PROTEOMICS OFBIOLOGICAL SYSTEMS

Protein Phosphorylation Using MassSpectrometry Techniques

Bryan M. Ham

~WILEYA JOHN WILEY & SONS, INC., PUBLICATION

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Preface

Acknowledgments

About the Author

CONTENTS

1 Posttranslational Modification (PTM) of Proteins

1.1 Over 200 Forms of PTM of Proteins 11.2 Three Main Types of PTM Studied by MS 21.3 Overview of Nano-Electrospray/Nanoflow LC-MS 2

1.3.1 Definition and Description of MS 21.3.2 Basic Design of Mass Analyzer

Instrumentation 31.3.3 ESI 71.3.4 Nano-ESI 11

1.4 Overview of Nucleic Acids 151.5 Proteins and Proteomics 20

1.5.1 Introduction to Proteomics 201.5.2 Protein Structure and Chemistry 221.5.3 Bottom-Up Proteomics: MS of Peptides 27

1.5.3.1 History and Strategy 271.5.3.2 Protein Identification through Product

Ion Spectra 301.5.3.3 High-Energy Product Ions 361.5.3.4 De Novo Sequencing 371.5.3.5 Electron Capture Dissociation

(ECD) 401.5.4 Top-Down Proteomics: MS of Intact Proteins 42

1.5.4.1 Background 421.5.4.2 GP Basicity and Protein Charging 421.5.4.3 Calculation of Charge State and

Molecular Weight 441.5.4.4 Top-Down Protein Sequencing 46

p

3 SuJfation of Proteins as posttranslational Modification 81

3.1 Glycosaminoglycan Sulfation 813.2 Cellular processes Involved in Sulfation 813.3 Brief Example of Phosphoryl ation 823.4 Sulfotr ansferase Class of Enzymes 823.5 Fragmentation Nomenclature for Carbohydrates 823.6 Sulfated Mucin Oligosaccharides 833.7 Tyrosine Sulfation 843.8 Tyrosylprotein Sulfotransferases TPSTl and

TPST2 873.9 O-Sulfated Human Proteins 893.10 Sulfated Peptide Product Ion Spectra 893.11 Use of Higher Energy Collisions 93

2 Glycosylation of Proteins 59

2.1 Production of a Glycoprotein 592.2 Biological Processes of Protein Glycosylation 592.3 N-Link ed and O-Linked Glycosylation 602.4 Carbohydrates 60

2.4.1 Ionization of Oligosaccharides 642.4.2 Carbohydrate Fragmentation 652.4.3 Complex Oligosaccharide Structural

Elucidation 702.5 Thr ee Objectives in Studying Glycoproteins 722.6 Glycosylation Study Approaches 72

2.6.1 MS of Glycopeptides 732.6.2 Mass Pattern Recognition 75

2.6.2.1 High Galactose GlycosylationPattern 75

2.6.3 Charge Sta te Determination 762.6.4 Diagnostic Fragment Ions 762.6.5 High-Resolution/High-Mass Accuracy

Measurement and Identification 762.6.6 Digested Bovine Fetuin 78

Reference 79

Systems Biology and Bioinformatics 48Biomarkers in Cancer 52

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1.5.51.5.6

Reference

viii CONTENTS

3.12 Electron Capture Dissociation (ECD) 943.13 Sulfation versus Phosphorylation 95Reference 97

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CONTENTS ix

4.1.5.24.1.5.34.1.5.4

4 Eukaryote PTM as Phosphorylation: Normal State Studies

4.1 Mass Spectral Measurement with Examples of HeLa CellPhosphoproteome 994.1.1 Introduction 994.1.2 Protein Phosphatase and Kinase 994.1.3 Hydroxy-Amino Acid Phosphorylation 1004.1.4 Traditional Phosphoproteomic Approaches 1024.1.5 Current Approaches 103

4.1.5.1 Phosphoproteomic EnrichmentTechniques 103IMAC 104MOAC 105Methylation of Peptides prior to IMACor MOAC Enrichment 107

4.1.6 The Ideal Approach 1074.1.7 One-Dimensional (l-D) Sodium Dodecyl Sulfate

(SDS) PAGE 1084.1.8 Tandem MS Approach 108

4.1.8.1 pS Loss of Phosphate Group 1094.1.8.2 pT Loss of Phosphate Group 1124.1.8.3 pY Loss of Phosphate Group 113

4.1.9 Alternative Methods: Infrared MultiphotonDissociation (IRMPD) and Electron CaptureDissociation (ECD) 115

4.1.10 Electron Transfer Dissociation (ETD) 1154.2 The He La Cell Phosphoproteome 118

4.2.1 Introduction 1184.2.2 Background of Study 1184.2.3 What is Covered 1194.2.4 Optimized Methods to Use for Phosphoproteomic

Studies 1194.2.4.1 Cell Culture 1194.2.4.2 Extraction of HeLa Cell

Proteins 1204.2.4.3 Trizol Extraction and Tryptic

Digestion 120

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4.2.4.4 Solid-Phase Extraction (SPE)Desalting 120

4.2.4.5 Converting Peptide Carboxyl Moieties toMethyl Esters 121

4.2.4.6 Roche Compl ete Lysis-M, EDTA-FreeExtraction 122

4.2.4.7 1-D SDS-PAGE Cleanup 1224.2.4.8 In-Gel Reduction, Alk ylation, Digestion,

and Extraction of Peptides 1224.2.4.9 Pho sphopeptide Enrichment Using

IMAC 1234.2.5 Description of Instrumental Analyses 123

4.2.5.1 RP/Nano-HPLC Separation 1234.2.5.2 MS An alysis 125

4.2.6 Current Approaches for Peptide Identificationand False Discovery Rate (FDR)Determination 125

4.2.7 Results of the Protein Extraction andPreparation 1264.2.7.1 Detergent Lysis,Trizol, and

Ultracentrifugation 1264.2.7.2 Nucleic Acid Removal with

SDS-PAG E 1274.2.8 HeLa Cell Pho sphoproteome Methodology

Comparison 1284.2.8.1 Roche In-Solution versus Trizol

Extraction 1294.2.8.2 In-Solution and In-Gel Dige sts

Phosphoproteome Coverage 129

4.2.9 Overall Conclusion 1344.3 Nonphosphoproteome HeLa Cell Analysis 135

4.3.1 IMAC Flow Through Peptide Analysis 1354.3.2 IMAC NaCI Wash Peptide Analysis 1364.3.3 IMAC Flow Through versus NaCI Wash

Compa rison 1384.3.4 Ge ne On tology Comparison 1384.3.5 IMAC Bed Nonspecific Binding Stud y 140

4.4 Reviewing Spectra Using the SpectrumLook SoftwarePackage 143

Reference 144

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CONTENTS xi

5 Eukaryote PTM as Phosphorylation: Perturbed State Studies 147

5.1 Study of the Phosphoproteome of He La Cells underPerturbed Conditions by Nano-High-Performance LiquidChromatography HPLC Electrospray Ionization (ESI)Linear Ion Trap (LTQ)-FT/Mass Spectrometry (MS) 1475.1.1 Introduction 1475.1.2 Ataxia Telangiectasia Mutated (ATM) and ATM

and Rad3-Related (ATR) 1495.1.3 Background of Study 149

5.1.3.1 PP5 1495.1.3.2 Functions of PP5 1515.1.3.3 DDR of PP5 151

5.1.4 Review of Optimized Approach to Study 1515.1.4.1 Producing Cell Cultures 1515.1.4.2 Protein Extraction 1525.1.4.3 Phosphopeptide Enrichment by

IMAC 1545.1.4.4 Reversed-Phase (RP)/Nano-HPLC

Separation 1555.1.4.5 LTQ-FT/MS/MS 1565.1.4.6 Protein Identification and False Discovery

Rate (FDR) Determination 1565.1.4.7 Phosphopeptide Quantitative Differential

Comparison 1575.1.4.8 Data Set Peak Matching and

Alignment 1575.1.4.9 Phosphopeptide Response

Normalization 1605.1.5 Phosphoproteome Gene Ontology (GO)

Comparison 1605.1.5.1 GO Cellular Component 162

5.1.6 Potential Regulated Target Proteins of PP5 1625.1.6.1 Analysis of Variance (ANOVA) 1625.1.6.2 Four Potential Target Proteins 166

5.1.7 GO Differential Comparison 1675.1.7.1 GO Cellular Component 1685.1.7.2 Influence of Classes or Categories of

Proteins 1685.1.7.3 Molecular Function Interacting

Modules 168

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Conclusion 175Reviewing Spectra Using the SpectrumLookSoftware Package 175

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5.1.85.1.9

Reference

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6 Prokaryotic Phosphorylation of Serine, Threonine,and Tyrosine

6.1 Introduction 1816.1.1 Serine (Ser)/Threonine (Thr)/Tyrosine (Tyr)

Phosphorylation 1816.1.2 Histidine (His) Phosphorylation 1816.1.3 Caulobacter crescentus 1816.1.4 Ser/Thr/Tyr Phosphorylation of C. crescentus 1836.1.5 Ser/Thr/Tyr Phosphorylation of Bacillus subtilis and

Escherichia coli 1846.1.6 C. crescentus as Cell Cycle Model 1856.1.7 Bacteria Starvation Response 1876.1.8 First Coverage of C. crescentus

Phosphoproteome 1886.2 Optimized Methodology for Phospho Ser/Thr/Tyr

Studies 1886.2.1 Bacterial Strain and Growth Conditions 1886.2.2 C. crescentus Cell Protein Extraction:

Phosphoproteomics 1896.2.3 Solid-Phase Extraction (SPE) Desalting 1906.2.4 In Vitro Methylation of Peptides 1906.2.5 Phosphopeptide Enrichment by IMAC 1916.2.6 Normal Proteomics 1926.2.7 pY Enrichment by IP 1926.2.8 RP/Nano-High-Performance Liquid

Chromatography (HPLC) Separation 1926.2.9 LC-Linear Ion Trap (LTQ) -Orbitrap MS/MS 1936.2.10 LTQ-Fourier Transform (FT)/MS/MS 1936.2.11 Peptide Identification and False Discovery Rate

(FDR) Determination 1936.2.12 Peptide Quantitative Comparison 194

6.3 Identification of the Components of the Ser/Thr/TyrPhosphoproteome in C. crescentus Grown in the Presenceand Absence of Glucose 1946.3.1 Total Phosphoprotein Identifications 1946.3.2 MSA Spectra 196

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crescenfUS 183811 if/lls subtilis and

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192MSfMS 193

193Vcry Rate

6.3.36.3.46.3.56.3.66.3.76.3.8

6.3.9

6.3.10

6.3.11

6.3.12

6.3.13

CONTENTS xiii

Phosphorylation Sites Identified 196Ser/Thr/Tyr Phosphoproteome of C. crescentus 205Phosphorylated His and Aspartate 213Cell Cycle His Kinase CckA 215Phosphoglutamate 216Enriched Tyr Phosphoproteome ofC. crescentus 2166.3.8.1 Sensor His Kinase KdpD 2166.3.8.2 TonB-Dependent Receptor Proteins 216

Carbon Environment-SharedPhosphoproteome 2176.3.9.1 Two-Component His Kinases 2176.3.9.2 Multiply Phosphorylated Kinases 2176.3.9.3 pTPLAALpSAQSRRAR Peptide as

Sensor His Kinase 2176.3.9.4 Aspartate Phosphorylated Tyr Kinase

DivL 217Carbon-Rich versus Carbon-Starved Class/Category 2256.3.10.1 Localization of Phosphoproteome of

C. crescentus 2256.3.10.2 Integral Membrane Proteins 2256.3.10.3 Function of Phosphoproteome of

C. crescentus 225Carbon-Rich versus Carbon-Starved UniquePhosphorylated Proteins 2276.3.11.1 Carbon-Rich Environment

Phosphorylated Proteins 2276.3.11.2 Carbon-Starved Environment

Phosphorylated Proteins 2276.3.11.3 Decreased Normal Activity 232

Confirmation of Decreased Energy Pathways 232

6.3.12.1 Carbon-Rich MitochondrialLocalization 232

6.3.12.2 Normal Proteome GlycolyticPathway 233

6.3.12.3 Starvation Survival Response 233Phosphopeptide Quantitative DifferentialComparison 2336.3.13.1 Upregulation in Phosphorylation 234

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7.1 Phosphohistidine as posttranslational Modification

(PTM) 2497.2 Bacterial Kinases and the Two-Component System 2507.3 Measurement of Phosphorylated His (pH) 251

7.3.1 Stabilities of Phosphorylated Amino Acids 2517.3.2 Immobilized Metal Affinity Chromatography

(IMAC) and Mass Spectrometry (MS) 2527.4 In Vitro and In Vivo Study of pH-Containing Peptides by

Nano-ESI Tandem MS 2557.4.1 Int roduction 2557.4.2 Background of Study 257

7.4.2.1 Bacteria Models of Ser/Thr/TyrPhosphorylation 257

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6.3.13.2 Adaptive Response withPhosphorylation 234

6.3.13.3 Upregulation NAD-DependentGDH 234

6.3.13.4 Downregulation of Flagellin Protein 235

6.3.14 Carbon-Rich versus Carbon-Starved NormalProteome Time Course Study 2356.3.14.1 Entire Proteome Localization and

Function 2356.3.14.2 Regulated Proteins 2376.3.14.3 Localization of Regulated Proteins 2376.3.14.4 Function of Regulated Proteins 2386.3.14.5 Normal Proteome Energy Pathways 2396.3.14.6 Overlap of Phosphorylated Proteins and

Regulated Normal Proteome 2396.3.14.7 Differences of Phosphorylated

Prot eins 2406.3.14.8 Localization of Phosphorylated

Proteins 2406.3.14.9 Dir ect Relationships Observed 240

6.3.15 Conclusions 2436.3.16 Supplementary Material 243

6.3.16.1 Reviewing Spectra Using theSpectrumLook Software Package 243

Reference 244

7 Prokaryotic Phosphorylation of Histidine

7.4.2.27.4.2.37.4.2.4

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7.4.3

7.4.47.4.5

7.4.6

7.4.7

7.4.8

CONTENTS xv

Prokaryotic Phosphorylation of His 258C crescentus 258Mass Spectral Measurement ofPhosphohistidine 258

Optimized Methodology for PhosphohistidineStudies 2597.4.3.1 In Vitro Selective pHis

Phosphorylation 2597.4.3.2 In Vitro Phosphorylation of Angio II

(Sar1Thr8) 261

7.4.3.3 In Vitro Methylation of Peptides 2627.4.3.4 C crescentus Cell Protein Extraction with

V-8 Protease Digestion 2627.4.3.5 1-D SDS-Polyacrylamide Gel

Electrophoresis (PAGE) 2637.4.3.6 Phosphohistidine Enrichment by Cu(II)­

Based IMAC 2647.4.3.7 Reversed-Phase (RP)/Nano-HPLC

Separation 2657.4.3.8 Nano-ESI Nano-HPLC MS 2667.4.3.9 Peptide Identification and False Discovery

Rate (FDR) Determination 268C18 RP LC Behavior 268Phosphohistidine Loses HP03 and H3P04 2707.4.5.1 Rational for H3P04 Loss 272Q-TOF/MS/MS Product Ion Spectra 2777.4.6.1 pH-Containing Peptide INpHDLR 2777.4.6.2 Doubly Charged (2+) Peptide

INpHDLR 2797.4.6.3 pH-Containing Peptide pHLGLAR 2797.4.6.4 Singly Charged (1+) Peptide

pHLGLAR 280Behavior of Monophosphohistidine andDiphosphohistidine Peptide 2817.4.7.1 Peptide Angio I as DRVYIHPFHL 281Behavior of Phosphotyrosine and PhosphohistidinePeptide 2857.4.8.1 Peptide Angio II as DRVpYIHPF 2857.4.8.2 Phosphorylated Angio II as

DRVpYlpHPF 285

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7.4.9 Behavior of Phosphotyrosine-, Phosphothreonine-,and Pho sphohistidin e-Containing Peptide 2877.4.9.1 Peptide Angio II (Sar1Thr

8) 287

7.4.10 Validation of Cu(II) -Based IMAC Phosphohistidin e

Enrichment 2917.4.10.1 Fe(III )-Based IMAC versus Cu(ll)

Based 2927.4.10.2 Cu(II) -Base d IMAC of An gio I 2927.4.10.3 Cu(II )-Based IMAC of Angio II 293

7.4.11 In Vivo Measurement of Phosphohistidine 2937.4.11.1 Time-Based Digestion Study 2937.4.11.2 Pho sphohistidine-Containing

Peptides 2947.4.11.3 Phosphohistidine Product Ion

Spect ra 2947.4.12 Gene Ontology of Phosphorylated Proteins 296

7.4.12.1 Localizat ion of PhosphorylatedProteins 296

7.4.12.2 Function of PhosphorylatedProteins 304

7.4.13 Predicted Regulatory Protein Motif Study 3077.4.14 Validation of Phosphohistidine-Containing

Proteins 3087.4.14.1 Phosphorylation Motif Stud y 3087.4.14.2 Phosphohistidine Kinase Mot if 309

7.4.15 The pDpH Motif 3107.4.16 Conclusions 311

7.5 Supplementary Material 3117.5.1 Reviewing Spectra Using the SpectrumLook

Software Package 311

Reference 313

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Glossary

Index

Appendix I Atomic Weights and Isotopic Compositions

Appendix II Periodic Table of the Elements

Appendix III Fundamental physical Constants