel electrophoresis (PAGE). Protein quantitation is achieved via imaging of the gel using different fluorescence excitation wavelengths. To determine the identity of those proteins which are either up- or down-regulated, gel spots are individually excised, subjected to enzymatic in-gel digestion then the resultant peptide mixture is subsequently analyzed by mass spectrometry. Although this technique overcomes many of the disadvantages associated with protein quantitation via traditional 2D PAGE, it still suffers from limited dynamic range (104), as well as a limited ability to resolve proteins with extremes of molecular weight and p1. Additionally, accurate quantitative analysis is precluded when two or more proteins are present in the same gel spot. The quantitative analysis of protein expression levels from different protein populations has also been achieved using “label free” mass spectrometry-based 52-55 approaches. In the label free approach, control and experimental samples are enzymatically digested and analyzed individually by LC-MS and MS/MS. Protein abundances are then determined by summing the extracted ion chromatographic peak areas,52’ 55 peptide identification scores obtained from database analysis53 or MS/MS spectral countsS4’ 55 for all the peptide ions identified for a single protein. However, in order to achieve accurate quantitation, highly reproducible LC-MS analysis is required to minimize shifts in retention time and fluctuations in MS signal intensity. Furthermore, protein quantitation may be precluded when peptides are present at low abundance or the MS signals of peptide ions with identical m/z values elute at the same retention time. Differential quantitative analysis has also been achieved via the incorporation of differential stable isotope labels between control and experimental samples. To date, the majority of stable isotope labeling methods have involved either in viva metabolic -58 32, 33, 35, 41-43, 58-60 labeling56 or in vitro chemical derivatization. In vivo metabolic labeling approaches such as SILAC (Stable Isotope Labeling by Amino acids in Cell culture)57 incorporate a differential stable isotope label by growing one population of cells in normal media and a second population of cells in media enriched with an isotopically encoded amino acid. Following protein extraction, the two protein populations are combined and enzymatically digested. Protein expression levels are then determined by MS analysis via comparison of the relative abundances of intact peptide precursor ions derived from the “light” and “heavy” isotopically labeled samples. In vitro chemical derivatization approaches employed for differential quantitative analysis either label all peptides within a proteolytically derived peptide mixture (i.e., at the N- or C-terminus), or target specific amino acid side chains or post-translational modifications. A general overview of commonly employed chemical derivatization strategies is shown in Figure 1.1. A more detailed discussion of each of these chemical 7 derivatization approaches can be found in a review article by Julka et al.58 and the references cited therein. Although numerous chemical derivatization and quantitative analysis strategies have now been described, one of the earliest of these involved use of the isotope coded affinity tag (ICAT) reagent.32 The first generation ICAT reagent, designed by Gygi et al., consisted of an iodoacetyl thiol-specific reactive group, a biotin tag and an oxyethylene linker region which contained either eight hydrogen atoms (light ICAT reagent) or eight deuterium atoms (heavy ICAT reagent).32 In this approach, all cysteine residues from control and experimental samples are labeled with the light and heavy ICAT reagent, respectively. After labeling, the two protein populations are combined, subjected to enzymatic digestion and then the ICAT labeled peptides are isolated from non-labeled peptides using avidin affinity chromatography. Relative protein expression levels are then determined by measming the peak ratios of peptide pairs in MS mode, while peptide identification is achieved by MS/MS analysis of individual peptide precursor ions. Using the ICAT approach, protein quantitation and a reduction in sample mixture complexity are achieved simultaneously. Figure 1.1 Generic summary of the stable isotope labeling strategies currently employed for mass spectrometry-based relative protein quantitation. All reactions shown to occur on the amino terminus also apply to the s-amino group of lysine residues. The numbers in parentheses indicate the respective references cited in the review article by Julka et al. J. Proteome Res. 2004, 3, 350-363. [Reproduced from reference 58] 2\ /2I NIz oNIo / NI 4 HA9: 23:69.83 IoNIose 8:33 ca IoNIo 8.3.3 89 010.139 MHz Nuw : N. O zNoce 0 N 0 I000 1 NI N :0 NIw 02 I000 NIOIZOOIOIZOQIWIZOOIWUIZO%I0IZOON IOIZIOIZOOIOIZOOIOZN I I wozm $9 / / 0 2I _ NIOOOIzm M/NI mooooom a: meson \I/ N / _ ozooozzm moiOOIm 83... F 3 oz NNIzoOIo I0 0 ANN. N a: O I a: so: Despite the initial success of this approach, a number of disadvantages associated with the first generation ICAT reagent have been noted. For example, the presence of deuterium atoms in the linker region of the heavy ICAT reagent may result in the chromatographic separation of light and heavy ICAT labeled peptides during reversed- phase chromatography, thereby precluding their accurate quantitation. To ensure that light and heavy labeled peptides co-elute, a second generation ICAT reagent was designed to include l3C rather than 2H in the linker region.33 In addition, fragmentation of the bulky biotin tag has often been observed during CID-MS/MS analysis, which complicates interpretation of the resultant MS/MS spectra for peptide identification. Thus, an acid-cleavable group, which connects the biotin moiety with the thiol-specific isotope tag, was also incorporated into the second generation ICAT reagent to cleave the biotin moiety from modified peptides.33 In contrast to the first generation ICAT reagent, the small size of the remaining tag results in minimal fragmentation following CID- MS/MS. As an alternative to the solution-phase ICAT approach, a number of solid-phase 33, 41-43 isotope labeling strategies have also been developed. Solid-phase covalent capture methods enable more stringent wash conditions to be employed in an effort to remove non-specifically bound peptides. In addition, peptide labeling and isolation can be achieved in a single step. Zhou et al. have described a method for solid-phase stable- isotope labeling of cysteine-containing peptides from Saccharamyces cerevisiae using controlled-pore glass beads containing an o-nitrobenzyl-based photocleavable linker, a stable isotope tag incorporating either seven hydrogen or seven deuterium atoms and a thiol-specific iodoacetyl group.41 Proteins from control and experimental samples were 11 individually proteolyzed and then cysteine-containing peptides were captured by do- or d7-beads, respectively. The beads were then combined, washed and photolytically cleaved to release the differentially labeled cysteine-containing peptides. Following LC- MS/MS analysis in a quadrupole ion trap it was determined that more proteins were able to be identified and quantified using the solid-phase isotope labeling approach than when the samples were prepared using the first generation solution-phase ICAT reagent. A similar solid-phase isotope labeling approach termed acid-labile isotope-coded extractants (ALICE) has also been developed by Qiu et al.42 In this approach, cysteine- containing peptides are captured using a nonbiological polymer which has been chemically modified to contain a maleimido thiol-reactive group and an acid-labile linker in both heavy and light isotope-encoded forms. For the majority of stable-isotope labeling approaches, including those described above, quantitative analysis is achieved in MS-mode. Limitations are encountered however, when one or both of the differentially labeled peptide ions are present at or below the level of chemical noise in the MS spectrum, thereby precluding quantitative analysis. Furthermore, accurate quantitation may be precluded when the m/z values of differentially labeled peptide ions overlap with non-labeled or other labeled components present in the peptide mixture. To address these challenges, several differential quantitative analysis strategies have been recently described whereby quantitation is performed by MS/MS, rather than by MS 61-67 The commercially available iTRAQ approach, which utilizes a multiplexed set of reagents to quantitate relative expression levels for multiple protein populations, is one example.64’ 65 The initial reagent employed in this approach consists of a reporter 12 group, a balance group and an amine specific peptide reactive group. Differential stable isotope labels are incorporated into the balance and reporter groups of four iTRAQ reagents in such a way that the tag generated upon reaction with a peptide has the same overall mass (+145.1 Da). To use this approach for quantitative analysis, peptide mixtures are individually labeled with one member of the multiplexed set after which the labeled peptide mixtures are combined and subjected to mass spectrometry analysis. In MS-mode, identical tagged peptides from each of the four samples are present at the same m/z value, therefore the MS-mode sensitivity is maximized. Upon CID-MS/MS of the peptide precursor ions, the balance group is lost as a neutral, while the reporter group retains a charge to generate low m/z product ions at m/z 114,1 115, 116 or 117, which are subsequently used for quantitative analysis. The product ions that can be used to identify the sequence of the labeled peptide may also be generated during the MS/MS experiment and remain isobaric. Compared to conventional MS-based approaches, increased sensitivity and greater specificity is achieved due to the reduction in chemical noise associated with the MS/MS experiment. To increase the number of protein populations which can be analyzed via this approach, an 8-p1ex version of the iTRAQ strategy was . 66 recently 1ntroduced. Recently, Li et al. described a similar MS/MS-based quantitative analysis strategy termed Cleavable Isobaric Labeled Affinity Tag or CILAT.67 Essentially a hybrid of the ICAT and iTRAQ approaches, the CILAT reagent includes an isobaric tag consisting of a reporter group and a balance group. The reagent also incorporates a biotin moiety for the enrichment of modified peptides via avidin affinity chromatography and an acid Cleavable linker to remove the biotin moiety prior to MS/MS analysis. The thiol group 13 employed in this reagent is used to modify tyrosine residues within peptides which have been converted to ortho-quinone via oxidation with tyrosinase. 1.3 Tandem Mass Spectrometry and Gas-Phase Chemistry of Protonated Peptide Ions The utility of the tandem mass spectrometry based approaches for protein identification and characterization described above, particularly for the analysis of peptides containing process-induced or post-translational modifications, is highly dependent on the ability to derive sufficient information from the peptide product ion spectrum to enable its subsequent database analysis, as well as on the ability to accurately predict in silica the types and abundances of the various product ions that will be generated.“ 69 The successful development and application of these methods, therefore, have been greatly facilitated by the concurrent development of an improved understanding of the mechanisms and other factors (e. g., precursor ion charge state, amino acid composition, peptide conformation, product ion structures, etc.) that influence the gas-phase fragmentation reactions of protonated peptide ions,70 and the development of methods for controlling the fragmentation reactions of peptide ions in the gas-phase. 1.3.1 Concepts and Terminologies Associated with the Gas-phase Fragmentation Reactions of Protonated Peptide Ions A central concept underlying the mechanisms currently proposed for the gas- phase fragmentation reactions of protonated peptide ions under low-energy collision induced dissociation (CID) conditions71 is that of the “mobile proton”, originally 14 72-75 described by the groups of Wysocki and Gaskell. “Proton mobility” has been divided into three categories.76 Peptides are classified as “mobile” when the total number of ionizing protons is greater than the total number of basic residues (i.e., combined number of arginine, lysine and histidine residues), and as “non-mobile” when the total number of ionizing protons is less than or equal to the number of arginine residues. Those peptides which fall outside of these two categories (i.e., when the total number of ionizing protons is greater than the number of arginine residues, but less than or equal to the total number of basic residues) are classified as “partially mobile”. Depending on the proton mobility of the precursor ion, the gas-phase fi'agmentation of protonated peptide ions may proceed via “charge-directed” (i.e., an ionizing proton initiates or is directly involved in the fragmentation reaction) or “charge- remote” (i.e., an ionizing proton is not directly involved in the fragmentation reaction) reaction mechanisms. The product ions formed from these reactions can be classified as “sequence ions” (i.e., those resulting from fragrnentations along the peptide backbone from which information regarding the amino acid sequence can be derived) or “non- sequence ions” (e.g., those resulting from fragmentation at the side chains of certain amino acids, from which diagnostic information regarding the composition of the peptide can be derived), and can be further classified as “non-selective” (i.e., those resulting from fragmentation at multiple sites within the peptide ion) or “selective” (i.e., those that result from fragmentation at a single site within the peptide ion). A variety of approaches have been employed to provide evidence for the mechanisms and other factors that influence the gas-phase fiagmentation reactions of protonated peptide ions. These include the use of multistage tandem mass spectrometry 15 (MSn) coupled with isotopic and structural labeling, comparison of the MS3 spectra of product ions with the MS/MS spectra of “authentic” product ion structures formed via independent synthesis, gas-phase ion-molecule reactions, determination of activation energies either through the measurement of dissociation kinetics or via energy-resolved dissociation experiments, theoretical molecular orbital modeling techniques, and the statistical analysis of large experimentally derived datasets.”85 1.3.2 Overview of the Mechanisms Responsible for the Formation of “Sequence” Ions from the Gas-Phase Fragmentation of Protonated Peptides The formation and types of sequence ions resulting from the gas-phase fragmentation of protonated peptide ions are highly dependent on the proton mobility of 76, 86, 87 their precursor ions. Under mobile proton conditions, both non-selective and selective sequence ions are typically observed. Non-selective fragmentation reactions typically result in the formation of a series of complementary b- and y-type sequence - 88, 89 mm, via cleavage of amide bonds along the peptide backbone (Scheme 1.1). This type of fragmentation is generally accepted to proceed via a charge-directed neighboring group participation mechanism, in which localization of an ionizing proton at either the carbonyl oxygen or the amide nitrogen of the amide bond initiates nucleophilic attack 77’ 90'” The pathway shown in Scheme 1.2 for from an adjacent amide carbonyl. dissociation of the second amide bond in a protonated tetrapeptide can be regarded as a general mechanism for the formation of 130122) and yn product ions. Dissociation of this bond results in an initial ion-molecule complex between the b-type product ion consisting l6 . . . . . . . 93-96 of a cyclic oxazolone structure contarnmg the two N-termrnal ammo acrd resrdues, and a truncated neutral peptide. Direct separation of this ion-molecule complex would result in observation of the b2 ion (pathway A of Scheme 1.2), while intermolecular proton transfer prior to its separation would yield the yz ion containing the two C- terminal amino acid residues (pathway B of Scheme 1.2). The relative abundances of these b- and y-type product ions have been shown to be dependent on the proton affinities of their respective N- and C-terrninal fragments,97 as well as the lifetime of the ion- molecule complex7. Subsequent fragmentation and rearrangement of the initial product ions can also occur, particularly for the oxazolone-containing b-type ions, thereby complicating the appearance of the resultant product ion spectrum.98 H+ 0, b1 c1 a2 b2 c2 a3 b3 03 R O R" O i I I i ll /’ \N ./’ \ // \N HZN \fi/ \T/ Kfi/ \OH O R' H O R'" x3 Y3 23 x2 Y2 22 3‘1 3’1 21 Scheme 1.1 Nomenclature for peptide fragment ions. Note that while Scheme 1.2 shows the amide bond fi'agmentation reaction 99-101 proceeding via an amide nitrogen protonated precursor, an alternative dissociation l7 mechanism initiated by protonation at the thermodynamically preferred carbonyl oxygen102 could also be considered, involving a simple intrarnolecular “proton shuttle” mechanism within the tetrahedral cyclic intermediate that would result from the initial nucleophilic attack process, analogous to that proposed for intrarnolecular proton transfer within protonated precursor ions, prior to amide bond cleavage. Indeed, previous results from the literature indicate that product ions formed via initial protonation at the carbonyl oxygen can be observed experimentally, at least under certain conditions. 103’ 104 18 i H i HzN (2"0 O HZNXCII/NYCKOH _ + R' O Rm _ PathwayA PathwayB H+ transfer b21011 yz ion R" H N ’0 O X H (II) 2 + N C mil-I H3N ('3’ Y \OH + R. O R!" R R" O H II )\ O O HzN/kc’Nj/C‘on HzN (If E ll 0 RI H N RI Scheme 1.2 Generally accepted mechanism for the formation of b- and y-type sequence product ions following CID-MS/MS. Selective fragmentation reactions can also be observed under mobile proton conditions, typically when proline residues are present within a protonated peptide ion. These reactions often result in the formation of dominant b- or y- type sequence ions by fragmentation of the imide bond N-terminal to the proline residue, presumably due to the higher local proton affinity of the imide bond, therefore leading to a greater degree of 19 localization of the ionizing proton, compared to that of a conventional amide bond.7’ 76’ 86, 87, 105 Under non-mobile proton conditions, selective fragmentation reactions typically dominate the appearance of the product ion spectrum. For example, dominant b- or y- type sequence ions are typically observed under these conditions as a result of selective cleavage C-terrninal to aspartic acid residues, via either a charge-remote or salt bridged mechanism, whereby the acidic proton of the aspartic acid side chain facilitates cleavage [06-113 of the amide bond.84’ Notably, it has been demonstrated that the specificity associated with the formation of selective b- and y-type sequence ions can be used to significantly enhance the utility of database search methods for the identification of aspartic acid-containing Peptides,' I4, 115 1.3.3 Overview of the Mechanisms for the Formation of “Sequence” and “Non- Sequence” Ions from the Gas-Phase Fragmentation of Protonated Peptides containing Post-Translational or Process-Induced Modifications The presence of post-translational or process-induced modifications of certain amino acids can also influence the observation of selective sequence ions fiom the fiagmentation of protonated peptides. For example, the oxidation of cysteine to cysteine sulfinic acid (Cys-SOZH) or to cysteine sulfonic acid (Cys-SO3H) has been observed to result in selective fragmentation C-terminal to the oxidized cysteine residue under 106, 116-120 conditions of limited proton mobility. Conversely, while the presence of histidine within a peptide sequence has previously been shown to result in some 20 . . . . . . . 86, 112 enhancement 1n selective cleavage C-termrnal to the h1st1drne re31due, a recent study by Bridgewater et al. has found that oxidation of histidine, presumably to 2- oxohistidine, eliminates this selective fiagmentation behavioum The dissociation reactions of protonated peptides containing post-translational or process-induced modifications under non-mobile proton conditions are also often observed to result in the formation of dominant non-sequence product ions via selective 2,123 fiagmentation of the side chains of the modified amino acid.12 Examples include the neutral loss of phosphoric acid (H3PO4, 98 Da) from phosphoserine- or , 124-126 phosphothreonine-containing peptides,80 the loss of HPO3 (80 Da) from 124-127 phosphotyrosine-containing peptides, the loss of S03 (80 Da) from O- 129 sulfonoserine-, O-sulfonothreonine-128 or thiosulfate- (-S-SO3H) containing peptides,130 the loss of a glycan moiety from O-linked N-acetylgalactosamine-containing 131,132 peptides, the loss of alkyl sulfenic acid (RSOH) from methionine sulfoxide- (i.e., CH3SOH, 64 Da)81 and S-alkyl cysteine sulfoxide-containing peptides)”135 the loss of 135, I36 H20 fi'om cysteine sulfenic acid (Cys-SOH)-containing peptides, the loss of 118-120 106,117-119 H2302 01' H2303 from cysteine sulfinic acid- and cysteine sulfonic acid- containing peptides, respectively, the loss of SOZ+NH3 from N-terrninal cysteine sulfinic acid-containing peptides,120 and the loss of H20 from peptides containing oxidized tryptophan.78 The formation of these non-sequence ions can often provide useful diagnostic information regarding the presence of a particular modified amino acid residue within a 21 peptide, and may {therefore be attractive for use as “targets” for selective proteomic analysis. However, their formation in high relative abundance can result in the loss of sequence information, potentially compromising peptide identification. Under such circumstances, isolation and further dissociation of this initial product ion (by MS3 in a quadrupole ion trap mass spectrometer, or by energy resolved “pseudo” MS3 in triple quadrupole or hybrid quadrupole-time of flight mass spectrometers), may be required in order to unambiguously identify the peptide or to localize the modification to a specific . . . . . . 25, 28, 137-140 ammo ac1d resrdue Awrthm the peptide sequence. 1.3.4 Alternative Dissociation Methods and Chemical Derivatization Strategies for Controlling the Formation of “Sequence” versus “Non-Sequence” Product Ions It is clear from the above discussion that the mechanisms responsible for the gas- phase fragmentation reactions of protonated peptide ions, and the appearance of the resultant product ion spectrum, are all highly dependent upon the proton mobility of the peptide precursor ion. This results in a generally limited ability to control or direct the low energy CID-MS/MS fragmentation reactions of protonated peptide ions toward a particular fragmentation pathway (e.g., sequence ion backbone fragmentation versus non- sequence ion side chain cleavage), thereby placing limitations on the ability of these methods to be selectively employed for either comprehensive or targeted proteome analysis. To address these limitations, alternative dissociation techniques have recently been developed in order to provide more extensive non-selective sequence ion information, while a number of chemical derivatization strategies have also been developed to control or direct the fragmentation reactions of protonated peptide ions 22 toward the formation of either non-selective sequence-ions, or selective non-sequence ions. 1.3.4.1 Alternative Dissociation Techniques The recently developed alternative dissociation methods of electron capture 129,135,141-146 120,129,147-152 dissociation (ECD) and electron transfer dissociation (ETD) have proven to be particularly successful for the characterization of protonated peptide ions containing post-translational or process-induced modifications. ECD involves the capture of an electron with near thermal, energy by a multiply protonated ([M+nH]n+) precursor ion, while ETD occurs via electron transfer to a multiply protonated precursor ion from an anion of low electron affinity. In both cases, an odd electron reduced charge state ([M+nH]("'l)+°) product is formed, that undergoes fragmentation to yield a series of non-selective c- and z-type sequence ions (Scheme 1.1). In contrast to the fragmentation processes typically observed by CID, the formation of product ions from ECD and ETD is generally indifferent to the proton mobility of the precursor ion, or to the presence of otherwise labile amino acid modifications, such as phosphorylation,l41’ 147’ 148’ '52 143,146,149 20,135 129 glycosylation, oxidation1 or sulfonylation. 1.3.4.2 Chemical Derivatization Strategies for Protein Identification and Characterization In an effort to improve peptide identification and characterization by de novo sequencing or database search algorithm strategies, numerous chemical derivatization approaches have been developed to direct the fragmentation reactions of peptides toward 23 the formation of a series of non-selective sequence product ions. One such approach involves chemical derivatization of the side chains of basic amino acid residues within a 153-156 peptide to alter proton affinity. For example, the reagents acetylacetonels3 and malondialdehyde'54 have been employed to chemically modify guanidino groups on the side chain of arginine residues in an effort to decrease the proton affinity of these sites. By decreasing the proton affinity of the arginine side chain, an ionizing proton is less likely to be sequestered and is therefore available to initiate cleavage of the amide bonds along the peptide backbone. It has been demonstrated that CID-MS/MS of these chemically modified arginine-containing peptides results in an increased number and intensity of b- and y-type sequence product ions compared to their non-derivatized counterparts, thereby improving peptide identification.154 In a similar approach, the e- arnino group of C-terrninal lysine residues have been converted to an imidazole derivative via chemical modification with 2-methoxy-4,5-dihydro-lH-imidazole.155’ 156 In contrast to producing both b- and y-type sequence ions, the increased proton affinity resulting from this chemical modification predominantly results in the formation of a series of y-type ions, yielding a simplified MS/MS product ion spectrum for interpretation, particularly for de nova peptide sequencing. The chemical derivatization of peptide N- or C-termini to incorporate a fixed positive or negative charge is yet another approach that has been extensively employed to direct peptide ion fragmentation toward the formation of a desired series of non-selective 146, 157-166 sequence product ions. For example, it has been shown that the chemical derivatization of peptide N-termini with S-pentafluorophenyl [N-tris(2,4,6- trimethoxyphenyl)phosphonium]acetate bromide (TMPP-AcSC6F5 bromide)‘58’ ‘59 or 24 with [tris(2,4,6-t1imethoxyphenyl)phosphonium]acetic acid N-hydroxysuccinimide ester (TMPP-Ac-OSu)160 to form [tris(2,4,6-trimethoxyphenyl)phosphonium]acetyl (TMPP- Ac) derivatives directs the gas-phase CID fragmentation reactions of protonated peptides toward the formation of a series of a- or b-type sequence product ions. Adamczyk et al. have also demonstrated that N-terminal b- and a-type ions are predominately formed following CID-MS3 analysis of the TMPP-Ac containing b-type ions produced in the first stage of tandem mass spectrometry analysis,'60 which can be particularly useful for the analysis of larger peptides, whereby incomplete sequence information is often obtained following CID-MS/MS alone. Recently, fixed-charge chemical derivatization of peptide N-termini with (N-succinimidyloxycarbonylmethyl)tris(2,4,6- trimethoxyphenyl)phosponium acetate to form an acetylphosphonium derivative has also been used to increase the sequence coverage obtained by electron capture dissociation of o‘ph051’horylated and o-glycosylated peptides.”6 Peptide N-termini have also been chemically modified with 4-sulfophenyl isothiocyanate and various other sulfonic acid derivatives to incorporate a fixed negative 163-166 charge. The introduction of a fixed negative charge on the N-terminus of the peptide, and the localization of an ionizing proton on the side chain of arginine or lysine residues contained within the peptide sequence, results in the formation of a neutral peptide molecule. To analyze the peptide via mass spectrometry, a second ionizing proton is therefore required. Since the arginine or lysine side chains are already protonated, the second ionizing proton is able to move along the peptide backbone and initiate cleavage of the amide bonds. Using this approach, a single series of y-type sequence product ions are generated. 25 Numerous chemical derivatization strategies have also been developed to direct the fragmentation reactions of protonated peptides toward the selective formation of 113, 167, 168 single characteristic sequence type product ions. For example, Surnmerfield and coworkers have demonstrated that N-terminal derivatization with phenylisothiocyanate to form the corresponding phenylthiocarbamoyl (PTC) derivative results in exclusive fragmentation of the amide bond between the first two amino acid residues under mobile proton conditions to generate a b1 ion and the complementary yn-1 ion.l67’ 168 The incorporation of a fixed-charge TMPP—Ac tag to the N-terminus of aspartic acid-containing peptides has also been employed to promote enhanced cleavage C-terminal to aspartic acid residues.1 '3 The specific information obtained regarding the presence and location of an aspartic acid residue within a peptide sequence has been shown to improve the specificity of database search analysis strategies employed for protein identification.l ‘4 Recent work in our laboratory has also examined the potential for controlling the formation of non-sequence product ions for use in selective protein identification and characterization via the introduction of fixed-charge derivatives to peptides containing 169-172 certain amino acid side chains. For example, it has been demonstrated that CID- MS/MS of peptide ions containing fixed-charge sulfonium ions on the side chains of methionine- or cysteine-containing peptides, formed by reaction with the alkylating 169-171 reagents phenacylbromide (BrCH2C0C6H5) or (3-[N-bromoacetamido]propyl)- methylphenacylsulfonium bromide (BAPMPS),172 respectively, results in exclusive fragmentation of the derivatized side chain and the formation of a single “diagnostic” 26 product ion via the neutral loss of methylphenacylsulfide (CH3SCH2COC6H5) (Scheme 1.3A and 1.3B, respectively). The mechanisms for these selective fi'agmentation reactions have been demonstrated, by using multistage tandem mass spectrometry of regioselectively deuterated sulfonium ion containing tryptic peptides and by molecular orbital calculations, to occur via SN2 neighboring group participation reactionsno’ 172 Notably, the selective fragmentation reactions of the side chain sulfonium ion derivatized peptides have also been shown to occur independently of the proton mobility of the precursor ion. Additional structural information on peptide ions initially identified from the selective fragmentation reactions is readily achieved by subjecting the characteristic MS/MS product ion to further dissociation by MS3 in a quadrupole ion trap mass spectrometer or by energy resolved “pseudo” MS3 in a triple quadrupole mass spectrometer. 27 EEEmNIo - I: + E EEEmNIo - I: + I: O I I HrZ\ + #051 I _ O ImNIo- All mngAHHU ImNIo- ‘IIIIIII mngAHO +555: + s: O = I O 2412\0 Z/OHI __ I I __ wIooouNIonI arZ\o 2/ > O _ I C w m I _ I Em fio mmim.\.v\+m/\/\7H/n__v\/VA mm o I\+/oNI +C+GVH=G + E“— O O __ I N o N “F er \o m/ 1. I08 Iona arZ\ m C. w I E fiO MN Am I\+/oNI Scheme 1.3 Gas-phase fragmentation reactions of phenacylsulfonium ion derivatized methionine- and cysteine-containing peptides. 28 A number of approaches described in the literature have also demonstrated that selective dissociation can be achieved by first generating a radical site within a peptide or 173-176 protein. For example, Ly et al. recently described an approach whereby reactive tyrosine residues within individual proteins were converted to 3-iodotyrosine under natively folded conditions.175 The modified tyrosine-containing proteins were ionized via ESI, introduced to a linear quadrupole ion trap and subsequently subjected to UV photodissociation which resulted in the formation of a radical site on the aromatic ring of the modified tyrosine residues. Following re-isolation and low-energy CID, radical- directed selective cleavage adjacent to the tyrosine residues was observed, resulting in the formation of a-type sequence product ions. Similar results were also obtained for proteins containing exposed histidine residues, which were also shown to be susceptible to iodination. The information generated via this site-specific fragmentation could potentially be used to reduce the computational time associated with database search analysis strategies. 1.3.4.3 Chemical Derivatization Strategies for Quantitative Analysis of Protein Expression Numerous chemical derivatization approaches have also been described to direct the fragmentation reactions of peptides toward the selective formation of isotopically encoded low mass product ions for quantitative peptide analysis (see Section 1.2.2). Although the MS/MS-based quantitative analysis strategies described in Section 1.2.2 above overcome the limitations associated with MS-based approaches, the low m/z product ions required for quantitation are generally observed below the low mass cut-off 29 introduced during MS/MS in quadrupole ion trap mass spectrometers, thereby precluding their use for quantitative analysis in this type of instrumentation. Thus, to date, the majority of proteomic experiments utilizing these reagents have been performed using quadrupole time-of-flight (QTOF), time-of-flight/time-of-flight (TOF/T OF), and to a lesser extent, hybrid triple quadrupole/linear ion trap mass spectrometersrn'181 However, a new “High Amplitude Short Time Excitation” (HASTE) dissociation technique (an analogous method is termed Pulsed Q Collision Induced Dissociation (PQD) in the commercially available ion trap mass spectrometry platforms available from Thermo Scientific), has been recently implemented which enables the low m/z product ions normally excluded fiom CID spectra to be observed.182 Using the iTRAQ approach coupled with PQD-MS/MS on a Thermo linear quadrupole ion trap, Meany and coworkers were able to successfully quantify carbonylated proteins enriched from rat skeletal muscle mitochondria.183 Griffin et al. also compared PQD-MS/MS in a linear quadupole ion trap mass spectrometer with MS/MS in a quadrupole time-of-flight mass spectrometer for the quantitative analysis of iTRAQ labeled peptides derived from a standard yeast lysate mixture.184 It was determined that similar quantitative accuracy could be achieved using both instrumentation platforms, although careful tuning of the relative collision energy was required for efficient fragmentation by PQD. Protein kinases extracted from cells which had been incubated with various drugs have also been quantified using the iTRAQ approach and PQD-MS/MS in a linear quadrupole-Orbitrap 1 85 mass spectrometer. It has also been demonstrated that the iTRAQ MS/MS based quantitative analysis strategy can be used in conjunction with the quadrupole ion trap by performing multiple 3O 3 186 stages of mass analysis (i.e., MS ). For example, chemical derivatization with the iTRAQ reagent not only labels the N-terminus of a peptide, but also the lysine side chain. Thus, tryptic peptides with a modified lysine residue present at the C-terminus will produce a y1 product ion at m/z 291 following CID-MS/MS. To generate the low m/z iTRAQ reporter ions required for quantitation, the y] product ion can be isolated and subjected to data dependent CID-MS3. Using this approach peptide identification is achieved in the MSMS scan, while quantitation is achieved via MS3. Regardless of the instrumentation platform employed for analysis, each of the quantitative MS/MS based derivatization strategies requires an ionizing proton to initiate cleavage of the stable-isotope containing label to generate the low m/z reporter ions required for quantitation. Therefore, the fragmentation reactions associated with these strategies are expected to be highly dependent upon the proton mobility of the precursor ion, such that the characteristic isotopically encoded low mass reporter ions may often be observed at sufficient levels in only a sub-set of the total peptide ions selected for MS/MS to enable their use for quantification. Another limitation is that the desired fragmentation pathway giving rise to the low m/z reporter ions of interest is typically only one of many dissociation channels, including those resulting in the formation of b- and y-type sequence product ions, thereby “diluting” the spectrum and limiting the dynamic range for quantitative analysis. To overcome some of these challenges, the SELECT approach described in Section 1.3.4.2 above has been applied to quantitative analysis, via the incorporation of light and heavy isotopically encoded labels into the fixed-charge chemical derivatization reagents. In the initial report by Reid et al., methionine-containing peptides were 31 alkylated with either “light” lH5-phenacylbromide or “heavy” 2H5-phenacylbromide to form a fixed-charge sulfonium ion on the side chain of methionine residues.169 The light and heavy phenacyl sulfonium ion fixed-charge containing peptides were subsequently combined and subjected to LC-MS/MS analysis. The relative abundances of the neutral loss product ions CH3SCH2COC6H5 (166 Da) and CH3SCH2COC62H5 (171) generated by CID-MS/MS were then used for quantitative analysis. In contrast to the MS/MS based quantitative analysis strategies described above, the neutral loss product ions required for quantitation via the SELECT approach are formed independent of the proton mobility of the precursor ion such that these product ions will be observed all of the time. Furthermore, formation of the neutral loss product ions at a m/z value close to that of the selected precursor ion circumvents the low mass cutoff limitation of the ion trap. Characterization of the identified and quantified methionine peptides may readily be achieved by subjecting the common neutral loss product ion formed from the light or heavy labeled peptides to MS3 analysis. One of the potential disadvantages to this initial approach, however is that some, albeit limited, chromatographic separation of the 1H5 and 2H5 forms of the fixed-charge sulfonium ion derivatives was observed following reversed-phase chromatography of these peptides. Although not investigated to date, light and heavy isotopically encoded labels could also be readily incorporated into the previously described alkylating reagent (3-[N-bromoacetamido]propyl)- methylphenacylsulfonium bromide172 to enable the selective MS/MS based quantitative analysis of cysteine-containing peptides. 32 1.4 Aims of this Dissertation The aims of this dissertation are: l. to examine the origin of ex vivo sample handling related oxidative modifications of methionine-, S-alkyl cysteine- and tryptophan-containing peptides obtained from typical in-solution or in-gel proteolytic digestion strategies and evaluate conditions for deliberately controlling the oxidation state of these peptides; 2. to investigate the mechanisms and proton mobility dependence to the fragmentation reactions of model S-alkyl cysteine sulfoxide-containing peptides; 3. to develop an automated electrospray ionization (BSD-HPLC neutral loss MS/MS and date dependent pseudo MS3 analysis method in a triple quadrupole mass spectrometer for the selective gas-phase identification, characterization and differential quantitative analysis of fixed-charge sulfonium ion-containing peptides, and; 4. to identify and quantify methionine oxidation in the Ca2+lcalmodulin dependent serine/threonine phosphatase calcineurin using a fixed-charge chemical derivatization and tandem mass spectrometry-based approach similar to that outlined in aim 3. 33 CHAPTER TWO Instrumentation 2.1 Mass Spectrometry Mass spectrometry is an analytical technique that involves the ionization of a sample to generate gas-phase ions, separation of the resultant ions based on their individual mass-to-charge (m/z) ratios, and measurement of the m/z and abundance of each ion reaching a detector. Optionally, individual gas-phase ions can be isolated and subjected to fragmentation, followed by mass analysis of the resultant product ions. The main components of a mass spectrometer are illustrated in Figure 2.1. Ion Formation Ion Separation Ion Detection Sample Ionization Mass [ Introduction ]:>[ Source ]=>[ Analyzer ]3[ Detector ] Vacuum Data Pump Handling Data System Data I I Output I Mass spectrum Figure 2.1 Components of a mass spectrometer (adapted from “What is mass spectrometry”. www.asms.org). 34 2.1.1 Ionization The ionization source is responsible for converting a sample of interest into gas- phase ions. Numerous ionization techniques are currently employed for mass spectrometry analysis including electron impact (EI), chemical ionization (CI), fast atom bombardment (FAB), electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). ESI and MALDI are generally used for the analysis of biological molecules, such as peptides and proteins, and therefore are described in more detail below. 2.1.1.1 Electrospray Ionization (ESI) Electrospray ionization (ESI), an atmospheric pressure ionization technique, was first introduced by Penn and coworkers in the late 1980’s.187 ESI results in minimal analyte fragmentation, is suitable for analyzing polar and ionic compounds, and produces multiply charged pseudomolecular ions ([M+nH+]"+), which enable high molecular weight species, such as peptides and proteins, to be readily analyzed by many types of mass analyzers. ESI can be performed in direct infirsion mode, whereby a sample of interest is dissolved in an appropriate liquid solvent and then introduced to the mass spectrometer via a syringe pump. However, ESI can also be readily coupled with high performance liquid chromatography (HPLC) to directly ionize an analyte as it elutes from a chromatographic column. The ability to couple ESI directly with HPLC is particularly advantageous for the analysis of complex mixtures, whereby separation prior to mass spectrometry analysis is required. When sample quantity is limited, the use of 35 nanoelectrospray ionization (nESI), which is typically operated at flow rates of less than 1 uL/min, may be beneficial. l 88 In ESI, a sample is initially pumped through a small diameter stainless steel or fused silica capillary tubing, the tip of which is maintained at atmospheric pressure in the source region of the mass spectrometer. A high potential is applied between the capillary tip and a counter electrode, which results in charge accumulation at the surface of the liquid. A combination of charge repulsion at the surface, and the presence of an electric field, enables the surface tension of the liquid to be overcome and the liquid subsequently expands into a Taylor cone. The tip of the Taylor cone then elongates into a liquid filament, which breaks to yield an electrostatic spray of charged droplets. Finally, solvent evaporation, in the presence of high temperature and/or a sheath gas, and charge repulsion, result in a series of coulomb fission events to yield gas-phase ions. The mechanism by which gas-phase ions are produced from the charged droplets has been explained by two competing theories, the charge residue model (CRM)189 and 190, 191 the ion evaporation model (IBM). In the CRM, successively smaller droplets are produced when the surface charge density of the droplet exceeds the Rayleigh stability limit (i.e., the force due to the repulsion of the surface charges becomes equal to the surface tension force of the liquid). The result of this process is the formation of droplets which contain only a single analyte molecule. A free gas-phase ion is then produced as the remaining solvent evaporates. In the IBM, it is also proposed that a series of Rayleigh instabilities produce successively smaller droplets. However, in contrast to the CRM, the surface electric field of the droplet becomes strong enough to overcome the solvation forces, causing direct emission of the analyte ions from the droplet surface into the gas- 36 phase. Experimental evidence suggests that large molecules, such as proteins, ionize according to the CRM, while small molecules ionize according to the IBM.192 2.1.1.2 Matrix-assisted Laser Desorption Ionization (MALDI) Matrix-assisted laser desorption ionization (MALDI) was developed by Karas and Hillencamp in the late 1980’s for the purpose of analyzing large molecular weight proteins.l93 MALDI is performed by dissolving an analyte of interest in an excess of matrix containing a UV absorbing chromophore. After the analyte-containing matrix has been spotted onto a metal target and allowed to crystallize, the target is placed under vacuum in the source region of the mass spectrometer. The target is then bombarded with short duration laser pulses, which induces rapid heating of the crystals and sublimation of both the analyte and matrix into the gas-phase. Although formation of analyte and matrix ions can occur at any time during this process, the exact mechanism of ionization is still . 194 the subject of much debate. Similar to ESI, MALDI results in limited analyte fragmentation and is suitable for analyzing polar and ionic compounds. Although MALDI is used for the analysis of high molecular weight species, the formation of singly charged pseudomolecular ions via this technique limits the types of mass analyzers that may be employed. Also, due to the nature of the ionization process, MALDI cannot be coupled on-line with HPLC. For this reason, MALDI was not explored as a potential ionization technique in the studies reported herein. 37 2.1.2 Mass Analyzers The mass analyzer component of the mass spectrometer separates gas-phase ions according to their individual m/z ratios. A number of mass analyzers are currently employed in the field of proteomics including the quadrupole, quadrupole ion trap, time- of-flight (TOF) and Fourier transform ion cyclotron resonance (FT-ICR) mass analyzerslgs. The quadrupole and quadrupole ion trap mass analyzers were exclusively used in the studies reported here and are described in more detail below. 2.1.2.1 The Quadrupole Mass Analyzer The ideal quadrupole mass analyzer consists of four parallel hyperbolic shaped electrodes; however, electrodes of circular cross-section are typically employed for both economic and practical reasons. Ions entering the quadrupole along the z-axis experience an electric field which is created by applying separate potentials to each pair of diagonally opposite electrodes. The applied potentials are expressed in equation (1), where U is the direct current (DC) potential, V is the zero-to-peak amplitude of the radio frequency (RF) voltage, (0 is the angular frequency (a) = 21:11, where u is the frequency of the RF field), and t is time. (D0 = + (U - V cosmt) and - (D0 = - (U - V coswt) (1) As the ions travel along the z-axis, the force induced by the electric field also accelerates the ions in the x and y dimensions. The force experienced by the ions along the x- and y- 38 axis is expressed in equations (2) and (3), respectively, where m is the mass of an ion, 2 is the number of charges on a given ion, and e is the charge of an electron (1.60 x 10'19 C). 2 Fx=mid—x=-ze29 (2) dt2 0x dzy 6CD Fy=m——=-ze-— (3) dt2 5y In equations (2) and (3), (I) is expressed as a function of (Do as shown by equation (4), where r0 is the distance from the center of the quadrupole mass analyzer to the electrodes. 2 2 x — U—Vcoscat ¢xy= the combined number of basic residues (Arg, Lys or His).76 For the methionine sulfoxide- and S-methyl cysteine sulfoxide-containing peptide M(ox)C(S-me)(ox)K, the neutral loss of CH3SOH could result from side chain cleavage of the S-methyl cysteine sulfoxide or methionine sulfoxide residues, or both. However, given the minimal loss of CH3SOH observed during MS/MS of the methionine sulfoxide- and S-carboxyamidomethyl cysteine sulfoxide-containing peptide M(ox)C(S-cam)(ox)K (where cam= CHZCONHZ), and the similar fragmentation behaviors of the S-methyl and S-carboxyamidomethyl cysteine sulfoxide-containing peptides C(S-me)(ox)K and C(S- carn)(ox)K (see below), it is expected that loss from the S-methyl cysteine sulfoxide residue was the dominant process. Essentially identical spectra to those discussed above for dissociation of the S- methyl cysteine sulfoxide-containing peptide ions were also obtained following CID- MS/MS of the singly, doubly or triply protonated precursor ions of the S- carboxyarnidomethyl cysteine sulfoxide-containing peptides C(S-carn)(ox)K, C(S- cam)(ox)R and M(ox)C(S-cam)(ox)K formed by reaction with iodoacetamide and oxidation with hydrogen peroxide (Table 5.1). Hydrogen/deuterium exchange followed 107 by MS/MS, as well as MS3 of the resultant [M+nH-H2NCOCHZSOH]nH+ product ions, confirmed that the side chain neutral loss of HzNCOCH2SOH (107 Da) from these peptides also occurred exclusively via the charge-remote cis-1,2 elimination reaction mechanism shown in pathway A of Scheme 5.1. As expected, the spectra obtained by MS3 dissociation of the [M+nH-H2NCOCH2SOH]"H+ product ions were identical to those from the S-methyl cysteine sulfoxide-containing peptides (data not shown). Notably, the CIR values calculated for the losses of CH3SOH or H2NCOCH2SOH from the doubly protonated (i.e., mobile proton) S-methyl and S-carboxyamidomethyl cysteine sulfoxide-containing derivatives of GAILCGAILK (CIR values of 3.9 and 2.5, respectively) and GAILCGAILR (CIR values of 4.0 and 3.1, respectively), were significantly higher than the CIR values calculated for the loss of CH3SOH from the doubly protonated precursor ions of the equivalent methionine sulfoxide-containing peptides GAILM(ox)GAILK (CIR 0.1) and GAILM(ox)GAILR (CIR 0.2) (Table 5.1). Furthermore, while the mechanism for the side chain fragmentation from the doubly protonated methionine sulfoxide derivative was previously demonstrated to occur via a charge-directed mechanism,81 the side chain losses from the S-alkyl cysteine sulfoxide- containing peptides were observed to occur via the charge-remote mechanism. These observations can be rationalized as being due to the increased acidity of the (it-hydrogen atom of the S-alkyl cysteine sulfoxide side chain compared to the B-hydrogen atoms of the methionine sulfoxide side chain. 108 Table 5.1 Cleavage intensity ratio (CIR) values for the “non-sequence” side chain fragmentation reactions of S-alkyl cysteine sulfoxide- and methionine sulfoxide- containing peptide ions. 8) “Non-mobile” proton [76, 81]. b’ “Partially mobile” proton [76, 81]. °’ “Mobile” proton [76, 81]. d) Contributions from sequential losses are included in the CIR calculation. 6) Corresponds to side chain cleavage from Cys(X)(ox) and/or Met(ox). f) Observed exclusively as a charged loss. g) Observed as both charged and neutral losses. 109 Precursor ion Calculated CIR value Peptide x= d) d) d) -CH3SOH from charge state -XSOH -(X-H) -COz Met(ox) GAILC x GAILK b ( )(OX) +1 ) 11.0 0 ‘ ' (C(X)(ox)K) CH3 ) +2c 3.9 0 — _ b) +1 10.8 0 ’ — CHZCONHZ c) +2 2.5 0 ‘ - b) +1 6.5 0 5.4 — +2 2.4 0 0 ‘ b) +1 5.4 6.6 — - CHZCHZCONHZ c) +2 3.9 2.6 - ‘ b) +1 10.0 2-0 — ' c) f) f +3“) 0 10.9 ) - - GAlw(X)(ox)GAILR +18) 11 o 0 (C(X)(ox)R) CH3 ) ' +26 4.0 0 ‘ - a) +1 10.9 0 ‘ _ +2 3.1 0 ‘ 7 a) +1 5.3 0 6-6 — CH2C02H C) +2 3.5 0 0 ‘ a) +1 4.9 6-9 " ' CH2CH2CONH2 c) +2 4.0 2.2 - ‘ a) +1 10.8 1.2 — - c) f) CH2CH2C5H4N +2 0 12.0 - - f +3“) 0 11.7 ) - - 110 Table 5.1 (continued) Precursor ior Calculated CIR value Pe tide X= I -CH SOH from p charge state -XSOHd) -(X-H)d) -C02d) 1:4 et(ox) VTM(ox)GHFC(X)(ox)NFGK b) e) +1 12 9 0 — — (M(OX)C(X)(0X)K) b ' CH3 +2 ) 12.66) 0 — - +3c) 5.76) 0 — — b +1 ) 12.2 0 — 0.2 b CH2(1)“le +2 ) 12.2 0 — 0.1 +3” 5.1 0 - 0.1 b +1 ) 7.0 0 6.9 0 b CH2C02H +2 ) 9.0 0 4.7 0 C +3 ) 4.0 0 6.3 0 b +1 ) 7.3 6.5 - 0.1 CH20120011112 +2” 9.3 4.6 — 0 +3” 3.7 8.0 - 0 b +1 ) 11.2 2.5 — 0.1 b CH2CH205H4N +2 ) 1.5 12.53) — 0 r +3” 0 14.0 ) — 0 GAILM(ox)GAILK b) +1 - - — 93 (M(OX)K) ' +2c) — — — 0.1 GAILM(ox)GAILR a) (M(ox)R) +1 ) - - - 10.9 +2c — - - 0.2 111 Further examples of the proton mobility dependence to the preferential side chain fragmentation behavior of S-carboxyamidomethyl cysteine sulfoxide residues are shown in Figure 5.2 and in Table 5.2. Figures 5.2A and 5.2B show selected mass spectra obtained by capillary HPLC-mass spectrometry analysis of an S- carboxyarnidomethylated and oxidized tryptic digest of bovine serum albumin, prepared by reduction and alkylation with iodoacetamide, followed by oxidation with hydrogen peroxide. The CID-MS/MS product ion spectra obtained by dissociation of the doubly (m/z 797.5) and triply (m/z 532.3) protonated precursor ions in Figure 5.2A are shown in Figures 5.2C and 5.2D, respectively. SEQUEST analysis of these spectra, followed by manual verification of the search results, enabled the peptide to be identified as the singly oxidized LKPDPNTLC(S-cam(ox))DEFK. It can be seen that the non-sequence loss of HzNCOCHZSOH dominated the product ion spectrum (82.4% total product ion abundance) for the doubly protonated precursor ion (i.e., partially mobile proton conditions). In contrast, the same loss (including the sequential loss of H20) was only observed at 20.5% total product ion abundance from dissociation of the triply protonated precursor ion (i.e., mobile proton conditions), with numerous abundant sequence product ions also being observed. The remaining doubly, triply and quadruply protonated ions in Figure 5.2A were found to correspond to the singly oxidized peptide LKPDPNTLC(S- cam(ox))DEFKADEK (Table 5.2). Interestingly, dissociation of the doubly protonated precursor ion at m/z 989.1 in Figure 5.2B resulted in the sequential loss of up to three H2NCOCH2SOH neutral molecules, with a combined total product ion abundance of 89.4% (see Figure 5.2B). This peptide was subsequently identified by SEQUEST analysis and manual verification of the 112 search results as the triply oxidized peptide C(S-cam(ox))C(S-cam(ox))AADDKEAC(S- cam(ox))FAVEGPK. Similar to that seen in Figures 5.2C and 5.2D however, dissociation of this peptide (Figure 5.2F) under mobile proton conditions (i.e., the triply protonated precursor ion at m/z 660.2 in Figure 5.2B) resulted in substantially less abundant non- sequence ions (combined total product ion abundance of 15.1%) and the formation of extensive sequence information. These data, as well as those obtained from the oxidized peptides in Table 5.2, indicate that the formation of abundant product ions corresponding to the loss of HzNCOCH2SOH under conditions of low proton mobility allows both the presence and number of S-carboxyamidomethyl cysteine sulfoxide residues within a given peptide to be readily determined. This information can be particularly useful to assist in identification of the peptide when sequence ions are present at low abundance. For example, automated SEQUEST analysis of the spectra obtained by CID-MS/MS of the triply protonated precursor ion of the peptide LKEC(S-cam(ox))C(S-cam(ox))DKPLLEK (Table 5.2) resulted in this peptide being included as only the second ranked candidate. However, manual interrogation of the spectrum enabled the observation of product ions corresponding to the loss of up to two HzNCOCHZSOH neutral molecules with a combined total product ion abundance of 92.8%, indicating that the peptide contained two S-carboxyamidomethyl cysteine sulfoxide residues. Given that the higher ranked candidate sequence corresponded to a peptide with only one oxidized cysteine residue, it could be immediately discounted, thereby bringing the correct sequence to the top of the candidate list. This is an approach that is directly analogous to those recently described 113 for identification of phosphoserine or phosphothreonine containing peptide ion product ion spectra that give rise to the dominant loss of H3PO4 upon CID-MS/MS.219’ 220 114 Figure 5.2 Capillary HPLC-mass spectrometry analysis of S—carboxyamidomethyl cysteine sulfoxide- (S-cam(ox)) containing peptides from an oxidized tryptic digest of bovine serum albumin. Mass spectra obtained from region 1 (28.2-29.2 minutes) and region 2 (22.5-23.5 minutes) of the LC-MS chromatogram (shown in the inset to panel A) are shown in panels A and B, respectively. The CID-MS/MS product ion spectra obtained from dissociation of the doubly (m/z 797.5) and triply (m/z 532.3) protonated precursor ions of the singly oxidized peptide LKPDPNTLC(S-cam(ox))DEFK in panel A are shown in panels C and D, respectively. The CID-MS/MS product ion spectra obtained from dissociation of the doubly (m/z 989.1) and triply (m/z 660.2) protonated precursor ions of the triply oxidized peptide C(S-cam(ox))C(S-cam(ox))AADDKEAC(S-— cam(ox))FAVEGPK in panel B are shown in panels E and F, respectively. Key: A = -H2NCOCH280H; O = -H20; * = -NH3. 115 r 8.2 _ 8.2 . 8.. So 8.. 8N 8.8. . 8mm 11 a 41 1 411 141 14 /0 ax PA w“ bra: OH 4 1 1 m. 2 4.; W. +N Q 1 m» 1 9 § NE » ..omNzoooszNo- 0V. :omNmooozN..- - - m lam—2+ m. a memN=8ozN=Y I N + o o; momNmoouzN..- m momNmoousz- Q a +N -8. 8N=+:0mN=oouzN=Y -8. EN... +83 83 coo. 83 com. cco— cow coo ccv coo. ccE ooN_ ccc~ cow coo ccv .11 - .1 .1 . . ....1 . 1. . p w -L 1.1 a . 11F . p W .. . q. -1; 11.11.11 % <4... N; NN... .. a <+N r m. Du <+~~§ m. \ momNmoooszm- W 6N:+=0mN=ooozN=v- AON=N+ N m mom .582 ..No- .. i m. memNmoooszN-m U m momNzooosz- too. :OmNzooozN..- -8. +m...N E38 co: co: co? co: coo 2.x. com com com— rooflt cog . co_: omo com com com p p p F b — - _ p . p — . . _ P b b n J 1 .1 . . : 1. . 1 - . rJLII /0 a _._ j A .88. fi .14 mo ..owo 1 9:5 2:: 53:83“ +nEN+Sa r m. SE»: 22 e +N 8 on 8 8 ON 2 o % _=N+2_ m. _l _ _ _ — _ m p . - p - .Ifl a 1?" I. a - . m. - w. r a .88 m . W. $.59: m. .. z .. m NNNW \ 1 9 N88 m . N . m. NEE... .8: < a +N_=n+2_ too. . a roo— 886 BR N .8. #5:): Bone 116 Table 5.2 Percent total product ion abundances for the “non-sequence” side chain neutral loss of H2NCOCH2SOH fi'om S-carboxyamidomethyl (S-cam) cysteine sulfoxide- containing peptide ions of an oxidized tryptic digest of bovine serum albumin. 93+ 3&8 snow - t 8:289.sz...xeosermouemzfiemo - can 39% 88.2. 1 22.389025422683982 - .02 em; 93.. i xmgumEroeermvofizEb... . i €98 ......NN - unmanxeoéoéufiznafii. i .....em 33.. i . ao9...6.89mUni:no:25.§9w6:6.§9m.05.:> - so: ......No - - v...1.488535389888265: - 1 ......me i i 2.1553588?028353682coaxecseoéumogoz> t i 98.8 9.8 . xomoz> t t .oNNn t - om.Qeoeeoaoufieéow.uofixeczootmmum; - - 9...: 9+8 1 v:em>tmon.5 [F] 3.0 kcal mol'l [T52] 17.8 kcal mol'l *‘o '"o' _e He a3. a5 .2: .54 °9 q \o .... co: “6' '8 .. as as ..M l .54 9. °< q <~> O a O <1: ‘58 fl .9 m Figure 5.5 Optimized precursor, transition state and product ion structures (at the B3LYP/6-31+G(d,p) + ZPVE level of theory) for (A) the loss of CH3SOH from the neutral model system CH3CONHCH(CHZS(O)CH3)CONHCH3 (equivalent to the loss of XSOH from the S-methyl cysteine sulfoxide—containing peptides) and (B) the loss of CH2CHCONH2 from the neutral model system HzNCOCHzCHZS(O)CH3 (equivalent to the loss of CHZCHX’ from the S-amidoethyl cysteine sulfoxide-containing peptides), via S-membered cis-1,2 elimination pathways. 127 Structure [TS]] in Figure 5.5A shows the predicted low energy transition state structure found for the loss of CH380H (equivalent to the loss of XSOH from the S-alkyl cysteine sulfoxide-containing peptides) from the model system CH3CONHCH(CHZS(O)CH3)CONHCH3 via a S-membered cis-l,2 elimination pathway. IRC calculations, followed by geometry optimizations, were performed to locate the precursor ion [B] and the intermediate product ion [C] associated with this reaction coordinate. A filrther lower energy precursor ion [A] was also located. The relative energy of the transition state (+23.1 kcal mol'l) was then calculated with respect to the energy of this precursor ion. Structure [T82] in Figure 5.5B represents the low energy transition state structure found for the neutral loss of CHzCHCONHz (equivalent to the loss of CHZCHX’ from the S-amidoethyl cysteine sulfoxide-containing peptides) from the model system HZNCOCHZCHZS(O)CH3. After IRC calculations were performed, the associated precursor ion [E], the intermediate product ion [F], and a lower energy precursor ion [D] were located. The energy of the transition state [T82] with respect to that of the low energy precursor ion was found to be +17.8 kcal mol'l. The total energies, zero point vibrational energies (ZPVE) and relative energies obtained for all the optimized transition structures are given in Table 5.3. The similar predicted activation barriers for the two fragmentation reactions (i.e., -XSOH and -CH2CHX’) are therefore consistent with the experimentally observed product ion abundances. 128 Table 5.3 Total energies (Etotal), zero point vibrational energies (ZPVE) and relative energies (Erel) computed for the precursor ions, transition states and product ion structures associated with each reaction pathway at the B3 LYP/6-31 + G (d,p) level of theory. Structure Em“ (Hartree) ZPVE (kcal mol") Em. (kcal mol")a) A 4008.5887922 137.44180 0.0 B 40085777340 137.34926 +63” T81 -1008.5459784 133.67822 +231") C 4008.5807054 135.69649 +3.3”) D -761.2438197 85.48194 0.0 E -761.2414516 85.01374 +1.06” TSZ -761.2090719 81.44402 +17.8” F -761.2348465 82.84947 +3.0“) G 839.6268943 111.59516 0.0 TS3 839.5874485 107.78148 +209“) H -839.6238461 109.49210 -02“) I 840.0063802 120.51 150 0.0 TS4 839.9849623 1 17.08063 +100“) J 840.0105422 118.44960 47°) 3’ Erel = total energy + (ZPVE). b) Energy relative to Structure A. c) Energy relative to Structure D. d) Energy relative to Structure G. c) Energy relative to Structure 1. MS3 dissociation of the neutral loss [M+H-CH2CHCONH2]+ product ion from Figure 5.4A resulted in the exclusive loss of H20 (Figure 5.4C). As shown in Scheme 5.3, the neutral loss of H20 may potentially occur via a charge-remote cis-1,2 elimination reaction (Scheme 5.3, pathway A) to yield an amino thiopropanoic acid containing 129 product ion (10). Alternatively, following intrarnolecular proton transfer from the peptide backbone to yield the protonated sulfenic acid intermediate (11), the loss of H20 could also potentially occur via a charge-directed E2 elimination reaction to yield (12) (Scheme 5.3, pathway B), or via SN2 neighboring group participation reactions involving the N- or C-terminal amide carbonyl groups adjacent to the side chain to yield a cyclic five- membered 1,2 oxathiolane product (13) (Scheme 5.3, pathway C) or a six-membered 1,3,5 oxathiazine product (structure not shown), respectively. Hydrogen/deuterium exchange can readily be used to differentiate between the charge-remote and charge- directed fragmentation pathways proposed in Scheme 5.3, which would result in the loss of H20 and HOD, respectively. MS3 dissociation of the [M+D-CH2CHCOND2]+ product ion formed by dissociation of the uniformly deuterated [M+D]+ precursor ion of the C(S-ae)(ox)K peptide resulted primarily in the neutral loss of D20 (100% relative abundance), with only minor loss of HOD observed (approx. 10% relative abundance) (data not shown). This suggested that the hydroxyl hydrogen in the sulfenic acid side chain (structure (4)) of the [M+D-CH2CHCOND2]+ MS/MS product ion underwent exchange (“scrambling”) with a deuterium on the peptide backbone prior to its dissociation by MS3. Under these conditions, the charge-remote mechanism would primarily result in the neutral loss of HOD, while the charge-directed mechanisms would primarily result in the loss of D20. Therefore, we propose that the loss of H20 from the [M+H-CH2CHCONH2]+ ion occurs via a charge-directed process. The MS4 spectrum obtained by dissociation of the [M+H-CH2CHCONH2-H20]+ product ion is shown in Figure 5.4D. Observation of the b4, b5 and y6 ions in this spectrum, corresponding to 130 cleavage of the amide bonds adjacent to the site of the cysteine side chain, suggests that the E2 elimination reaction had occurred. However, based on our recent study on the mechanisms responsible for the side chain fragmentation reactions of methionine fixed charge sulfonium ion-containing peptides, where formation of a mixture of cyclic five- and six-membered product ions were found to be favored,170 the 8N2 neighboring group participation reactions cannot be ruled out. 131 o w m +m+cerm\ /mia m\ o a: 33.2 of- 2 32.3.: + F m o\ /o.._4 G 5.355: a: / __ 9:23 S O m / PV— 2 m o z \ ../ is m N .. O C U O O H.— A an A: = Alll E+§Ym\ /n_w._9_. m2 0 «35¢ 0 08¢ . n c ._ .. .... . o that «a m ( 3.2: a: \ ”mow a n e _). m m _ E 335...: o 2 3355: 132 Scheme 5.3 Proposed mechanisms for the loss of H20 from cysteine sulfenic acid- containing peptide ions. In contrast to the data shown in Figures 5.4A and 5.4B, and in Table 5.1, where the product ions formed via the losses of HzNCOCHzCHZSOH and CHZCHCONHZ (and/or CHZCHCONH2+H20) from the S-amidoethyl cysteine sulfoxide-containing peptides were observed at similar abundances for all the precursor ion charge states examined (i.e., independently of the proton mobility of the peptide ions), the product ion abundances of the side chain losses from S-pyridylethyl cysteine sulfoxide-containing peptides were observed to vary significantly depending on the proton mobility of the precursor ion. For example, Figure 5.6 shows the product ion spectra obtained by CID- MS/MS of the singly, doubly and triply protonated precursor ions of the S-pyridylethyl cysteine sulfoxide-containing peptide VTMGHFCNFGK (M(ox)C(S-pe)(ox)K) (where pe= CH2CH2C5H4N) (Figures 5.6A, 5.6B and 5.6C, respectively). The CIR values calculated for the loss of pyridylethyl sulfenic acid (-XSOH, 155 Da, where X= CHzCH2C5H4N) and vinylpyridine (-CH2CHX’, 105 Da, where X’=C5H4N) from these ions, as well as those from the singly and doubly protonated S-pyridylethyl cysteine sulfoxide-containing peptides GAILCGAILK (C(S-pe)(ox)K) and GAILCGAILR (C(S- pe)(ox)R) are shown in Table 5.1. Note that the CIR values shown in Table 5.1 for the loss of CHZCHX’ also take into account the abundance of product ions formed via the combined losses of CHZCHC5H4N and H20 (CHzCHX’+H20, 123 Da). It can be seen from Figure 5.6A that CID-MS/MS of the singly protonated precursor ion of M(ox)C(S- pe)(ox)K resulted primarily in the loss of NC5H4CH2CH280H (XSOH), with the losses of CHzCHC5H4N (CHZCHX’) or CHZCHC5H4N+HZO (CH2CHX’+H20) observed at somewhat lower abundances. In contrast, the losses of CHZCHC5H4N (CHZCHX’) and 133 CHzCHC5H4N+H20 (CHZCHX’+H20) from the doubly and triply protonated precursors (Figures 5.6B and 5.6C, respectively), were observed as the most abundant products. Furthermore, the observation of these losses as charged (protonated) species was observed to increase with increasing charge state (proton mobility). Similar trends were also observed from dissociation of the C(S-pe)(ox)K and C(S—pe)(ox)R peptide ions (Table 5.1). 134 0100‘ A -NC5H4CH2CHZSOH o _— a "g -(CH2CHC5H4N+H20) D _. ..D < -(NC5H4CH2CHZSOH+H20) 'CHzCHCsH4N S»; _ -CH3SOH :3 _ -(NC5H4CH2CHZSOH+CH3SOH) [M+H]+ =4 \ | z O\° I I I I‘ I I I I I II ‘ I‘ I I 400 600 800 1000 1200 1400 100- -CHCHCHNHO 8 B ( 2 5 4 + 2 ) g _ -CH2CHC5H4N “U {:1 :5 _ .0 <1: g -(CH2CHC5H4N+H++HZO) é fi-NC5H4CH2CHZSO [M+2H]2+ -(CH2CHC5H4N+H+) . .1 °\° ‘1 I l I I I I 1 1 1 I l 1 I 200 400 600 800 1000 1200 1400 100 ‘ - + o C (CHZCHC5H4N+H ) O c: _ <6 "G :1 :s _ .0 <5, _ -(CH2CHC5H4N+H++H20) > *5 [M+3H]3+ T) m i °\° I l I‘ I I I l I l I I I l I 200 400 600 800 1000 1200 1400 m/z Figure 5.6 CID-MS/MS product ion spectra of the methionine sulfoxide- and S- pyridylethyl cysteine sulfoxide-containing peptide VTMGHFCNFGK (M(ox)C(S- pe)(ox)K). (A) [M+H]+ ion. (B) [M+2H]2+ ion. (C) [M+3H] 135 + . 1011. The differences in fragmentation behavior observed between the S-pyridylethyl and S-amidoethyl cysteine sulfoxide-containing peptides can be rationalized by taking into consideration the expected sites of protonation in the singly and multiply protonated precursor ions of each of the peptides, and the effect of the site of protonation on the transition state barriers for the loss of CHZCHX’ via the charge remote cis-1,2 elimination pathway. The proton affinity of an appropriate model for the S-pyridylethyl cysteine sulfoxide side chain is 4-ethylpyridine (227.3 kcal mol“).245 Although this is lower than the proton affinities ofarginine (251.2 kcal mol'l) and lysine (238 kcal mol'l), it is significantly higher than that of appropriate models for the S-amidoethyl cysteine sulfoxide side chain, i.e., propionamide (209.4 kcal mol'l), and the amide backbone (i.e., 4 245 ). N—methyl acetamide (212.4 kcal mol Therefore, the site of protonation for all the singly charged precursor ions examined here is expected to be at either the terminal lysine or arginine residues of the peptide sequence. In contrast, while the addition of a second ionizing proton would be expected to result in protonation along the amide backbone for the S-amidoethyl cysteine sulfoxide-containing peptides, thereby allowing backbone fragmentation processes to be observed (for example, see Figure 5.4B), a second ionizing proton added to the S-pyridylethyl cysteine sulfoxide-containing peptides would be expected to localize on the side chain pyridylethyl moiety. The effect of this localized proton on the predicted barriers for loss of the side chain CHZCHX’ is shown in Figure 5.7. Similar to that shown in Figure 5.58 for the neutral loss of CHzCHC5H4N, the barrier for this loss fiom the model system NC5H4CH2CH2$(O)CH3 (equivalent to the loss of CHgCHX’ from the S-pyridylethyl cysteine sulfoxide-containing peptides) 136 was predicted to be 20.9 kcal mol"1 (structure [TS3] in Figure 5.7A). In contrast, protonation of the pyridylethyl side chain resulted in a predicted transition state activation barrier of only 10.0 kcal mol'1 (structure [TS4] in Figure 5.78), resulting in the loss of CHZCHC5H4NH+. These data are therefore entirely consistent with the experimentally observed product ion abundances shown in Figure 5 .6 and in Table 5.1. 137 E E are ”'6 H O O l—l—M Hg 9! .4. O V I I '5 '25 8'8 an HO [_‘O ...-$4 _.34 91 O. O O N v— E '2 '_lv—I F-l—q $2.8 :8 M Ad 0. o, O O Figure 5.7 Optimized precursor, transition state and product ion structures (at the B3 LYP/6-31+G(d,p) + ZPVE level of theory) for (A) the loss of CH2CHC5H4N from the neutral model system NC5H4CH2CH2S(O)CH3 and (B) the loss of CH2CHC5H4NH+ fiom the protonated model system HNC5H4CH2CH2S(O)CH3+ (equivalent to the loss of CHZCHX’ from the S-py1idy1ethy1 cysteine sulfoxide-containing peptides). 138 Loss of CHzCHX’ from the side chain of the doubly and triply protonated precursor ions of the M(ox)C(S-pe)(ox)K peptide (Figures 5.6B and 5.6C) as predominantly a neutral versus protonated species, respectively, can be readily rationalized by the expected differences in proton affinity between the singly and doubly protonated peptide product ions and the neutral 4-vinylpyridine side chain cleavage product (i.e., the order of proton affinities is expected to be [M+H-CH2CHC5H4N]+ > CHgCHC5H4N > [M+2H-CH2CHC5H4N]2+). Thus, for dissociation of the doubly protonated precursor ion, the proton affinity of the peptide product ion is higher than that of CHzCHC5H4N, such that proton transfer within the initially formed ion-ion complex results in loss of the side chain 4-vinylpyridine product as a neutral species (Scheme 5.4, pathway A), while for dissociation of the triply protonated precursor ion, the proton affinity of the peptide product ion is lower than that of CHZCHC5H4N, such that loss of the 4-vinylpyridine product is observed as the protonated species (Scheme 5.4, pathway B). 139 N l \ / / (14) H 11.. I \ S) / _I H I. N I \ S) / (5) :5 73 ESE Est—3 8:3 + I; g + g E) + e i; 8 8 as h C: <——— + ——> t: 8:» e’ 9‘ if g mz\ :z :12 1” 213—”) 0:0 g—m \U=O m \ E o—m o=o ‘—‘< \——< \_,’ 5 J g ..D 0 <1 0‘ L O °\° f I .3. 7 1200 1400 (U 2 .. e\° _ I I 10 20 30 40 50 Retention time (min) Ratio = 0.91 Ratio = 0.13 2.35137 " 374.9 308137 580,] 13 l c o - 8. a - '8 3 _ < 71.0 _ 369{ 358.9 l382.7 n n A 384.7 . 340 350 360 370 380 390 400 530 540 550 560 570 580 590 Ratio = 0.85 Ratio = 1.05 3.2056 " 369.1 5.74E6 ' 553,] D F 0 O 5 ‘ ‘ 5 .o _ _ <2 . 1.4 l - L n .1 A. a. L I I I I I I 1 I I I 340 350 360 370 380 390 400 530 540 550 560 570 580 590 3.75E6 - 371.1 5.49E6 - 556.0 E G _ 4 D 8 _ _ 8 5 - _ .0 <1 A]; AI I I I ‘I :L‘A ILA Al I“‘ I 340 350 360 370 380 390 400 530 540 550 560 570 580 590 m/z m/z 157 10 I 10 I A +2 MS C +2 Neutral loss 8 - 8 - :5, 6 6 ~ 2 4 _ y=0.004x+0.391 4 _ y=0.090x+0.207 a R2 = 0.403 R2 = 0.998 E 2 - 2 ~ 3 12345678910 12345678910 E, 10‘} 10] .2 B +3 MS D +3 Neutral loss 5 8 4 8 . '8 i: 6 6 d) (I) 3 4 - 4 - y = 0.069x + 0.295 y = 0.065x + 0.293 2 - ' R2=0.992 2 . - R2=0.993 12345678910 12345678910 Theoretical Ratio [MK(AP)/MK(AP'3C6)] Theoretical Ratio [MK(AP)/MK(AP13C6)] Figure 6.3 Observed versus theoretical abundance ratios for 0.121, 05:], 1:1, 1:05 and 1:01 pmol mixtures of phenacylsulfonium and 13C6 phenacylsulfonium ion derivatives of GAILMGAILK in a tryptic digest mixture of seven proteins (1 pmol each) obtained by MS analysis of the doubly and triply charged precursor ions (panels A and B, respectively) and by neutral loss scan mode MS/MS analysis (83.0 and 86.0 m/z for doubly charged ions and 55.3 and 57.3 m/z for triply charged ions) (panels C and D, respectively). 6.5 Conclusions The results presented here demonstrate the utility of an automated approach for the selective gas—phase identification, characterization and differential quantitative analysis of fixed-charge methionine-containing peptides, based on neutral loss MS/MS and data dependent “pseudo M83” scans in a triple quadrupole mass spectrometer. Using this approach, a synthetic fixed-charge methionine-containing peptide was successfully ‘extracted’ in the gas-phase, and selectively identified and characterized from within a 158 complex seven protein tryptic digest mixture. By incorporating l3C isotopic labels into the alkylating reagent employed for fixed-charge derivatization, differential quantitative analysis was achieved for a low abundance fixed-charge methionine containing peptides spiked into a complex seven protein tryptic digest mixture, by measurement of the abundances of the characteristic product ions formed by neutral loss scan mode MS/MS. In contrast to MS-based quantitative analysis strategies, the neutral loss scan mode MS/MS method employed here was able to achieve accurate quantitation for amounts ranging from 100 frnol to 1 pmol and abundance ratios ranging from 0.1 to 10. It is expected that use of the gas-phase methionine-containing peptide “enrichment” approach described here would improve the specificity of current database search analysis strategies, by enabling searches against only a subset of the peptides contained within a protein sequence database (i.e., those containing methionine). 159 CHAPTER SEVEN Quantitative Analysis of Calcineurin Methionine Oxidation via Fixed-Charge Chemical Derivatization and Tandem Mass Spectrometry 7.1 Introduction Calcineurin (protein phosphatase 2B, PP2B) is a Ca2+/calmodulin-activated serine/threonine phosphatase which is found in all mammalian tissues, but is particularly abundant in the brain.248 Calcineurin consists of two subunits, A and B, the amino acid sequences of which are depicted in Schemes 7.1 and 7.2, respectively. Calcineurin-A contains a regulatory region (amino acid residues 381-521) depicted by the underlined amino acid residues in Scheme 7.1, as well as a catalytic region. The regulatory region of calcineurin-A contains the calmodulin binding domain which is shown in Scheme 7.1 by the double underlined amino acid residues. Calmodulin, an acidic protein with 148 amino acid residues, consists of an N- terrninal and C-terminal domain, each of which can bind up to two calcium ions.249 Binding to calcium induces a conformational change within calmodulin which subsequently exposes a hydrophobic surface. The resultant hydrophobic surface enables calmodulin to bind calcineurin, therefore resulting in an activated calcineurin-calmodulin complex. This activated calcineurin-calmodulin complex has been shown to contribute to hypertrophy in cardiac muscle cells,250 to memory formation in neurons,251 and to aid in the growth and differentiation of T-cells involved in immune response.252 160 MSEPKAIDPKLSTTDRWKAVPFPPSHRLTAKEVFDNDGKPRVDILKAHLM KEGRLEESVALRIITEGASIL RQEKNLLDIDAPVTVCGDIHGQFFDLMQQKLFEVGGSPANTRYLFLGDYVDRGYFSIECVLYLWALKILYPK TLFLLRGNHECRHLTEYFTFKQECKIKYSERWDACMDAFDCLPLAALMNQQFLCVHGGLSPEINTLDDIR KLDRFKEPPAYGPMTNCDILWSDPLEDFGNEKTQEHFI'HNTVRGCSYFYSYPAVCEFLQHNNLLSILRAH EAQDAGYMYRKSQTTGFPSLITIFSAPNYLDWNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFMDVFI' WSLPFVGEKVTEM354LVNVLNICSDDELGSEEDGFDGATAAARKEVIRN KIRAIGKMNGARVFSVLREESE SVLTLKG LTPTGM431 LPSGVLSGGKQTLQSATVEAIEADEAIKGFS PQHKITSFEEAKGLDRINERM483PPR RDAM490PSDANLNSINKALTSETNGTDSNGSNSSNIQ Scheme 7.1 Amino acid sequence of the calcineurin A subunit. The amino acid residues comprising the regulatory region are underlined. The predicted calmodulin binding domain is indicated by the double underline. Methionine residues identified both in this study and the study by Carruthers et al. are in bold, while the methionine residues identified exclusively in the study by Carruthers et al. are in italics. MGNEASYPLEM,1CSHFDADEIKRLGKRFKKLDLDNSGSLSVEEFMMSLPELQQNPLVQRVIDIFDTDGN GEVDFKEFIEGVSQFSVKGDKEQKLRFAFRIYDM101 DKDGYISNGELFQVLKMMVGNNLKDTQLQQIVDK TIINADKDGDGRISFEEFCAWGGLDIHKKMWDV Scheme 7.2 Amino acid sequence of the calcineurin B subunit. Methionine residues identified both in this study and the study by Carruthers et al. are in bold, while the methionine residues identified exclusively in the study by Carruthers et al. are in italics. It has been previously demonstrated that Met406, the only methionine residue residing in the calmodulin binding domain of calcineurin-A, is involved in stabilizing the calcineurin-calmodulin complex.253 Thus, it has been predicted that Met405 is highly solvent accessible and, as a result, potentially susceptible to oxidation by various reactive oxygen or nitrogen species. A recent study by Carruthers et al. showed that Met406 is in fact highly susceptible to oxidation following treatment of purified calcineurin with low millimolar concentrations of hydrogen peroxide in vitro.202 More importantly, it was demonstrated that Met406 oxidation results in a decreased affinity of calcineurin for calmodulin and therefore disrupts calcineurin activation. In addition to Met406, several 161 other methionine residues located within subunits A and B of calcineurin were found to be susceptible to oxidation, albeit to varying extents. The results from this initial investigation by Carruthers et al. have suggested that a decrease in the affinity of calcineurin for calmodulin in response to oxidation may play a role in modulating calmodulin—dependent signaling as well as enable calmodulin to bind to and activate other calmodulin-dependent enzymes. Thus, the ability to accurately identify and quantify calcineurin methionine oxidation is of importance. In the study by Carruthers et al., a mass spectrometry-based analysis strategy was employed to quantify calcinemin methionine oxidation. In this approach, native calcineurin, and calcineurin treated with 12 mM hydrogen peroxide for varying reaction times, was subjected to enzymatic digestion and the resultant peptide mixtures were then individually analyzed by LC-MS and MS/MS. The extent of methionine oxidation was then determined using a peptide mapping procedure whereby the extracted ion chromatographic peak areas of reduced and oxidized forms of methionine-containing peptides of interest were calculated. Using this approach, 10 of the 19 methionine residues located within subunits A and B of calcineurin were identified (Schemes 7.1 and 7.2). However, the extent of methionine oxidation could only be quantified unambiguously for five of the methionine residues due to interference from co-eluting peptides. Despite the initial success of the peptide mapping procedure in quantifying calcineurin methionine oxidation, there are a number of limitations associated with this experimental strategy. For example, in an effort to achieve accurate quantitation, highly reproducible LC-MS analysis is required to minimize shifts in retention time and limit fluctuations in MS signal intensity. Protein quantitation may also be precluded when 162 peptides are present at low abundance or the MS signals of peptide ions with identical m/z values elute at the same retention time. Furthermore, the reduced and oxidized forms of a methionine-containing peptide may exhibit different ionization efficiencies and may experience differences in ionization suppression because they elute at different chromatographic retention times. Here, as an extension of the initial study by Carruthers et al., and in an effort to address the limitations associated with the peptide mapping procedure described above, a fixed-charge chemical derivatization and tandem mass spectrometry-based analysis strategy, similar to that described in Chapter 6 for the identification, characterization and quantification of methionine-containing peptides, was applied to the identification and quantification of calcineurin methionine oxidation. Using this approach, the extent of methionine oxidation was determined indirectly by measuring the decrease in phenacylbromide labeling that occurred when oxidizable or partially oxidizable methionine residues of purified calcineurin were exposed to hydrogen peroxide under non-denaturing conditions. The results obtained using the fixed-charge chemical derivatization approach were then compared to those obtained using the peptide mapping procedure described in Carruthers et al. 7.2 Results Obtained for the Quantitative Analysis of Calcineurin Methionine Oxidation Using Different Measurement Strategies In an initial effort to demonstrate the utility of the fixed-charge chemical derivatization and tandem mass spectrometry-based approach for the quantitative analysis of calcineurin methionine oxidation, purified Wildtype calcineurin was subjected to 163 oxidation in triplicate with 12 mM hydrogen peroxide (1-1202) at room temperature for 0, l, 2, 4 or 10 hours under non-denaturing conditions, followed by proteolytic digestion using LysC. LysC was chosen in this study as opposed to trypsin due to the abundance of arginine and lysine residues in the calmodulin-binding domain of calcineurin-A. The resultant peptide mixtures were then individually alkylated with phenacylbromide then analyzed in triplicate by on-line capillary RP-HPLC nano-ESI-MS using a linear quadrupole ion trap mass spectrometer. The oxidation of individual methionine residues was then indirectly calculated by measuring the extent to which methionine alkylation with phenacylbromide had occurred in native versus oxidized samples. This approach is based on the fact that as the degree of methionine oxidation increases for an individual methionine residue, the extent of methionine alkylation decreases. To measure the decrease in methionine alkylation, and therefore calculate the proportion of each methionine containing peptide remaining in the reduced form, both an MS- and MS/MS- based approach were employed. In the MS-based approach, extracted ion chromatograms were used to determine the precursor ion abundance of individual fixed-charge methionine-containing peptides. In the MS/MS-based approach, the abundances of the characteristic [M++nH-CH3SCH2COC6H5]("+m neutral loss product ions resulting from selective fragmentation of the sulfonium ion derivative at the methionine side chain following CID-MS/MS analysis were determined from the resultant product ion spectra. For comparison, the native and oxidized calcineurin peptide mixtures were also subjected to LC-MS and MS/MS analysis prior to alkylation with phenacylbromide and the extent of methionine oxidation was calculated using the peptide mapping procedure described in 164 Carruthers et al. For a more detailed description of the experimental procedure employed in this study please refer to Chapter 3. A representative example of the fixed-charge chemical derivatization MS-based quantitative analysis strategy is shown in Figure 7.1 for the methionine-containing peptide M406ARVFSVLREESESVLTLK (Met406) derived from subunit A of calcineurin. This peptide contains the Met406 residue located in the calmodulin binding domain. Extracted ion chromatograms for the [M++3H]4+ precursor ion (m/z 579.2) of the methionine-containing phenacylsulfonium ion derivative of the Met406 peptide are shown in Figures 7.1A, 7.13 and 7.1C following treatment of calcineurin with 12 mM H202 for 0, 1 and 4 hours, respectively. Mass spectra obtained from the 27.0-27.3 minute region of the extracted ion chromatograms shown in Figures 7.1A, 7.1B and 7.1C are depicted in Figures 7.1D, 7.1E and 7.1F, respectively. Using this approach, the proportion of the Met406 peptide remaining in the reduced form was calculated as 0.58 and 0.34 following treatment of calcineurin with 12 mM H202 for l and 4 hours. Figures 7.2A, 7.2B and 7.2C depict the CID-MS/MS product ion spectra of the [M++3H]4+ precursor ion (m/z 579.2) of the methionine-containing phenacylsulfonium ion derivative of the Me1406 peptide following treatment of calcineurin with 12 mM H202 for 0, l and 4 hours, respectively. Using the fixed-charge chemical derivatization MS/MS-based quantitative analysis strategy, the proportion of the Met406 peptide remaining in the reduced form was calculated as 0.71 and 0.50 following treatment of calcineurin with 12 mM H202 for l and 4 hours. Using the peptide mapping procedure described in Carruthers et al., the 165 proportion of the Met406 peptide remaining in the reduced form was calculated as 0.66 and 0.39 following treatment of calcineurin with 12 mM H202 for 1 and 4 hours, respectively. A plot of the proportion of the Met406 peptide in the reduced form as a function of the time of exposure to H202 is depicted in Figure 7.3. The data shown are the average of three replicate experiments each analyzed in triplicate where the error bars represent the standard deviation of the experimental replicates. 166 7.19135 — 100‘ lM++3H]4+ o D 579.3 .1 U - .. 3 0 _ g _ 5 .3 [M++2ri]3+ 5 - o - 772.1 .0 _> < .3 610.7 I I I L 1 I A If 1 1A 1 1 1 I I 1 0 10 20 30 40 50 300 500 700 900 l 100 4 15135 — 1007 [M++3H]4+ 8 B 579.3 ‘ Ratio=0.58 a ‘ 8 5 - E [M++2H]3+ g g o - 772.0 '3: s? 610.7 a — 2 7 391.3 [ 814.0 - L I 2 1 - . -f - T I f r l I I I T T ' l 0 10 20 30 40 50 300 500 700 900 1100 2.48E5 " 100- F [M++3H]4+ - Ratio=0.34 3 - 579-3 ” 5 U ..I + g g [M++2H]3+ g _ g . 772.1 < .5 3913 610.8 In - -— - 814.0 0 [ a: 0 10 20 30 40 50 300 500 700 900 1 100 Retention time (min) m/z Figure 7.1 Extracted ion chromatograms of the quadruply charged ([M++3H]4+) precursor ion (m/z 579.2) of the fixed-charge methionine-containing peptide M406ARVFSVLREESESVLTLK (Met406) following treatment of calcineurin with 12 mM H202 for 0 (panel A), 1 (panel B) or 4 (panel C) h, respectively. Mass spectra obtained from the 27.0-27.3 minute region of the extracted ion chromatograms in panels A-C are shown in panels DR 167 1.14135 - -CH3SAP A 537.7 d) O a .. (U '0 § _ .0 <1 I l l l —l 200 400 600 800 1000 8.10134 - -CH33AP B 537.7 ‘ Ratio = 0.71 d) o —1 5 "O S - ..D < 1 [___T—_—l—_'I 200 400 600 800 1000 5.67E4 - -CH33AP C 537.7 — Ratio = 0.50 (D 0 g -1 “U I: :3 .. ..D < 1 1 ' 1 1 i 200 400 600 800 1000 m/z Figure 7.2 CID-MS/MS product ion spectra of the [M++3H]4+ precursor ion of the fixed- charge methionine-containing peptide M406ARVFSVLREESESVLTLK (Met406) following treatment of calcineurin with 12 mM H202 for 0 (panel A), 1 (panel B) or 4 (panel C) h. 168 1.0 k 0.9 T 0.8 ~ 0.7 - 0.6 4 0.5 . 0.4 . 0.3 - . 1 0.1 « 0.0 . v . . . . 0 2 4 6 8 10 12 Time(h) PHI-EH I-O-Dtlli m—I l-fil Fraction of peptide in reduced form Figure 7.3 Fraction of the calcineurin-A peptide M406ARVFSVLREESESVLTLK remaining in the reduced form following treatment of calcineurin with 12 mM H202 for 0, 1, 2, 4 or 10 hcalculated using either the peptide mapping procedure described in Carruthers et al. (A), the fixed-charge chemical derivatization-MS (O) or the fixed-charge chemical derivatization-MS/MS (I) approach. All data are the mean of three experiments each analyzed in triplicate. Error bars are i: the standard deviation. In addition to the Met406 peptide, the extent of methionine oxidation occurring within the calcineurin-A peptides GLTPTGMLPSGVLSGGK (Met431), EPPAYGPMCDILWSDPLEDFGNEK (Met227) and VTEMLVNVLNICSDDELGSEEDGFDGATAAARK (Met364), as well as the calcineurin-B peptide LRFAFRIYDMDK (Metlm), was also determined (Figures 7.4- 7.7). 169 1.0 0.9 ~ 0.8 - 0.7 ~ 0.6 - 0.5 - 0.4 - _ o... l 0.2 - 0.1 - 0.0 Nib—l i-D-ifl Fraction of peptide in reduced form IE O 2 4 6 8 1O 12 Time (h) Figure 7.4 Fraction of the calcineurin-A peptide GLTPTGM431LPSGVLSGGK remaining in the reduced form following treatment of calcineurin with 12 mM H202 for 0, 1, 2, 4 or 10 h calculated using either the peptide mapping procedure described in Carruthers et al. (A), the fixed-charge chemical derivatization-MS (O) or the fixed-charge chemical derivatization-MS/MS (I) approach. All data are the mean of three experiments each analyzed in triplicate. Error bars are d: the standard deviation. 1.2 - 1.0 ‘ i 0.8 - ' 0.6 . 0.4 - 0.2 - Fraction of peptide in reduced form 0.0 . . . . T . 0 2 4 6 8 10 12 Time (h) Figure 7.5 Fraction of the calcineurin-A peptide EPPAYGPM227CDILWSD- PLEDFGNEK remaining in the reduced form following treatment of calcineurin with 12 mM H202 for 0, 1, 2, 4 or 10 h calculated using either the peptide mapping procedure described in Carruthers et al. (A), the fixed-charge chemical derivatization-MS (O) or the fixed-charge chemical derivatization-MS/MS (I) approach. All data are the mean of three experiments each analyzed in triplicate. Error bars are :1: the standard deviation. 170 1.0 k 0.9 0.8 4 I i I I 0.7 - . i i i i 0.5 - 0.4 - 0.3 - 0.2 5 0.1 1 0.0 . fl . . . . 0 2 4 6 8 10 12 Time(h) Fraction of peptide in reduced form Figure 7.6 Fraction of the calcineurin-A peptide VTEM364LVNVLNICSDDELGSE- EDGFDGATAAARK remaining in the reduced form following treatment of calcineurin with 12 mM H202 for 0, 1, 2, 4 or 10 h calculated using either the peptide mapping procedure described in Carruthers et al. (A), the fixed-charge chemical derivatization-MS (O) or fixed-charge chemical derivatization-MS/MS (I) approach. All data are the mean of three experiments each analyzed in triplicate. Error bars are :i: the standard deviation. 1.0 I 0.9 - ‘ g 0.8 « § 0.7 - 0.6 - f 0.5 - 0.4 4 0.3 l 0.2 - 0.1 - 0.0 Fraction of peptide in reduced form 0 2 4 6 8 10 1 2 Time (h) Figure 7.7 Fraction of the calcineurin-B peptide LRFAFRlYDMlmDK remaining in the reduced form following treatment of calcineurin with 12 mM H202 for 0, 1, 2, 4 or 10 h calculated using either the peptide mapping procedure described in Carruthers et al. (A), the fixed-charge chemical derivatization-MS (O) or the fixed-charge chemical derivatization-MSfMS (I) approach. All data are the mean of three experiments each analyzed in triplicate. Error bars are :1: the standard deviation. 171 7.3 Comparison of the Results Obtained for the Quantitative Analysis of Methionine Oxidation Using Different Measurement Strategies In general, there was a very good correlation between the results obtained using the abundances of the fixed-charge methionine—containing precursor ions (MS-based quantitation) and the abundances of the characteristic neutral loss product ions (MS/MS- based quantitation) to calculate methionine oxidation as demonstrated in Figure 7.4 for the Met431 peptide derived from subunit A of calcineurin. However, it is important to note that a discrepancy was observed between the results obtained using the fixed-charge MS- and MS/MS-based approaches for the Met406 peptide (Figure 7.3). One potential explanation for this discrepancy could be ionization suppression resulting from slight changes in the chromatographic retention time from run to run, therefore precluding accurate quantitative analysis by MS. Furthermore, the isolation width used to isolate the fixed-charge methionine containing precursor ion for fragmentation could also have played a role in the observed differences. The isolation width of 2.0 used in this study may have been too narrow to account for slight changes in the m/z value of the precursor ion during isolation and subsequent fragmentation. Although likely not an issue in this particular study, where a relatively simple peptide mixture was subjected to analysis, the isolation width may have been too wide therefore resulting in interference from co- eluting peptides with nearly identical m/z values. There was also a very good correlation between the results obtained using either of the fixed-charge chemical derivatization approaches and those obtained using the peptide mapping procedure described in Carruthers et al. with the exception of the Met364 calcineurin-A peptide (Figure 7.6). A possible explanation for the observed differences 172 could be that the reduced and oxidized forms of the Met364 peptide, the abundances of which were used to calculate the extent of methionine oxidation using the peptide mapping procedure, may exhibit different ionization efficiencies. Also, the reduced and oxidized forms of the Met364 peptide may experience differences in ionization suppression given that they elute at different chromatographic retention times. 7.4 Conclusions and Future Directions The results fiom this initial investigation indicate that the fixed-charge chemical derivatization and tandem mass spectrometry-based approach holds promise for the quantitative analysis of calcineurin methionine oxidation. In addition, it also addresses several of the limitations associated with the peptide mapping procedure described in Carruthers et al. It is important to note that despite the initial success of this approach, several of the methionine-containing peptides identified in the study by Carruthers et al. were not identified here. However, this is likely attributed to differences in the chromatographic separation used in each individual study as opposed to the experimental approach. As an extension of this initial investigation, it would be beneficial to subject the native and oxidized peptide mixtures to alkylation with 12C6- and 13C‘s-phenacylbromide, respectively so that these samples could be combined prior to LC-MS and MS/MS analysis. Analyzing the native and oxidized peptide mixtures in a single run would eliminate differences caused by sample preparation, shifts in retention time and fluctuations in MS signal intensity. It would also be beneficial to analyze native and oxidized calcineurin using the automated neutral loss MS/MS and data dependent energy- 173 resolved pseudo MS3 analysis method as described in Chapter 6 in an effort to increase protein sequence coverage and to identify/quantify additional methionine residues, particularly those present at low abundance. 174 APPENDIX 175 Cartesian Coordinates of Optimized Precursor, Transition State and Product Ion Structures Chapter 5 B3LYP/6—31 + G(d,p) Optimized Structures 3.336 -2.137 1.136 2.114 -1.250 0.990 1.076 -l.771 0.278 2.067 -0.115 1.473 -0.204 -l.096 0.175 -l.210 -2.150 -0.364 -0.094 0.170 -0.712 -l.149 1.546 -0.074 -0.840 -3.314 -0.561 -2.463 -l.7l9 -0.579 -2.609 1.090 -0.216 -0.845 2.709 -l.453 -3.495 -2.622 -l.068 3.509 -2.318 2.201 3.241 -3.096 0.620 4.208 -1.603 0.748 1.052 -2.745 -0.013 -0.526 -0.794 1.181 0.929 0.551 -0.660 -0.374 -0.025 -1.752 -2.691 -0.731 -0.422 -1.458 3.591 -l.256 -1.149 2.246 -2.395 0.213 2.985 -1.467 -3.211 -3.058 -2.032 -4.419 -2.056 —l.186 -3.661 -3.443 -0.364 3:13:12EZIIEIEIEIIOOOZOWOOOOZOO 11> 176 LIIEICEEEEEEEEZEIOOOZOWOOOOZOO w 1'! Sl OOOZOVJOOOOZOO 2.437 1.369 1.415 0.508 0.612 -3.229 -1.146 -2.153 -l.077 -1.328 0.023 -2.026 -1.943 -0.108 0.138 -0.889 -0.343 0.402 0.827 0.880 -1.022 1.710 2.414 -0.417 -1.688 0.582 0.251 -1.233 -1.552 0.903 2.493 0.247 -2.616 3.137 3.000 1.965 2.236 0.995 1.974 0.822 3.334 0.192 -1.904 1.188 -2.976 -1.950 -3.348 -0.215 -4.178 -1.409 -1.372 0.612 0.375 1.050 1.477 0.455 -1.786 -0.114 1.189 -1.480 -0.533 -2.279 0.890 0.637 4.178 0.764 -0.379 2.691 0.813 -0.325 3.693 -0.669 -3.137 -1.020 1.560 -2.639 -2.688 1.950 -3.l37 -2.259 0.290 2.571 1.402 1.576 0.381 0.637 -2.471 -2.213 -l.620 -1.741 -0.999 -0.522 -1.498 -2.408 -0.066 0.061 -0.399 -0.590 1.012 0.219 1.039 -0.746 1.217 2.752 0.267 -1.248 0.166 1.504 -0.314 -1.907 1.349 1.970 1.747 1.256 -0.220 3.320 1.233 -1.291 -2.543 2.217 177 IIEIEEEEIIEEEE SEEEEOOWEEEEEIIIEEOZOOOOOZOO O 3.011 3.354 2.192 2.481 0.640 -0.813 0.299 1.465 0.139 -2.001 -3.099 -2.603 -1.461 -3.450 -2.517 -1.102 —0.080 1.131 -1.741 1.363 -0.653 -2.486 0.794 0.738 0.952 3.918 2.073 -0.786 2.449 1.573 -0.827 3.943 0.568 -1.610 -1.824 2.972 -0.834 -3.407 2.707 -2.179 -2.875 1.663 3.753 3.100 1.784 3.701 0.967 -3.320 -0.664 -2.023 -1.111 -1.866 -0.707 -1.193 -1.777 -0.717 -0.877 -0.514 -0.983 -0.847 1.418 0.525 -l.103 -1.346 -0.123 -0.540 -0.899 -2.254 -1.l64 -2.311 4.530 3.058 4.244 1.425 -2.605 -1.269 -3.085 0.070 -4.040 -0.220 -3.777 -1.526 -2.570 -0.076 2.476 0.737 -l.172 0.709 1.332 -1.244 -0.227 -2.837 -1.646 -2.838 -2.240 -0.386 -2.400 -3.691 -1.323 -2.777 -2.155 -2.153 -2.652 3.341 -0.898 -l.250 2.640 -0.266 -3.6413.541 0.614 -1.343 1.664 -0.312 -3.099 4.141 1.350 -3.920 2.575 1.046 -4.551 4.076 0.317 178 EIIEIEEIZZOOOOWOO U EEIEEIEIEZOOOOWOO I!) 0.848 0.665 -0.701 0.938 -1.558 -1.488 1.872 0.089 1.527 1.257 -0.342 1.377 -1.591 -2.477 0.676 -0.438 -0.746 -1 .002 1.498 -1.867 0.418 1.825 0.179 0.011 0.846 -1.442 -2.650 -0.152 -l.918 0.682 1.364 -0.878 -0.901 0.895 2.232 -0.894 2.560 -0.664 -1.239 0.240 0.672 1.061 0.664 -0.137 -2.635 0.648 -0.678 2.063 -1.481 1.066 0.419 -1.187 -l.415 -1.815 -2.007 -1.224 -1.993 1.173 1.761 -1.237 -0.596 -0.022 -0.088 1.778 1.772 0.979 -0.9151.711 0.842 -1.262 -1.371 -0.567 0.142 1.419 -0.309 1.561 1.789 -2.366 0.955 0.600 -1.306 0.912 -1.975 -1.718 -1.345 -0.495 0.206 -0.862 0.746 1.346 -1.039 2.509 1.578 -1.741 1.725 0.126 1.044 -2.402 1.362 -0.604 -2.947 1.265 179 T82 0.337 1.043 0.600 0.222 1.627 -0.986 -0.920 0.807 0.783 -0.750 1.964 1.553 -1.225 -0.899 -1.748 1.761 0.316 EIEEEEEEEZOOOOWOO 1:: 1.539 2.004 1.162 -0.012 1.799 2.144 0.535 3.011 2.758 1.277 0.145 -l.13l -0.534 -0.089 2.269 -0.541 -1.143 0.987 1.846 0.600 2.473 0.337 -0.755 1.261 -2.161 1.694 0.178 -2.121 0.098 —1.124 -1.044 -0.369 0.661 0.539 1.552 0.876 3.324 1.015 2.682 -0.422 -2.444 1.538 -2.523 2.497 0.848 -1.408 0.015 -0.469 -1.111 0.051 -1.271 -0.273 -1.950 0.921 1.661 -1.794 0.722 -1.804 0.129 -0.073 -1.823 1.199 -2.723 1.310 IEEEOOWEIEIEZOOOO -1.586 2.548 -0.166 2.328 -2.506 1.039 0.455 1.826 -1.977 0.144 -2.693 0.999 -3.464 1.115 0.353 1.264 0.784 0.710 0.446 1.860 0.258 180 EIEIIEIEIEEZOOOOOOOWOO C) 1-1 S3 IEEIIEEZOOOOOOOWOO 1.352 0.175 1.108 0.178 -0.127 -0.044 -1.091 1.191 1.813 0.740 -1.164 -0.220 -0.407 2.865 -0.358 2.901 1.858 1.401 -2.067 0.844 -0.280 -1.551 -1.451 0.137 -2.788 -1.561 0.773 -3.282 0.631 0.376 -0.544 0.901 -3.653 1.400 2.295 0.403 0.137 3.611 3.004 3.056 -1.826 -0.904 -3.103 -3.993 1.406 0.054 2.350 -1.023 -0.595 0.200 -0.938 0.802 1.833 2.798 1.015 1.824 -2.322 -2.513 1.448 0.097 0.551 1.671 -0.21 1 0.937 2.371 3.207 2.638 -0.809 -0.975 -2.342 -0.l68 -3.344 -0.873 -1.880 —1.645 -3.138 -1.611 1.671 -0.801 2.032 0.279 -0.265 1.031 0.245 1.647 2.568 2.737 3.451 3.625 4.129 2.106 0.649 -0.682 —1.684 -1 .784 0.569 1.946 2.080 -0.679 0.061 1.197 0.484 -0.810 -0.825 0.583 -0.137 0.725 -0.708 1.023 -0.616 0.050 1.613 1.150 -0.259 -1.678 -1.751 -0.014 -1.588 -0.310 -0.967 181 21:52:12.1: EIIEOOWEEIIEEIZOOOOOOO I _ 53200000005000 0.175 -2.586 -1.048 0.407 1.477 -1.506 -4.367 -0.845 -0.322 -1.718 2.264 2.537 1.562 0.169 2.011 1.088 -0.666 -0.238 1.253 3.068 3.578 -0.261 3.074 1.430 -1.744 -1.597 -1.184 -2.817 -1.731 -3.716 -2.376 -3.085 1.560 1.121 1.743 -0.358 0.689 3.346 -1.297 -2.240 1.383 0.294 -0.658 -0.540 -1753 -2.653 -1.495 -2544 1.681 2.015 0.061 0.279 -1905 -3.505 -1.417 3.704 2.176 3.219 2.022 2.791 2.521 4.144 0.582 -0.547 2.239 -0.846 2.246 1.943 0.096 2.510 -1.425 -0.691 -0.130 -0.300 0.623 1.157 0.273 0.994 -1.686 -1.786 -0.469 -0.868 0.794 1.741 0.147 -1.439 -2.078 -0.184 -2.863 -0.633 0.533 0.339 1 .006 0.040 0.162 0.063 -0.944 -0.666 -0.405 -0.839 -2.083 0.534 -2.195 -2.352 0.532 -2.643 -0.212 -0.396 -3.055 -1.417 0.069 0.829 0.748 1.803 182 33322122122322.1321: ..3 S4 IEI‘EI‘EEII‘EEIZOOOOOOOWOO E1 0000 2.519 1.675 1.381 3.299 3.520 4.134 0.351 -1.459 -0.270 1.044 2.813 1.871 -0.948 1.059 -0.158 -2.843 0.901 -2.623 -3.284 0.879 -3.414 0.464 -4.050 -1.626 0.068 1.928 0.449 -0.356 -0.244 1.480 0.281 -0.990 -1.284 -1.302 0.087 -0.774 -0.748 -0.452 -0.480 2.552 1.397 0.245 -0.392 -1.035 0.380 1.110 1.919 0.628 2.951 0.025 2.228 -1.567 -l.326 1.636 -1.756 -1.268 0.029 -2.594 -1.965 0.858 -0.848 -2.261 2.434 -2.134 -2.452 2.043 2.348 2.402 0.026 0.432 2.134 3.101 3.880 1.038 -1.083 -0.463 -0.058 1.024 2.100 3.286 1.794 -1.429 -2.146 -0.895 —3.632 -2.l65 0.621 -0.566 -2.676 3.394 -2.766 -2.967 2.647 0.829 -1.344 0.242 -1.428 -1.466 0.142 -2.039 -1.107 -1.708 1.995 -0.911 -1.483 -0.264 -2.050 -1.143 -1.504 -1.486 -0.617 -1.698 -0.100 -0.389 183 EIIIOOWIEIEIIIEZOOO -2.581 -2.353 -0.313 -3.764 -1.856 0.185 -2.898 0.352 0.108 -3.899 -0.523 0.383 0.888 1.708 -0.263 -1.851 -0.254 -3. l 31 -0.921 0.630 -2.487 -3.422 -0.467 -4.618 -2.477 0.432 -3.103 1.398 0.300 -4.777 -0.167 0.749 1.913 0.539 1.439 0.692 1.356 0.514 2.257 2.302 2.205 3.674 2.784 4.614 3.454 3.761 -1.393 -1.866 -1.258 -0.597 0.114 -0.913 1.201 -1.676 0.651 1.739 1.925 184 REFERENCES 185 10. REFERENCES Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Correlation between Protein and mRNA Abundance in Yeast. Mol. Cell. Biol. 1999, 19, 1720-1730. Aebersold, R.; Mann, M. Mass Spectrometry- -based Proteomics. Nature 2003, 422, 198- 207. Simpson, R. J .; Connelly, L. M.; Eddes, J. S.; Pereira, J. J.; Moritz, R. L.; Reid, G. E. Proteomic Analysis of the Human Colon Carcinoma Cell Line (LIM 1215): Development of a Membrane Protein Database. Electrophoresis 2000, 21, 1707- 1732. Reid, G. E.; Rasmussen, R. K.; Dorow, D. 8.; Simpson, R. J. Capillary Column Chromatrography Improves Sample Preparation for Mass Spectrometric Analysis: Complete Characterization of Human a-Enolase from Two-Dimensional Gels Following In Situ Proteolytic Digestion. Electrophoresis 1998, 19, 946-955. Zugaro, L. M.; Reid, G. E.; Ji, H.; Eddes, J. S.; Murphy, A. C.; Burgess, A. W.; Simpson, R. J. Characterization of Rat Brain Stathmin Isoforms by Two- Dimensional Gel Electrophoresis-Matrix Assisted Laser Desorption/Ionization and Electrospray Ionizationion Trap Mass Spectrometry. Electrophoresis 1998, 19, 867- 876. Rasmussen, R. K.; Ji, H.; Eddes, J. S.; Moritz, R. L.; Reid, G. E.; Simpson, R. J .; Dorow, D. S. Two-Dimensional Electrophoretic Analysis of Mixed Lineage Kinase 2 N-terminal Domain Binding Proteins. Electrophoresis 1998, 19, 809- 817. Hunt, D. R; Yates, J. R., III; Shabanowitz, J .; Winston, S.; Hauer, C. R. Protein Sequencing by Tandem Mass Spectrometry. Proc. Natl. Acad. Sci. USA. 1986, 83, 6233-6237. Steen, H.; Mann, M. The ABC's (and XYZ's) of Peptide Sequencing. Nat. Rev. Mol. Cell Biol. 2004, 5, 699-711. Eng, J. K.; McCormack, A. L.; Yates, J. R. An Approach to Correlate Tandem Mass Spectral Data of Peptides with Amino Acid Sequences in a Protein Database. J. Am. Soc. Mass Spectrom. 1994, 5, 976-989. Perkins, D. N.; Pappin, D. J. C.; Creasy, D. M.; Cottrell, J. S. Probability-Based Protein Identification by Searching Sequence Databases using Mass Spectrometry Data. Electrophoresis 1999, 20, 3551-3567. 186 11. 12. 13. 14. 15. 16. 17. 18. 19. MacCoss, M. J .; Wu, C. C.; Yates, J. R. Probability-Based Validation of Protein Identifications Using a Modified SEQUEST Algorithm. Anal. Chem. 2002, 74, 5593-5599. Creasy, D. M.; Cottrell, J. S. Error Tolerant Searching of Uninterpreted Tandem Mass Spectrometry Data. Proteomics 2002, 2, 1426-1434. Washbum, M. P.; Walters, D.; Yates, J. R. Large-Scale Analysis of the Yeast Proteome by Multidimensional Protein Identification Technology. Nat. Biotechnol. 2001, 19, 242-247. Spahr, C. S.; Susin, S. A.; Bures, E. J .; Robinson, J. H.; Davis, M. T.; McGinley, M. D.; Kroemer, G.; Patterson, S. D. Simplification of Complex Peptide Mixtures for Proteomic Analysis: Reversible Biotinylation of Cysteinyl Peptides. Electrophoresis 2000, 21, 1635-1650. Spahr, C. S.; Davis, M. T.; McGinley, M. D.; Robinson, J. H.; Bures, E. J.; Beierle, J .; Mort, J .; Courchesne, P. L.; Chen, K.; Walrl, R. C.; Yu, W.; Luethy, R.; Patterson, S. D. Towards Defining the Urinary Proteome using Liquid Chromatography-Tandem Mass Spectrometry I.Profiling an Unfractionated Tryptic Digest. Proteomics 2001, 1, 93-107. Davis, M. T.; Spahr, C. S.; McGinley, M. D.; Robinson, J. H.; Bures, E. J.; Beierle, J .; Mort, J .; Yu, W.; Luethy, R.; Patterson, S. D. Towards Defining the Urinary Proteome using Liquid Chromatography-Tandem Mass Spectrometry II.Limitations of Complex Mixture Analyses. Proteomics 2001, I, 108-117. Wang, N. ; Li, L. Exploring the Precursor Ion Exclusion Feature of Liquid Chromatography-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry for Improving Protein Identification in Shotgun Proteome Analysis. Anal. Chem. 2008, 80, 4696-4710. Ahmed, N.; Barker, G.; Oliva, K.; Garfin, D.; Talmadge, K.; Georgiou, H.; Quinn, M.; Rice, G. An Approach to Remove Albumin for the Proteomic Analysis of Low Abundance Biomarkers in Human Serum. Proteomics 2003, 3, 1980-1987. Maccarrone, G.; Mifay, D.; Birg, 1.; Rosenhagen, M.; Holsboer, F.; Grimm, R.; Bailey, J .; Zolotarjova, N.; Turck, C. W. Mining the Human Cerebrospinal Fluid Proteome by Immunodepletion and Shotgun Mass Spectrometry. Electrophoresis 2004, 25, 2402-2412. 187 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. Bjdrhall, K.; Miliotis, T.; Davidsson, P. Comparison of Different Depletion Strategies for hnproved Resolution in Proteomic Analysis of Human Serum Samples. Proteomics 2005, 5, 307-317. Thulasiraman, V.; Lin, S.; Gheorghiu, L.; Lathrop, J .; Lomas, L.; Hammond, D.; Boschetti, E. Reduction of the Concentration of Proteins in Biological Fluids Using a Library of Combinatorial Ligands. Electrophoresis 2005, 26, 3561-3571. Guenier, L.; Claverol, S.; Fortis, F.; Rinalducci, S.; Timperio, A. M.; Antonioli, P.; Jandrot-Perrus, M.; Boschetti, E.; Righetti, P. G. Exploring the Platelet Proteome via Combinatorial, Hexapeptide Ligand Libraries. J. Proteome Res. 2007, 6, 4290-4303. Shores, K. S.; Udugarnasooriya, D. G.; Kodadek, T.; Knapp, D. R. Use of Peptide Analogue Diversity Library Beads for Increased Depth of Proteomic Analysis: Application to Cerebrospinal Fluid. J. Proteome Res. 2008, 7, 1922-1931. Ndassa, Y. M.; Orsi, C.; Marto, J. A.; Chen, 8.; Ross, M. M. Improved Immobilized Metal Affinity Chromatography for Large-Scale Phosphoproteomics Applications. J. Proteome Res. 2006, 5, 2789-2799. Lee, J .; Xu, Y.; Chen, Y.; Sprung, R.; Kim, S. C.; Xie, S.; Zhao, Y. Mitochondrial Phosphoproteome Revealed by an Improved IMAC Method and MS/MS/MS. Mol. Cell. Proteomics 2007, 6, 669-676. ch, D.; Penner, N. A.; Slentz, B. E.; Mirzaei, H.; Regnier, F. Evaluating Immobilized Metal Affinity Chromatography for the Selection of Histidine- Containing Peptides in Comparative Proteomics. J. Proteome Res. 2003, 2, 321- 329. Rafiery, M. J. Enrichment by Organomercurial Agarose and Identification of Cys- Containing Peptides from Yeast Cell Lysates. Anal. Chem. 2008, 80, 3334-3341. Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M. Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks. Cell 2006, 12 7, 635-648. Kweon, H. K.; Hakansson, K. Selective Zirconium Dioxide-Based Enrichment of Phosphorylated Peptides for Mass Spectrometric Analysis. Anal. Chem. 2006, 78, 1743-1749. 188 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. Li, Y.; Xu, X.; Qi, D.; Deng, C.; Yang, P.; Zhang, X. Novel Fe3O4@T102 Core- Shell Microspheres for Selective Enrichment of Phosphopeptides in Phosphoproteome Analysis. J. Proteome Res. 2008, 7, 2526-2538. Qiu, R.; Regnier, F. E. Use of Multidimensional Lectin Affinity Chromatography in Differential Glycoproteomics. Anal. Chem. 2005, 77, 2802-2809. Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Quantitative Analysis of Complex Protein Mixtures using Isotope-Coded Affinity Tags. Nat Biotech 1999, I 7, 994-999. Hansen, K. C.; Schmitt-Ulms, G.; Chalkley, R. J.; Hirsch, J.; Baldwin, M. A.; Burlingarne, A. L. Mass Spectrometric Analysis of Protein Mixtures at Low Levels Using Cleavable l3'C-Isotope-coded Affinity Tag and Multidimensional Chromatography. Mol. Cell. Proteomics 2003, 2, 299-314. Bernhard, O. K.; Kapp, E. A.; Simpson, R. J. Enhanced Analysis of the Mouse Plasma Proteome Using Cysteine-Containing Tryptic Glycopeptides. J. Proteome Res. 2007, 6, 987-995. Qian, W.-J.; Goshe, M. 3.; Camp, D. G.; Yu, L.-R.; Tang, K.; Smith, R. D. Phosphoprotein Isotope-Coded Solid-Phase Tag Approach for Enrichment and Quantitative Analysis of Phosphopeptides from Complex Mixtures. Anal. Chem. 2003, 75, 5441-5450. Jalili, P. R.; Ball, H. L. Novel Reversible Biotinylated Probe for the Selective Enrichment of Phosphorylated Peptides from Complex Mixtures. J. Am. Soc. Mass Spectrom. 2008, I 9, 741-750. Jalili, P. R.; Sharma, D.; Ball, H. L. Enhancement of Ionization Efficiency and Selective Enrichment of Phosphorylated Peptides fi'om Complex Protein Mixtures Using a Reversible Poly-Histidine Tag. J. Am. Soc. Mass Spectrom. 2007, 18, 1007-1017. Ren, D.; Julka, S.; Inerowicz, H. D.; Regnier, F. E. Enrichment of Cysteine- Containing Peptides from Tryptic Digests Using a Quaternary Amine Tag. Anal. Chem. 2004, 76, 4522-4530. Brittain, S. M.; Ficarro, S. B.; Brock, A.; Peters, E. C. Enrichment and Analysis of Peptide Subsets using Fluorous Affinity Tags and Mass Spectrometry. Nat. Biotechnol. 2005, 23, 463-468. 189 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. Weinberger, S. R.; Viner, R. 1.; Ho, P. Tagless Extraction-Retentate Chromatography: A New Global Protein Digestion Strategy for Monitoring Differential Protein Expression. Electrophoresis 2002, 23, 3182-3192. Zhou, H.; Ranish, J. A.; Watts, J. D.; Aebersold, R. Quantitative Proteome Analysis by Solid-Phase Isotope Tagging and Mass Spectrometry. Nat. Biotechnol. 2002, 20, 512-515. Qiu, Y.; Sousa, E. A.; Hewick, R. M.; Wang, J. H. Acid-Labile Isotope-Coded Extractants: A Class of Reagents for Quantitative Mass Spectrometric Analysis of Complex Protein Mixtures. Anal. Chem. 2002, 74, 4969-4979. Shi, Y.; Xiang, R.; Horvath, C.; Wilkins, J. A. Quantitative Analysis of Membrane Proteins from Breast Cancer Cell Lines BT474 and MCF7 Using Multistep Solid Phase Mass Tagging and 2D LC/MS. J. Proteome Res. 2005, 4, 1427-1433. Wang, H.; Qian, W.-J.; Chin, M. H.; Petyuk, V. A.; Barry, R. C.; Liu, T.; Gritsenko, M. A.; Mottaz, H. M.; Moore, R. J .; Camp, D. G.; Khan, A. H.; Smith, D. J .; Smith, R. D. Characterization of the Mouse Brain Proteome Using Global Proteomic Analysis Complemented with Cysteinyl-Peptide Enrichment. J. Proteome Res. 2006, 5, 361-369. Lansdell, T. A.; Tepe, J. J. Isolation of Phosphopeptides using Solid Phase Enrichment. Tetrahedron Lett. 2004, 45, 91-93. Shen, M.; Guo, L.; Wallace, A.; Fitzner, J .; Eisenman, J .; Jacobson, E.; Johnson, R. S. Isolation and Isotope Labeling of Cysteine- and Methionine-Containing Tryptic Peptides: Application to the Study of Cell Surface Proteolysis. Mol. Cell. Proteomics 2003, 2, 315-324. Gevaert, K.; Ghesquiere, B.; Staes, A.; Martens, L.; Van Damme, J .; Thomas, G. R.; Vandekerckhove, J. Reversible Labeling of Cysteine-Containing Peptides Allows their Specific Chromatographic Isolation for Non-Gel Proteome Studies. Proteomics 2004, 4, 897-908. Gevaert, K.; Pinxteren, J.; Demol, H.; Hugelier, K.; Staes, A.; Damme, J. V.; Martens, L.; Vandekerckhove, J. Four Stage Liquid Chromatographic Selection of Methionyl Peptides for Peptide-Centric Proteome Analysis: The Proteome of Human Multipotent Adult Progenitor Cells. J. Proteome Res. 2006, 5, 1415-1428. Unlu, M.; Morgan, M. E.; Minden, J. S. Differential Gel Electrophoresis: A Single Gel Method for Detecting Changes in Protein Extracts. Electrophoresis 1997, 18, 2071-2077. 190 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. Tonge, R.; Shaw, J.; Middleton, B.; Rowlinson, R.; Rayner, 8.; Young, J.; Pognan, F.; Hawkins, E.; Cunie, 1.; Davison, M. Validation and Development of Fluorescence Two-Dimensional Differential Gel Electrophoresis Proteomics Technology. Proteomics 2001, I, 377-396. Zhou, G.; Li, H.; DeCamp, D.; Chen, S.; Shu, H.; Gong, Y.; Flaig, M.; Gillespie, J. W.; Hu, N.; Taylor, P. R.; Emmert-Buck, M. R.; Liotta, L. A.; Petricoin, E. F ., III; Zhao, Y. 2D Differential In-gel Electrophoresis for the Identification of Esophageal Scans Cell Cancer-specific Protein Markers. Mol. Cell. Proteomics 2002, 1, 117-123. Chelius, D.; Zhang, T.; Wang, G.; Shen, R.-F. Global Protein Identification and Quantification Technology Using Two-Dimensional Liquid Chromatography Nanospray Mass Spectrometry. Anal. Chem. 2003, 75, 6658-6665. Colinge, J .; Chiappe, D.; Lagache, S.; Moniatte, M.; Bougueleret, L. Differential Proteomics via Probabilistic Peptide Identification Scores. Anal. Chem. 2005, 77, 596-606. Liu, H.; Sadygov, R. G.; Yates, J. R. A Model for Random Sampling and Estimation of Relative Protein Abundance in Shotgun Proteomics. Anal. Chem. 2004, 76, 4193-4201. Old, W. M.; Meyer-Arendt, K.; Aveline-Wolf, L.; Pierce, K. G.; Mendoza, A.; Sevinsky, J. R.; Resing, K. A.; Ahn, N. G. Comparison of Label-free Methods for Quantifying Human Proteins by Shotgun Proteomics. Mol. Cell. Proteomics 2005, 4, 1487-1502. Oda, Y.; Huang, K.; Cross, F. R.; Cowbum, D.; Chait, B. T. Accurate Quantitation of Protein Expression and Site-Specific Phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 6591-6596. Ong, S.-E.; Blagoev, B.; Kratchmarova, 1.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics. Mol. Cell. Proteomics 2002, I, 376-386. Julka, S.; Regnier, F. Quantification in Proteomics through Stable Isotope Coding: A Review. J. Proteome Res. 2004, 3, 350-363. Kuhn, K.; Prinz, T.; Schéifer, J .; Baumann, C.; Scharfke, M.; Kienle, S.; Schwarz, J .; Steiner, S.; Hamon, C. Protein Sequence Tags: A Novel Solution for Comparative Proteomics. Proteomics 2005, 5, 2364-2368. 191 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. Simons, B. L.; Wang, G.; Shen, R.-F.; Knepper, M. A. In Vacuo Isotope Coded Alkylation Technique (IVICAT); An N-terminal Stable Isotopic Label for Quantitative Liquid Chromatography/Mass Spectrometry Proteomics. Rapid Commun. Mass Spectrom. 2006, 20, 2463-2477. Shi, Y.; Yao, X. Oxygen Isotopic Substitution of Peptidyl Phosphates for Modification-Specific Mass Spectrometry. Anal. Chem. 2007, 79, 8454-8462. Thompson, A.; Schafer, J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Neumann, T.; Hamon, C. Tandem Mass Tags: A Novel Quantification Strategy for Comparative Analysis of Complex Protein Mixtures by MS/MS. Anal. Chem. 2003, 75, 1895-1904. Dayon, L.; Hainard, A.; Licker, V.; Turck, N.; Kuhn, K.; Hochstrasser, D. F.; Burkhard, P. R.; Sanchez, J .-C. Relative Quantification of Proteins in Human Cerebrospinal Fluids by MS/MS Using 6-P1ex Isobaric Tags. Anal. Chem. 2008, 80, 2921-2931. Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents. Mol. Cell. Proteomics 2004, 3, 1154-1169. Sachon, E.; Mohammed, S.; Bache, N.; Jensen, 0. N. Phosphopeptide Quantitation using Amine-Reactive Isobaric Tagging Reagents and Tandem Mass Spectrometry: Application to Proteins Isolated by Gel Electrophoresis. Rapid Commun. Mass Spectrom. 2006, 20, 1127-1134. Choe, L.; D'Ascenzo, M.; Relkin, N. R.; Pappin, D.; Ross, P.; Williamson, B.; Guertin, S.; Pribil, R; Lee, K. H. 8-Plex Quantitation of Changes in Cerebrospinal Fluid Protein Expression in Subjects Undergoing Intravenous Immunoglobulin Treatment for Alzheimer's Disease. Proteomics 2007, 7, 3651-3660. Li, S.; Zeng, D. CILAT-A New Reagent for Quantitative Proteomics. Chem. Commun. 2007, 21, 2181-2183. Hernandez, P.; Miiller, M.; Appel, R. D. Automated Protein Identification by Tandem Mass Spectrometry: Issues and Strategies. Mass Spectrom. Rev. 2006, 25, 235-254. Sadygov, R. G.; Cociorva, D.; Yates, J. R. Large-Scale Database Searching using Tandem Mass Spectra: Looking up the Answer in the Back of the Book. Nat. Methods 2004, I, 195-202. 192 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. Paizs, B.; Suhai, S. Fragmentation Pathways of Protonated Peptides. Mass Spectrom. Rev. 2005, 24, 508-548. McLuckey, S. A. Principles of Collisional Activation in Analytical Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1992, 3, 599-614. Dongre, A. R.; Jones, J. L.; Somogyi, A.; Wysocki, V. H. Influence of Peptide Composition, Gas-Phase Basicity, and Chemical Modification on Fragmentation Efficiency: Evidence for the Mobile Proton Model. J. Am. Chem. Soc. 1996, 118, 8365-8374. Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. Mobile and Localized Protons: A Framework for Understanding Peptide Dissociation. J. Mass Spectrom. 2000, 35, 1399-1406. Cox, K. A.; Gaskell, S. J .; Morris, M.; Whiting, A. Role of the Site of Protonation in the Low-Energy Decompositions of Gas-Phase Peptide Ions. J. Am. Soc. Mass Spectrom. 1996, 7, 522-531. Polfer, N. C.; Oomens, J.; Suhai, S.; Paizs, B. Infrared Spectroscopy and Theoretical Studies on Gas-Phase Protonated Leu-enkephalin and Its Fragments: Direct Experimental Evidence for the Mobile Proton. J. Am. Chem. Soc. 2007, 129, 5887-5897. Kapp, E. A.; Schutz, F.; Reid, G. E.; Eddes, J. S.; Moritz, R. L.; O'Hair, R. A. J .; Speed, T. P.; Simpson, R. J. Mining a Tandem Mass Spectrometry Database To Determine the Trends and Global Factors Influencing Peptide Fragmentation. Anal. Chem. 2003, 75, 6251-6264. O'Hair, R. A. J. The Role of Nucleophile-Electrophile Interactions in the Unimolecular and Birnolecular Gas-Phase Ion Chemistry of Peptides and Related Systems. J. Mass Spectrom. 2000, 35, 1377-1381. Lioe, H.; O'Hair, R. A. J.; Reid, G. E. A Mass Spectrometric and Molecular Orbital Study of H20 Loss from Protonated Tryptophan and Oxidized Tryptophan Derivatives. Rapid Commun. Mass Spectrom. 2004, 18, 978-988. Reid, G. E.; Simpson, R. J.; O'Hair, R. A. J. Leaving Group and Gas Phase Neighboring Group Effects in the Side Chain Losses from Protonated Serine and its Derivatives. J. Am. Soc. Mass Spectrom. 2000, II, 1047-1060. 193 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 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. Reid, G. E.; Roberts, K. D.; Kapp, E. A.; Simpson, R. J. Statistical and Mechanistic Approaches to Understanding the Gas-Phase Fragmentation Behavior of Methionine Sulfoxide Containing Peptides. J. Proteome Res. 2004, 3, 751-759. Green, M. K.; Lebrilla, C. B. Ion-Molecule Reactions as Probes of Gas-Phase Structures of Peptides and Proteins. Mass Spectrom. Rev. 1997, 16, 53-71. Polce, M. J .; Beranova, S.; Nold, M. J .; Wesdemiotis, C. Characterization of Neutral Fragments in Tandem Mass Spectrometry: A Unique Route to Mechanistic and Structural Information. J. Mass Spectrom. 1996, 31, 1073-1085. Bailey, T. H.; Laskin, J .; Futrell, J. H. Energetics of Selective Cleavage at Acidic Residues Studied by Time- and Energy-Resolved Surface-Induced Dissociation in FT-ICR MS. Int. J. Mass Spectrom. 2003, 222, 313-327. Ve’key, K.; Somogyi, A.; Wysocki, V. H. Average Activation Energies of Low- energy Fragmentation Processes of Protonated Peptides Determined by a New Approach. Rapid Commun. Mass Spectrom. 1996, 10, 911-918. Tabb, D. L.; Smith, L. L.; Breci, L. A.; Wysocki, V. H.; Lin, D.; Yates, J. R. Statistical Characterization of Ion Trap Tandem Mass Spectra from Doubly Charged Tryptic Peptides. Anal. Chem. 2003, 75, 1155-1163. Huang, Y.; Triscari, J. M.; Tseng, G. C.; Pasa-Tolic, L.; Lipton, M. S.; Smith, R. D.; Wysocki, V. H. Statistical Characterization of the Charge State and Residue Dependence of Low-Energy CID Peptide Dissociation Patterns. Anal. Chem. 2005, 77, 5800-5813. Roepstorff, P.; Fohlman, J. Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides. Biomed. Mass Spectrom. 1984, I I, 601. Biemann, K.; Papayannopoulos, I. A. Amino Acid Sequencing of Proteins. Acc. Chem. Res. 1994, 27, 370-378. Schlosser, A.; Lehmann, W. D. Five-Membered Ring Formation in Unimolecular Reactions of Peptides: A Key Structural Element Controlling Low-Energy Collision-Induced Dissociation of Peptides. J. Mass Spectrom. 2000, 35, 1382- 1390. 194 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. Polce, M. J .; Ren, D.; Wesdemiotis, C. Dissociation of the Peptide Bond in Protonated Peptides. J. Mass Spectrom. 2000, 35, 1391-1398. Paizs, B.; Suhai, S. Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 1: Mechanism of Amide Bond Cleavage. J. Am. Soc. Mass Spectrom. 2004, 15, 103-113. Yalcin, T.; Khouw, C.; Csizmadia, I. G.; Peterson, M. R.; Harrison, A. G. Why Are B Ions Stable Species in Peptide Spectra? J. Am. Soc. Mass Spectrom. 1995, 6. 1165-1174. Yalcin, T.; Csizmadia, I. G.; Peterson, M. R.; Harrison, A. G. The Structure and Fragmentation of B, (n >= 3) Ions in Peptide Spectra. J. Am. Soc. Mass Spectrom. 1996, 7, 233-242. Harrison, A. G.; Csizmadia, I. G.; Tang, T.-H. Structure and Fragmentation of b2 Ions in Peptide Mass Spectra. J. Am. Soc. Mass Spectrom. 2000, 11, 427-43 6. Polfer, N. C.; Oomens, J .; Suhai, S.; Paizs, B. Spectroscopic and Theoretical Evidence for Oxazolone Ring Formation in Collision-Induced Dissociation of Peptides. J. Am. Chem. Soc. 2005, 127, 17154-17155. Morgan, D. G.; Bursey, M. M. A Linear Free-Energy Correlation in the Low- Energy Tandem Mass Spectra of Protonated Tripeptides Gly-Gly-Xxx. Org. Mass Spectrom. 1994, 29, 354-359. Harrison, A. G.; Young, A. B.; Bleiholder, C.; Suhai, S.; Paizs, B. Scrambling of Sequence Information in Collision-Induced Dissociation of Peptides. J. Am. Chem. Soc. 2006, 128, 10364-10365. Vékey, K.; Giimiiry, A. Theoretical Modelling of Mass Spectrometric Behaviour of Peptides: Singly and Doubly Protonated Tetraglycine. Rapid Commun. Mass Spectrom. 1996, 10, 1485-1496. Paizs, B.; Lendvay, G.; Vékey, K.; Suhai, S. Formation of b; Ions from Protonated Peptides: An ab initio Study. Rapid Commun. Mass Spectrom. 1999, 13, 525-533. Csonka, I. P.; Paizs, B.; Lendvay, G.; Suhai, S. Proton Mobility in Protonated Peptides: A Joint Molecular Orbital and RRKM Study. Rapid Commun. Mass Spectrom. 2000, 14, 417-43 1 . 195 102. 103. 104. 105. 106. 107. 108. 109. 110. Somogyi, A.; Wysocki, V. H.; Mayer, I. The Effect of Protonation Site on Bond Strengths in Simple Peptides: Application of ab initio and Modified Neglect of Differential Overlap Bond Orders and Modified Neglect of Differential Overlap Energy Partitioning. J. Am. Soc. Mass Spectrom. 1994, 5, 704-717. Reid, G.; Simpson, R.; O'Hair, R. J. A Mass Spectrometric and ab initio Study of the Pathways for Dehydration of Simple Glycine and Cysteine-Containing Peptide [M+H]+ Ions. J. Am. Soc. Mass Spectrom. 1998, 9, 945-956. Reid, G. E.; Simpson, R. J .; O'Hair, R. A. J. Probing the Fragmentation Reactions of Protonated Glycine Oligomers via Multistage Mass Spectrometry and Gas Phase Ion Molecule Hydrogen/Deuterium Exchange. Int. J. Mass Spectrom. 1999, 190-191, 209-230. Breci, L. A.; Tabb, D. L.; Yates, J. R.; Wysocki, V. H. Cleavage N-Terminal to Proline: Analysis of a Database of Peptide Tandem Mass Spectra. Anal. Chem. 2003, 75, 1963-1971. Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W.; Futrell, J. H.; Surnmerfield, S. G.; Gaskell, S. J. Influence of Secondary Structure on the Fragmentation of Protonated Peptides. J. Am. Chem. Soc. 1999, 121, 5142-5154. Yu, W.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Identification of the Facile Gas-Phase Cleavage of the Asp-Pro and Asp-Xxx Peptide Bonds in Matrix- Assisted Laser Desorption Time-of-Flight Mass Spectrometry. Anal. Chem. 1993, 65, 3015-3023. ' Surnmerfield, S. G.; Whiting, A.; Gaskell, S. J. Intra-Ionic Interactions in Electrosprayed Peptide Ions. Int. J. Mass Spectrom. Ion Processes 1997, 162, 149-161. Paizs, 13.; Suhai, s.; Hargittai, B.; Hruby, v. J.; Somogyi, A. Ab Initio and MS/MS Studies on Protonated Peptides Containing Basic and Acidic Amino Acid Residues: I. Solvated Proton vs. Salt-Bridged Structures and the Cleavage of the Terminal Amide Bond of Protonated RD-NHz. Int. J. Mass Spectrom. 2002, 219, 203-232. Qin, J.; Chait, B. T. Preferential Fragmentation of Protonated Gas-Phase Peptide Ions Adjacent to Acidic Amino Acid Residues. J. Am. Chem. Soc. 1995, 117, 5411-5412. 196 111. 112. 113. 114. 115. 116. 117. 118. 119. Tsaprailis, G.; Somogyi, A.; Nikolaev, E. N.; Wysocki, V. H. Refining the Model for Selective Cleavage at Acidic Residues in Arginine-Containing Protonated Peptides. Int. J. Mass Spectrom. 2000, 195-196, 467-479. Huang, Y.; Wysocki, V. H.; Tabb, D. L.; Yates, J. R. The Influence of Histidine on Cleavage C-terminal to Acidic Residues in Doubly Protonated Tryptic Peptides. Int. J. Mass Spectrom. 2002, 219, 233-244. Gu, C.; Tsaprailis, G.; Breci, L.; Wysocki, V. H. Selective Gas-Phase Cleavage at the Peptide Bond C-Terminal to Aspartic Acid in Fixed-Charge Derivatives of Asp-Containing Peptides. Anal. Chem. 2000, 72, 5804-5813. Sullivan, A. G.; Brancia, F. L.; Tyldesley, R.; Bateman, R.; Sidhu, K.; Hubbard, S. J .; Oliver, S. G.; Gaskell, S. J. The Exploitation of Selective Cleavage of Singly Protonated Peptide Ions Adjacent to Aspartic Acid Residues using a Quadrupole Orthogonal Time-of-F light Mass Spectrometer Equipped with a Matrix-Assisted Laser Desorption/Ionization Source. Int. J. Mass Spectrom. 2001, 210-211, 665- 676. Sidhu, K. S.; Sangvanich, P.; Brancia, F. L.; Sullivan, A. G.; Gaskell, S. J.; Wolkenhauer, 0.; Oliver, S. G.; Hubbard, S. J. Bioinforrnatic Assessment of Mass Spectrometric Chemical Derivatisation Techniques for Proteome Database Searching. Proteomics 2001, I, 1368-1377. Burlet, 0.; Yang, C.-Y.; Gaskell, S. J. Influence of Cysteine to Cysteic Acid Oxidation on the Collision-Activated Decomposition of Protonated Peptides: Evidence for Intraionic Interactions. J. Am. Soc. Mass Spectrom. 1992, 3, 337- 344. Surnmerfield, S. G.; Cox, K. A.; Gaskell, S. J. The Promotion of d-type Ions during the Low Energy Collision-Induced Dissociation of Some Cysteic Acid- Containing Peptides. J. Am. Soc. Mass Spectrom. 1997, 8, 25-31. Wang, Y.; Vivekananda, S.; Men, L.; Zhang, Q. Fragmentation of Protonated Ions of Peptides Containing Cysteine, Cysteine Sulfinic Acid, and Cysteine Sulfonic Acid. J. Am. Soc. Mass Spectrom. 2004, 15, 697-702. Men, L.; Wang, Y. Further Studies on the Fragmentation of Protonated Ions of Peptides Containing Aspartic Acid, Glutamic Acid, Cysteine Sulfinic Acid, and Cysteine Sulfonic Acid. Rapid Commun. Mass Spectrom. 2005, 19, 23-30. 197 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. Srikanth, R.; Wilson, J .; Bridgewater, J. D.; Numbers, J. R.; Lim, J .; Olbris, M. R.; Kettani, A.; Vachet, R. W. Improved Sequencing of Oxidized Cysteine and Methionine Containing Peptides Using Electron Transfer Dissociation. J. Am. Soc. Mass Spectrom. 2007, 18, 1499-1506. Bridgewater, J. D.; Srikanth, R.; Lim, J .; Vachet, R. W. The Effect of Histidine Oxidation on the Dissociation Patterns of Peptide Ions. J. Am. Soc. Mass Spectrom. 2007, 18, 553-562. Swiderek, K. M.; Davis, M. T.; Lee, T. D. The Identification of Peptide Modifications Derived from Gel-Separated Proteins Using Electrospray Triple Quadrupole and Ion Trap Analyses. Electrophoresis 1998, 19, 989-997. Mann, M.; Jensen, 0. N. Proteomic Analysis of Post-Translational Modifications. Nat Biotech 2003, 21, 255-261. Arman, R. S.; Carr, S. A. Phosphopeptide Analysis by Matrix-Assisted Laser Desorption Time-of-F light Mass Spectrometry. Anal. Chem. 1996, 68, 3413-3421. DeGnore, J. P.; Qin, J. Fragmentation of Phosphopeptides in an Ion Trap Mass Spectrometer. J. Am. Soc. Mass Spectrom. 1998, 9, 1175-1188. Tholey, A.; Reed, J .; Lehmann, W. D. Electrospray Tandem Mass Spectrometric Studies of Phosphopeptides and Phosphopeptide Analogues. J. Mass Spectrom. 1999, 34, 117-123. Metzger, S.; Hoffinann, R. Studies on the Dephosphorylation of Phosphotyrosine- Containing Peptides during Post-Source Decay in Matrix-Assisted Laser Desorption/Ionization. J. Mass Spectrom. 2000, 35, 1165-1177. Medzihradszky, K. F.; Darula, Z.; Perlson, E.; Fainzilber, M.; Chalkley, R. J .; Ball, H.; Greenbaum, D.; Bogyo, M.; Tyson, D. R.; Bradshaw, R. A.; Burlingarne, A. L. O-Sulfonation of Serine and Threonine: Mass Spectrometric Detection and Characterization of a New Posttranslational Modification in Diverse Proteins Throughout the Eukaryotes. Mol. Cell. Proteomics 2004, 3, 429-440. Medzihradszky, K. F.; Guan, S.; Maltby, D. A.; Burlingarne, A. L. Sulfopeptide Fragmentation in Electron-Capture and Electron-Transfer Dissociation. J. Am. Soc. Mass Spectrom. 2007, 18, 1617-1624. Raftery, M. J. Selective Detection of Thiosulfate-Containing Peptides using Tandem Mass Spectrometry. Rapid Commun. Mass Spectrom. 2005, 19, 674-682. 198 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. Hanisch, F.-G.; Green, B. N.; Bateman, R.; Peter-Katalinic, J. Localization of 0- glycosylation Sites of MUCl Tandem Repeats by QTOF ESI Mass Spectrometry. J. Mass Spectrom. 1998, 33, 358-362. Rademaker, G. J.; Pergantis, S. A.; Blok-Tip, L.; Langridge, J. 1.; Kleen, A.; Thomas-Oates, J. E. Mass Spectrometric Determination of the Sites of O-Glycan Attachment with Low Picomolar Sensitivity. Anal. Biochem. 1998, 25 7, 149-160. Steen, H.; Mann, M. Similarity Between Condensed Phase and Gas Phase Chemistry: Fragmentation of Peptides Containing Oxidized Cysteine Residues and its Implications for Proteomics. J. Am. Soc. Mass Spectrom. 2001, 12, 228- 232. Yagiie, J.; Nufiez, A.; Boix, M.; Esteller, M.; Alfonso, P.; Ignacio Casal, J. Oxidation of Carboxyamidomethyl Cysteine may add Complexity to Protein Identification. Proteomics 2005, 5, 2761-2768. Chowdhury, S. M.; Munske, G. R.; Ronald, R. C.; Bruce, J. E. Evaluation of Low Energy CID and ECD Fragmentation Behavior of Mono-Oxidized Thio-Ether Bonds in Peptides. J. Am. Soc. Mass Spectrom. 2007, 18, 493-501. Shetty, V.; Spellman, D. S.; Neubert, T. A. Characterization by Tandem Mass Spectrometry of Stable Cysteine Sulfenic Acid in a Cysteine Switch Peptide of Matrix Metalloproteinases. J. Am. Soc. Mass Spectrom. 2007, 18, 1544-1551. Gruhler, A.; Olsen, J. V.; Mohammed, S.; Mortensen, P.; Faergeman, N. J.; Mann, M.; Jensen, 0. N. Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway. Mol. Cell. Proteomics 2005, 4, 310-327. Chang, E. J .; Archambault, V.; McLachlin, D. T.; Krutchinsky, A. N.; Chait, B. T. Analysis of Protein Phosphorylation by Hypothesis-Driven Multiple-Stage Mass Spectrometry. Anal. Chem. 2004, 76, 4472-4483. Schroeder, M. J.; Shabanowitz, J.; Schwartz, J. C.; Hunt, D. F.; Coon, J. J. A Neutral Loss Activation Method for Improved Phosphopeptide Sequence Analysis by Quadrupole Ion Trap Mass Spectrometry. Anal. Chem. 2004, 76, 3590-3598. Wolschin, F.; Lehmann, U.; Glinski, M.; Weckwerth, W. An Integrated Strategy for Identification and Relative Quantification of Site-Specific Protein Phosphorylation using Liquid Chromatography Coupled to MSz/MS3. Rapid Commun. Mass Spectrom. 2005, 19, 3626-3632. 199 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. Stensballe, A.; Jensen, 0. N.; Olsen, J. V.; Haselmann, K. F.; Zubarev, R. A. Electron Capture Dissociation of Singly and Multiply Phosphorylated Peptides. Rapid Commun. Mass Spectrom. 2000, 14, 1793-1800. Bakhtiar, R.; Guan, Z. Electron Capture Dissociation Mass Spectrometry in Characterization of Post-Translational Modifications. Biochem. Biophys. Res. Commun. 2005, 334, 1-8. Mirgorodskaya, E.; Roepstorff, P.; Zubarev, R. A. Localization of O- Glycosylation Sites in Peptides by Electron Capture Dissociation in a Fourier Transform Mass Spectrometer. Anal. Chem. 1999, 71, 4431-4436. Cooper, H. J.; Hakansson, K.; Marshall, A. G. The Role of Electron Capture Dissociation in Biomolecular Analysis. Mass Spectrom. Rev. 2005, 24, 201-222. Bakhtiar, R.; Guan, Z. Electron Capture Dissociation Mass Spectrometry in Characterization of Peptides and Proteins. Biotechnol. Lett. 2006, 28, 1047-1060. Chamot-Rooke, J .; van der Rest, G.; Dalleu, A.; Bay, S.; Lernoine, J. The Combination of Electron Capture Dissociation and Fixed Charge Derivatization Increases Sequence Coverage for O-Glycosylated and O-Phosphorylated Peptides. J. Am. Soc. Mass Spectrom. 2007, 18, 1405-1413. Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J .; Hunt, D. F. Peptide and Protein Sequence Analysis by Electron Transfer Dissociation Mass Spectrometry. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 9528-9533. Gunawardena, H. P.; Emory, J. F.; McLuckey, S. A. Phosphopeptide Anion Characterization via Sequential Charge Inversion and Electron-Transfer Dissociation. Anal. Chem. 2006, 78, 3788-3793. Zhang, Q.; Frolov, A.; Tang, N.; Hoffmann, R.; van de Goor, T.; Metz, T. 0.; Smith, R. D. Application of Electron Transfer Dissociation Mass Spectrometry in Analyses of Non-Enzymatically Glycated Peptides. Rapid Commun. Mass Spectrom. 2007, 21, 661-666. Hogan, J. M.; Pitteri, S. J .; Chrisman, P. A.; McLuckey, S. A. Complementary Structural Information from a Tryptic N-Linked Glycopeptide via Electron Transfer Ion/Ion Reactions and Collision-Induced Dissociation. J. Proteome Res. 2005, 4, 628-632. 200 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. O'Connor, P. B.; Coumoyer, J. J .; Pitteri, S. J .; Chrisman, P. A.; McLuckey, S. A. Differentiation of Aspartic and Isoaspartic Acids Using Electron Transfer Dissociation. J. Am. Soc. Mass Spectrom. 2006, 17, 15-19. Chi, A.; Huttenhower, C.; Geer, L. Y.; Coon, J. J.; Syka, J. E. P.; Bai, D. L.; Shabanowitz, J .; Burke, D. J .; Troyanskaya, O. G.; Hunt, D. F. Analysis of Phosphorylation Sites on Proteins from Saccharomyces cerevisiae by Electron Transfer Dissociation (ETD) Mass Spectrometry. Proc. Natl. Acad. Sci. USA 2007, 104, 2193-2198. Dikler, 8.; Kelly, J. W.; Russell, D. H. hnproan Mass Spectrometric Sequencing of Arginine-Containing Peptides by Derivatization with Acetylacetone. J. Mass Spectrom. 1997, 32, 1337-1349. Foettinger, A.; Leitner, A.; Lindner, W. Derivatisation of Arginine Residues with Malondialdehyde for the Analysis of Peptides and Protein Digests by LC-ESI- MS/MS. J. Mass Spectrom. 2006, 41, 623-632. Peters, E. C.; Horn, D. M.; Tully, D. C.; Brock, A. A Novel Multifunctional Labeling Reagent for Enhanced Protein Characterization with Mass Spectrometry. Rapid Commun. Mass Spectrom. 2001, 15, 2387-2392. Conrotto, P.; Hellman, U. Lys Tag: An Easy and Robust Chemical Modification for Improved de novo Sequencing with a Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-F light Mass Spectrometer. Rapid Commun. Mass Spectrom. 2008, 22, 1823-1833. Roth, K. D. W.; Huang, Z.-H.; Sadagopan, N.; Watson, J. T. Charge Derivatization of Peptides for Analysis by Mass Spectrometry. Mass Spectrom. Rev. 1998, 17, 255-274. Huang, Z.-H.; Wu, J.; Roth, K. D. W.; Yang, Y.; Gage, D. A.; Watson, J. T. A Picomole-Scale Method for Charge Derivatization of Peptides for Sequence Analysis by Mass Spectrometry. Anal. Chem. 1997, 69, 137-144. Sadagopan, N.; Watson, J. T. Investigation of the Tris(trimethoxyphenyl)phosphonium Acetyl Charged Derivatives of Peptides by Electrospray Ionization Mass Spectrometry and Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2000, 11, 107-119. Adamczyk, M.; Gebler, J. C.; Wu, J. Charge Derivatization of Peptides to Simplify their Sequencing with an Ion Trap Mass Spectrometer. Rapid Commun. Mass Spectrom. 1999, 13, 1413-1422. 201 161. 162. 163. 164. 165. 166. 167. 168. 169. Kuyarna, H.; Sonomura, K.; Shima, K.; Nishimura, 0.; Tsunasawa, S. An Improved Method for de novo Sequencing of Arginine-Containing, Na-his(2,4,6- trimethoxyphenyl)phosphonium-acetylated Peptides. Rapid Commun. Mass Spectrom. 2008, 22, 2063-2072. Munchbach, M.; Quadroni, M.; Miotto, G.; James, P. Quantitation and Facilitated de Novo Sequencing of Proteins by Isotopic N-Terminal Labeling of Peptides with a Fragmentation-Directing Moiety. Anal. Chem. 2000, 72, 4047-4057. Lee, Y. H.; Kim, M.-S.; Choie, W.-S.; Min, H.-K.; Lee, S.-W. Highly Informative Proteome Analysis by Combining Improved N-terminal Sulfonation for de novo Peptide Sequencing and Online Capillary Reverse-Phase Liquid Chromatography/Tandem Mass Spectrometry. Proteomics 2004, 4, 1684-1694. Keough, T.; Youngquist, R. S.; Lacey, M. P. A Method for High-Sensitivity Peptide Sequencing using Postsource Decay Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 7131- 7136. Bauer, M. D.; Sun, Y.; Keough, T.; Lacey, M. P. Sequencing of Sulfonic Acid Derivatized Peptides by Electrospray Mass Spectrometry. Rapid Commun. Mass Spectrom. 2000, 14, 924-929. Keough, T.; Lacey, M. P.; Fieno, A. M.; Grant, R. A.; Sun, Y.; Bauer, M. D.; Begley, K. B. Tandem Mass Spectrometry Methods for Definitive Protein Identification in Proteomics Research. Electrophoresis 2000, 21, 2252-2265. Summerfield, S. G.; Bolgar, M. S.; Gaskell, S. J. Promotion and Stabilization of b1 Ions in Peptide Phenythiocarbarnoyl Derivatives: Analogies with Condensed- phase Chemistry. J. Mass Spectrom. 1997, 32, 225-231. Summerfield, S. G.; Steen, H.; O'Malley, M.; Gaskell, S. J. Phenyl Thiocarbamoyl and Related Derivatives of Peptides: Edman Chemistry in the Gas Phase. Int. J. Mass Spectrom. 1999, 188, 95-103. Reid, G. E.; Roberts, K. D.; Simpson, R. J.; O'Hair, R. A. J. Selective Identification and Quantitative Analysis of Methionine Containing Peptides by Charge Derivatization and Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2005, 16,1131-1150. 202 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. Amunugama, M.; Roberts, K. D.; Reid, G. E. Mechanisms for the Selective Gas- Phase Fragmentation Reactions of Methionine Side Chain Fixed Charge Sulfonium Ion Containing Peptides. J. Am. Soc. Mass Spectrom. 2006, 17, 1631- 1642. Sierakowski, J .; Amunugama, M.; Roberts, K. D.; Reid, G. E. Substituent Effects on the Gas-Phase Fragmentation Reactions of Sulfonium Ion Containing Peptides. Rapid Commun. Mass Spectrom. 2007, 21, 1230-1238. Roberts, K. D.; Reid, G. E. Leaving Group Effects on the Selectivity of the Gas- Phase Fragmentation Reactions of Side Chain Fixed-Charge-Containing Peptide Ions. J. Mass Spectrom. 2007, 42, 187-198. Chu, I. K.; Rodriguez, C. F.; Hopkinson, A. C.; Siu, K. W. M.; Lau, T.-C. Formation of Molecular Radical Cations of Enkephalin Derivatives via Collision- Induced Dissociation of Electrospray-Generated Copper (11) Complex Ions of Amines and Peptides. J. Am. Soc. Mass Spectrom. 2001, 12, 1114-1119. Yin, H.; Chacon, A.; Porter, N. A.; Masterson, D. S. Free Radical-Induced Site- Specific Peptide Cleavage in the Gas Phase: Low-Energy Collision-Induced Dissociation in ESI- and MALDI Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2007, 18, 807-816. Ly, T.; Julian, R. R. Residue-Specific Radical-Directed Dissociation of Whole Proteins in the Gas-Phase. J. Am. Chem. Soc. 2008, 130, 351-358. Diedrich, J. K.; Julian, R. R. Site-Specific Radical Directed Dissociation of Peptides at Phosphorylated Residues. J. Am. Chem. Soc. 2008, 130, 12212-12213. Jones, A. M. E.; Bennett, M. H.; Mansfield, J. W.; Grant, M. Analysis of the Defence Phosphoproteome of Arabidopsis thaliana using Differential Mass Tagging. Proteomics 2006, 6, 4155-4165. Champion, P. A. D.; Stanley, S. A.; Champion, M. M.; Brown, E. J .; Cox, J. S. C- Terminal Signal Sequence Promotes Virulence Secretion in Mycobacterium Tuberculosis. Science 2006, 313, 1632-1636. Corvey, C.; Koetter, P.; Beckhaus, T.; Hack, J .; Hofrnann, S.; Harnpel, M.; Stein, T.; Karas, M.; Entian, K.-D. Carbon Source-Dependent Assembly of the Snflp Kinase Complex in Candida albicans. J. Biol. Chem. 2005, 280, 25323-25330. 203 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. Williamson, B. L.; Marchese, J .; Monice, N. A. Automated Identification and Quantification of Protein Phosphorylation Sites by LC/MS on a Hybrid Triple Quadrupole Linear Ion Trap Mass Spectrometer. Mol. Cell. Proteomics 2006, 5, 337-346. Wolf-Yadlin, A.; Hautaniemi, S.; Lauffenburger, D. A.; White, F. M. Multiple Reaction Monitoring for Robust Quantitative Proteomic Analysis of Cellular Signaling Networks. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 5860-5865. Cunningham, J. C.; Glish, G. L.; Burinsky, D. J. High Amplitude Short Time Excitation: A Method to Form and Detect Low Mass Product Ions in a Quadrupole Ion Trap Mass Spectrometer. J. Am. Soc. Mass Spectrom. 2006, 17, 81-84. Meany, D. L.; Xie, H.; Thompson, L. V.; Arriaga, E. A.; Griffin, T. J. IdentificatiOn of Carbonylated Proteins from Enriched Rat Skeletal Muscle Mitochondria using Affinity Chromatography-Stable Isotope Labeling and Tandem Mass Spectrometry. Proteomics 2007, 7, 1150-1163. Griffin, T. J.; Xie, H.; Bandhakavi, S.; Popko, J.; Mohan, A.; Carlis, J. V.; Higgins, L. iTRAQ Reagent-Based Quantitative Proteomic Analysis on a Linear Ion Trap Mass Spectrometer. J. Proteome Res. 2007, 6, 4200-4209. Bantscheff, M.; Eberhard, D.; Abraham, Y.; Bastuck, S.; Boesche, M.; Hobson, S.; Mathieson, T.; Perrin, J.; Raida, M.; Rau, C.; Reader, V.; Sweetrnan, G.; Bauer, A.; Bouwrneester, T.; Hopf, C.; Kruse, U.; Neubauer, G.; Rarnsden, N.; Rick, J .; Kuster, B.; Drewes, G. Quantitative Chemical Proteomics Reveals Mechanisms of Action of Clinical ABL Kinase Inhibitors. Nat Biotech 2007, 25, 1035-1044. Chang, B.; Unlii, M.; Clauser, K.; Carr, S. A., iTRAQ-IT: Implementation of iTRAQ Quantitation Tags on Ion Trap Instruments via MS3. In 53rd ASMS Conference on Mass Spectrometry, San Antonio, Texas, 2005. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray Ionization for Mass Spectrometry of Large Biomolecules. Science 1989, 246, 64-71. Wilm, M. 3.; Mann, M. Electrospray and Taylor Cone Theory, Dole's Beam of Macromolecules at Last? Int. J. Mass Spectrom. Ion Processes 1994, I36, 167- 180. Gomez, A.; Tang, K. Charge and Fission of Droplets in Electrostatic Sprays. Phys. Fluids 1994, 6, 404-414. 204 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. Iribarne, J. V.; Thompson, B. A. On the Evaporation of Small Ions from Charged Droplets. J. Chem. Phys. 1976, 64, 2287-2294. Thompson, B. A.; Iribarne, J. V. Field Induced Ion Evaporation from Liquid Surfaces at Atmospheric Pressure. J. Chem. Phys. 1979, 71, 4451-4463. Kebarle, P. A Brief Overview of the Present Status of the Mechanisms Involved in Electrospray Mass Spectrometry. J. Mass Spectrom. 2000, 35, 804-817. Karas, M.; Hillencamp, F. Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10 000 Daltons. Anal. Chem. 1988, 60, 2299-2301. Zenobi, R.; Knochenmuss, R. Ion Formation in MALDI Mass Spectrometry. Mass Spectrom. Rev. 1998, I 7, 337-366. de Hoffmann, E. D.; Stroobant, V., Mass Spectrometry Principles and Applications. 2nd ed.; John Wiley and Sons: New York, 2002. March, R. E. An Introduction to Quadrupole Ion Trap Mass Spectrometry. J. Mass Spectrom. 1997, 32, 351-369. Hager, J. W. A New Linear Ion Trap Mass Spectrometer. Rapid Commun. Mass Spectrom. 2002, 16, 512-526. Schwartz, J. C.; Senko, M. W.; Syka, J. E. A Two-Dimensional Quadrupole Ion Trap Mass Spectrometer. J. Am. Soc. Mass Spectrom. 2002, 13, 659-669. Finkielsztein, L. M.; Aguirre, J. M.; Lantafio, B.; Alesso, E. N.; Iglesias, G. Y. M. ZnIz/NaCNBH3 as an Efficient Reagent for Regioselective Ring Opening of the Benzylic Epoxide Moiety. Synth. Commun. 2004, 34, 895-901. Shechter, Y. Selective Oxidation and Reduction of Methionine Residues in Peptides and Proteins by Oxygen Exchange Between Sulfoxide and Sulfide. J. Biol. Chem. 1986, 261, 66-70. 205 201. 202. 203. 204. 205. 206. 207. 208. 209. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A.; Stratrnan, R. E.; Burant, J. C.; Dapprich, S.; Millarn, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, 0.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J .; Peterson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J .; Oritz, J. V.; Baboul, A. G.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, 1.; Gomperts, R.; Martin, R. L.; Fox, D. J .; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, Revision A. 7, Gaussian Inc.: Pittsburg PA, 1998. Carruthers, N. J .; Stemmer, P. M. Methionine Oxidation in the Calmodulin- Binding Domain of Calcineurin Disrupts Calmodulin Binding ad Calcineurin Activation. Biochemistry 2008, 47, 3085-3095. Clauser, K. R.; Hall, S. C.; Smith, D. M.; Webb, J. W.; Andrews, L. E.; Tran, H. M.; Epstein, L. B.; Burlingame, A. L. Rapid Mass Spectrometric Peptide Sequencing and Mass Matching for Characterization of Human Melanoma Proteins Isolated by Two-Dimensional PAGE. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 5072-5076. Qin, J .; Chait, B. T. Identification and Characterization of Posttranslational Modifications of Proteins by MALDI Ion Trap Mass Spectrometry. Anal. Chem. 1997, 69, 4002-4009. Cohen, S. L. Ozone in Ambient Air as a Source of Adventitious Oxidation. A Mass Spectrometric Study. Anal. Chem. 2006, 78, 4352-4362. Morand, K.; Talbo, G.; Mann, M. Oxidation of Peptides During Electrospray Ionization. Rapid Commun. Mass Spectrom. 1993, 7, 738-743. Sommer, S.; Fakata, K. L.; Swanson, S. A.; Stemmer, P. M. Modulation of the Phosphatase Activity of Calcineurin by Oxidants and Antioxidants in vitro. Eur. J. Biochem. 2000, 267, 2312-2322. Cross, J. V.; Templeton, D. J. Regulation of Signal Transduction Through Protein Cysteine Oxidation. Antioxid. Redox Signaling 2006, 8, 1819-1827. Baty, J. W.; Hampton, M. B.; Winterboum, C. C. Proteomic Detection of Hydrogen Peroxide-Sensitive Thiol Proteins in Jurkat Cells. Biochem. J. 2005, 389, 785-795. 206 210. 211. 212. 213. 214. 215. 216. 217. 218. 219. Midwinter, R. G.; Cheah, F.-C.; Moskovizt, J .; Vissers, M. C.; Winterbourn, C. C. Ich is a Sensitive Target for Oxidation by Cell-Permeable Chlorarrrines: Inhibition of NF-KB Activity by Glycine Chloramine Through Methionine Oxidation. Biochem. J. 2006, 396, 71-78. Setlruraman, M.; McComb, M. E.; Heibeck, T.; Costello, C. E.; Cohen, R. A. Isotope-Coded Affinity Tag Approach to Identify and Quantify Oxidant-Sensitive Protein Thiols. Mol. Cell. Proteomics 2004, 3, 273-278. Shechter, Y.; Rubinstein, M.; Patchornik, A. Selective Covalent Binding of Methionyl-Containing Peptides and Proteins to Water Insoluble Polymeric Reagent and their Regeneration. Biochemistry 1977, 16, 1424-1430. Degen, J .; Kyte, J. The Purification of Peptides which Contain Methionine Residues. Anal. Biochem. 1978, 89, 529-539. Gevaert, K.; Van Damme, J.; Goethals, M.; Thomas, G. R.; Hoorelbeke, B.; Demol, H.; Martens, L.; Puype, M.; Staes, A.; Vandekerckhove, J. Chromatographic Isolation of Methionine-Containing Peptides for Gel-Free Proteome Analysis: Identification Of More Than 800 Escherichia Coli Proteins. Mol. Cell. Proteomics 2002, I, 896-903. Kuyama, H.; Watanabe, M.; Toda, C.; E., A.; Tanaka, K.; Nishimura, 0. An Approach to Quantitative Proteome Analysis by Labeling Tryptophan Residues. Rapid Commun. Mass Spectrom. 2003, 17, 1642-1650. Lagerwerf, F. M.; van de Weert, M.; Heerrna, W.; Haverkarnp, J. Identification of Oxidized Methionine in Peptides. Rapid Commun. Mass Spectrom. 1996, 10, 1905-1910. Jiang, X.; Smith, J. 3.; Abraham, E. C. Identification of a MS-MS Fragment Diagnostic for Methionine Sulfoxide J. Mass Spectrom. 1996, 31, 1309-1310. Betancourt, L.; Takao, T.; Gonzalez, J.; Reyes, 0.; Besada, V.; Padron, G.; Shimonishi, Y. The Metastable Decomposition of a Peptide Containing Oxidized Methionine(s) in Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 1999, 13, 1075-1076. Yang, F.; Stenoien, D. L.; Strittmatter, E. F.; Wang, J .; Ding, L.; Lipton, M. 8.; Monroe, M. E.; Nicora, C. D.; Gristenko, M. A.; Tang, K.; Fang, R.; Adkins, J. N.; Camp, D. G.; Chen, D. J .; Smith, R. D. Phosphoproteome Profiling of Human Skin Fibroblast Cells in Response to Low- and High-Dose Irradiation. J. Proteome Res. 2006, 5, 1252-1260. 207 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 230. 231. 232. Beausoleil, S. A.; Villen, J.; Gerber, S. A.; Rush, J.; Gygi, S. P. A Probability- Based Approach for High-Throughput Protein Phosphorylation Analysis and Site Localization. Nat. Biotechnol. 2006, 24, 1285-1292. Madesclaire, M. Reduction of Sulfoxides to Thioethers. Tetrahedron 1988, 44, 6537-6580. Houghten, R. A.; Li, C. H. Reduction of Sulfoxides in Peptides and Proteins. Anal. Biochem. 1979, 98, 36-46. Faucher, A.; Grand-Maine, C. Tris(2-carboxyethyl)phosphine (TCEP) for the Reduction of Sulfoxides, Sulfonylchlorides, N-oxides and Azides. Synth. Commun. 2003, 33, 3503-3511. Savige, W. E.; F ontana, A. Interconversion of Methionine and Methionine Sulfoxide. Methods Enzymol. 1977, 4 7, 453-459. Toennies, G.; Callan, T. P. A Comparison of Oxidative Reactions of Methionine, Cysteine, and Cystine. Determination of Methionine by Hydrogen Peroxide Oxidation. J. Biol. Chem. 1939, 129, 481-490. Caldwell, K. A.; Tappel, A. L. Reactions of Seleno- and Sulfoamino Acids with Hydroperoxides. Biochemistry 1964, 3, 1643-1647. Knowles, J. R. The Role of Methionine in a-Chymotrypsin-Catalyzed Reactions. Biochem. J. 1965, 95, 180-190. Atassi, M. Z. Periodate Oxidation of Sperm-Whale Myoglobin and the Role of the Methionine Residues in the Antigen-Antibody Reaction. Biochem. J. 1967, 102, 478-487. Clamp, J. R.; Hough, L. The Periodate Oxidation of Amino Acids with Reference to Studies on Glycoproteins. Biochem. J. 1965, 94, 17-24. Shechter, Y.; Burstein, Y.; Patchorrrik, A. Selective Oxidation of Methionine Residues in Proteins. Biochemistry 1975, 14, 4497-4503. Savige, W. E.; Fontana, A., New Procedures for the Chemical Modification of Tryptophan and Sulfur Amino Acids in Peptides and Proteins. In Peptides, Proceedings, 14th European Peptide Symposium, 1976; p 135. Burstein, Y.; Patchomik, A. Selective Chemical Cleavage of Trytophanyl Peptide Bonds in Peptides and Proteins. Biochemistry 1972, 11, 4641-4650. 208 233. 234. 235. 236. 237. 238. 239. 240. 241. 242. Hachimori, Y.; Horinishi, H.; Kurihara, K.; Shibata, K. States of Amino Acid Residues in Proteins: Different Reactivities with H202 of Tryptophan Residues in Lysozyme, Proteinases and Zymogens. Biochim. Biophys. Acta 1964, 93, 346- 360. van de Weert, M.; Lagerwerf, F. M.; Haverkamp, J.; Heerma, W. Mass Spectrometric Analysis of Oxidized Tryptophan. J. Mass Spectrom. 1998, 33, 884-891. Uchida, K.; Kawakishi, S. Identification of Oxidized Histidine Generated at the Active Site of Cu,Zn-Superoxide Dismutase Exposed to H202:Selective Generation of 2-Oxo-Histidine at the Histidine 118. J. Biol. Chem. 1994, 269, 2405-2410. Elias, J. E.; Gibbons, F. D.; King, 0. D.; Roth, F. P.; Gygi, S. P. Intensity-Based Protein Identification by Machine Learning from a Library of Tandem Mass Spectra. Nat. Biotechnol. 2004, 22, 214-219. Gibbons, F. D.; Elias, J. E.; Gygi, S. P.; Roth, F. P. SILVER Helps Assign Peptides to Tandem Mass Spectra using Intensity-Based Scoring. J. Am. Soc. Mass Spectrom. 2004, 15, 910-912. Zhang, Z. Prediction of Low-Energy Collision-Induced Dissociation Spectra of Peptides. Anal. Chem. 2004, 76, 3908-3922. Zhang, Z. Prediction of Low-Energy Collision-Induced Dissociation Spectra of Peptides with Three or More Charges. Anal. Chem. 2005, 77, 6364-6373. Narasimhan, C.; Tabb, D. L.; VerBerkmoes, N. C.; Thompson, M. R.; Hettich, R. L.; Uberbacher, E. C. MASPIC: Intensity-Based Tandem Mass Spectrometry Scoring Scheme That Improves Peptide Identification at High Confidence. Anal. Chem. 2005, 7 7, 7581-7593. Sun, S.; Meyer-Arendt, K.; Eichelberger, B.; Brown, R.; Yen, C.-Y.; Old, W. M.; Pierce, K.; Cios, K. J.; Ahn, N. G.; Resing, K. A. Improved Validation of Peptide MS/MS Assignments Using Spectral Intensity Prediction. Mol. Cell. Proteomics 2007, 6, 1-17. Sechi, S.; Chait, B. T. Modification of Cysteine Residues by Alkylation. A Tool in Peptide Mapping and Protein Identification. Anal. Chem. 1998, 70, 5150-5158. 209 243. 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. Moritz, R. L.; Eddes, J. 8.; Reid, G. E.; Simpson, R. J. S-Pyridylethylation of Intact Polyacrylamide Gels and In-Situ Digestion of Electrophoretically- Separated Proteins: A Rapid Mass Spectrometric Method for Identifying Cysteine-Containing Peptides. Electrophoresis 1996, I 7, 907-917. Shevchenko, A.; Wilm, M.; Vorrn, 0.; Mann, M. Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels. Anal. Chem. 1996, 68, 850- 858. Hunter, E. P.; Lias, S. G. Evaluated Gas Phase Basicities and Proton Affinities of Molecules: An Update. J. Phys. Chem. Ref Data 1998, 27, 413-656. Zhang, R.; Sioma, C. S.; Thompson, R. A.; Xiong, L.; Regnier, F. E. Controlling Deuterium Isotope Effects in Comparative Proteomics. Anal. Chem. 2002, 74, 3662-3669. Lehmann, W. D.; Kruger, R.; Salek, M.; Hung, C.-W.; Wolschin, F.; Weckwerth, W. Neutral Loss-Based Phosphopeptide Recognition: A Collection of Caveats. J. Proteome Res. 2007, 6, 2866-2873. Rusnak, F.; Mertz, P. Calcineurin: Form and Function. Physiol. Rev. 2000, 80, 1483-1521. Chin, D.; Means, A. R. Calmodulin: A Prototypical Calcium Sensor. Trends Cell Biol. 2000, 10, 322-328. Heineke, J .; Molkentin, J. D. Regulation of Cardiac Hypertrophy by Intracellular Signalling Pathways. Nat. Rev. Mol. Cell Biol. 2006, 7, 589-600. Fujiwara, A.; Kakizawa, S.; Iino, M. Induction of Cerebellar Long-Term Depression Requires Activation of Calcineurin in Purkinje Cells. Neuropharmacology 2007, 52, 1663-1670. Kissinger, C. R.; Parge, H. E.; Knighton, D. R.; Lewis, C. T.; Pelletier, L. A.; Tempczyk, A.; Kalish, V. J .; Tucker, K. D.; Showalter, R. E.; Moomaw, E. W.; Gastrinel, L. N.; Habuka, N.; Chen, X.; Maldonado, F.; Barker, J. E.; Bacquet, R.; Villafranca, J. E. Crystal Structures of Human Calcineurin and the Human FKBP12-FK506-Calcineurin Complex. Nature 1995, 378, 641-644. Ye, Q.; Li, X.; Wong, A.; Wei, Q.; Jia, Z. Structure of Calmodulin Bound to a Calcineurin Peptide: A New Way of Making an Old Binding Mode. Biochemistry 2006, 45, 738-745. 210 1111111111111111111111111111Ill11H 3 1 9252 293 02956