Preface xvii Acknowledgments xxi About the Author xxiii 1 Posttranslational Modification (PTM) of Proteins 1 1.1 Over 200 Forms of PTM of Proteins 1 1.2 Three Main Types of PTM Studied by MS 2 1.3 Overview of Nano-Electrospray/Nanofl ow LC-MS 2 1.3.1 Defi nition and Description of MS 2 1.3.2 Basic Design of Mass Analyzer Instrumentation 3 1.

3.3 ESI 7 1.3.4 Nano-ESI 11 1.4 Overview of Nucleic Acids 15 1.5 Proteins and Proteomics 20 1.5.1 Introduction to Proteomics 20 1.

5.2 Protein Structure and Chemistry 22 1.5.3 Bottom-Up Proteomics: MS of Peptides 27 1.5.3.1 History and Strategy 27 1.5.

3.2 Protein Identifi cation through Product Ion Spectra30 1.5.3.3 High-Energy Product Ions 36 1.5.3.4 De Novo Sequencing 37 1.

5.3.5 Electron Capture Dissociation (ECD) 40 1.5.4 Top-Down Proteomics: MS of Intact Proteins 42 1.5.4.1 Background 42 1.

5.4.2 GP Basicity and Protein Charging 42 1.5.4.3 Calculation of Charge State and Molecular Weight 44 1.5.4.

4 Top-Down Protein Sequencing 46 1.5.5 Systems Biology and Bioinformatics 48 1.5.6 Biomarkers in Cancer 52 Reference 56 2 Glycosylation of Proteins 59 2.1 Production of a Glycoprotein 59 2.2 Biological Processes of Protein Glycosylation 59 2.3 N-Linked and O-Linked Glycosylation 60 2.

4 Carbohydrates 60 2.4.1 Ionization of Oligosaccharides 64 2.4.2 Carbohydrate Fragmentation 65 2.4.3 Complex Oligosaccharide Structural Elucidation 70 2.5 Three Objectives in Studying Glycoproteins 72 2.

6 Glycosylation Study Approaches 72 2.6.1 MS of Glycopeptides 73 2.6.2 Mass Pattern Recognition 75 2.6.2.1 High Galactose Glycosylation Pattern 75 2.

6.3 Charge State Determination 76 2.6.4 Diagnostic Fragment Ions 76 2.6.5 High-Resolution/High-Mass Accuracy Measurement andIdentification 76 2.6.6 Digested Bovine Fetuin 78 Reference 79 3 Sulfation of Proteins as Posttranslational Modification81 3.

1 Glycosaminoglycan Sulfation 81 3.2 Cellular Processes Involved in Sulfation 81 3.3 Brief Example of Phosphorylation 82 3.4 Sulfotransferase Class of Enzymes 82 3.5 Fragmentation Nomenclature for Carbohydrates 82 3.6 Sulfated Mucin Oligosaccharides 83 3.7 Tyrosine Sulfation 84 3.8 Tyrosylprotein Sulfotransferases TPST1 and TPST2 87 3.

9 O-Sulfated Human Proteins 89 3.10 Sulfated Peptide Product Ion Spectra 89 3.11 Use of Higher Energy Collisions 93 3.12 Electron Capture Dissociation (ECD) 94 3.13 Sulfation versus Phosphorylation 95 Reference 97 4 Eukaryote PTM as Phosphorylation: Normal State Studies99 4.1 Mass Spectral Measurement with Examples of HeLa CellPhosphoproteome 99 4.1.1 Introduction 99 4.

1.2 Protein Phosphatase and Kinase 99 4.1.3 Hydroxy-Amino Acid Phosphorylation 100 4.1.4 Traditional Phosphoproteomic Approaches 102 4.1.5 Current Approaches 103 4.

1.5.1 Phosphoproteomic Enrichment Techniques 103 4.1.5.2 IMAC 104 4.1.5.

3 MOAC 105 4.1.5.4 Methylation of Peptides prior to IMAC or MOAC Enrichment107 4.1.6 The Ideal Approach 107 4.1.7 One-Dimensional (1-D) Sodium Dodecyl Sulfate (SDS) PAGE108 4.

1.8 Tandem MS Approach 108 4.1.8.1 pS Loss of Phosphate Group 109 4.1.8.2 pT Loss of Phosphate Group 112 4.

1.8.3 pY Loss of Phosphate Group 113 4.1.9 Alternative Methods: Infrared Multiphoton Dissociation(IRMPD) and Electron Capture Dissociation (ECD) 115 4.1.10 Electron Transfer Dissociation (ETD) 115 4.2 The HeLa Cell Phosphoproteome 118 4.

2.1 Introduction 118 4.2.2 Background of Study 118 4.2.3 What is Covered 119 4.2.4 Optimized Methods to Use for Phosphoproteomic Studies119 4.

2.4.1 Cell Culture 119 4.2.4.2 Extraction of HeLa Cell Proteins 120 4.2.4.

3 Trizol Extraction and Tryptic Digestion 120 4.2.4.4 Solid-Phase Extraction (SPE) Desalting 120 4.2.4.5 Converting Peptide Carboxyl Moieties to Methyl Esters121 4.2.

4.6 Roche Complete Lysis-M, EDTA-Free Extraction 122 4.2.4.7 1-D SDS-PAGE Cleanup 122 4.2.4.8 In-Gel Reduction, Alkylation, Digestion, and Extractionof Peptides 122 4.

2.4.9 Phosphopeptide Enrichment Using IMAC 123 4.2.5 Description of Instrumental Analyses 123 4.2.5.1 RP/Nano-HPLC Separation 123 4.

2.5.2 MS Analysis 125 4.2.6 Current Approaches for Peptide Identification and FalseDiscovery Rate (FDR) Determination 125 4.2.7 Results of the Protein Extraction and Preparation 126 4.2.

7.1 Detergent Lysis, Trizol, and Ultracentrifugation 126 4.2.7.2 Nucleic Acid Removal with SDS-PAGE 127 4.2.8 HeLa Cell Phosphoproteome Methodology Comparison 128 4.2.

8.1 Roche In-Solution versus Trizol Extraction 129 4.2.8.2 In-Solution and In-Gel Digests Phosphoproteome Coverage129 4.2.9 Overall Conclusion 134 4.3 Nonphosphoproteome HeLa Cell Analysis 135 4.

3.1 IMAC Flow Through Peptide Analysis 135 4.3.2 IMAC NaCl Wash Peptide Analysis 136 4.3.3 IMAC Flow Through versus NaCl Wash Comparison 138 4.3.4 Gene Ontology Comparison 138 4.

3.5 IMAC Bed Nonspecifi c Binding Study 140 4.4 Reviewing Spectra Using the SpectrumLook Software Package143 Reference 144 5 Eukaryote PTM as Phosphorylation: Perturbed State Studies147 5.1 Study of the Phosphoproteome of HeLa Cells under PerturbedConditions by Nano-High-Performance Liquid Chromatography HPLCElectrospray Ionization (ESI) Linear Ion Trap (LTQ)-FT/MassSpectrometry (MS) 147 5.1.1 Introduction 147 5.1.2 Ataxia Telangiectasia Mutated (ATM) and ATM andRad3-Related (ATR) 149 5.

1.3 Background of Study 149 5.1.3.1 PP5 149 5.1.3.2 Functions of PP5 151 5.

1.3.3 DDR of PP5 151 5.1.4 Review of Optimized Approach to Study 151 5.1.4.1 Producing Cell Cultures 151 5.

1.4.2 Protein Extraction 152 5.1.4.3 Phosphopeptide Enrichment by IMAC 154 5.1.4.

4 Reversed-Phase (RP)/Nano-HPLC Separation 155 5.1.4.5 LTQ-FT/MS/MS 156 5.1.4.6 Protein Identifi cation and False Discovery Rate (FDR)Determination 156 5.1.

4.7 Phosphopeptide Quantitative Differential Comparison157 5.1.4.8 Data Set Peak Matching and Alignment 157 5.1.4.9 Phosphopeptide Response Normalization 160 5.

1.5 Phosphoproteome Gene Ontology (GO) Comparison 160 5.1.5.1 GO Cellular Component 162 5.1.6 Potential Regulated Target Proteins of PP5 162 5.1.

6.1 Analysis of Variance (ANOVA) 162 5.1.6.2 Four Potential Target Proteins 166 5.1.7 GO Differential Comparison 167 5.1.

7.1 GO Cellular Component 168 5.1.7.2 Infl uence of Classes or Categories of Proteins 168 5.1.7.3 Molecular Function Interacting Modules 168 5.

1.8 Conclusion 175 5.1.9 Reviewing Spectra Using the SpectrumLook Software Package175 Reference 176 6 Prokaryotic Phosphorylation of Serine, Threonine, andTyrosine 181 6.1 Introduction 181 6.1.1 Serine (Ser)/Threonine (Thr)/Tyrosine (Tyr)Phosphorylation 181 6.1.

2 Histidine (His) Phosphorylation 181 6.1.3 Caulobacter crescentus 181 6.1.4 Ser/Thr/Tyr Phosphorylation of C. crescentus 183 6.1.5 Ser/Thr/Tyr Phosphorylation of Bacillus subtilis and Escherichia coli 184 6.

1.6 C. crescentus as Cell Cycle Model 185 6.1.7 Bacteria Starvation Response 187 6.1.8 First Coverage of C. crescentus Phosphoproteome188 6.

2 Optimized Methodology for Phospho Ser/Thr/Tyr Studies188 6.2.1 Bacterial Strain and Growth Conditions 188 6.2.2 C. crescentus Cell Protein Extraction:Phosphoproteomics 189 6.2.3 Solid-Phase Extraction (SPE) Desalting 190 6.

2.4 In Vitro Methylation of Peptides 190 6.2.5 Phosphopeptide Enrichment by IMAC 191 6.2.6 Normal Proteomics 192 6.2.7 pY Enrichment by IP 192 6.

2.8 RP/Nano-High-Performance Liquid Chromatography (HPLC)Separation 192 6.2.9 LC-Linear Ion Trap (LTQ)-Orbitrap MS/MS 193 6.2.10 LTQ-Fourier Transform (FT)/MS/MS 193 6.2.11 Peptide Identification and False Discovery Rate (FDR)Determination 193 6.

2.12 Peptide Quantitative Comparison 194 6.3 Identifi cation of the Components of the Ser/Thr/TyrPhosphoproteome in C. crescentus Grown in the Presence andAbsence of Glucose 194 6.3.1 Total Phosphoprotein Identifications 194 6.3.2 MSA Spectra 196 6.

3.3 Phosphorylation Sites Identifi ed 196 6.3.4 Ser/Thr/Tyr Phosphoproteome of C. crescentus 205 6.3.5 Phosphorylated His and Aspartate 213 6.3.

6 Cell Cycle His Kinase CckA 215 6.3.7 Phosphoglutamate 216 6.3.8 Enriched Tyr Phosphoproteome of C. crescentus 216 6.3.8.

1 Sensor His Kinase KdpD 216 6.3.8.2 TonB-Dependent Receptor Proteins 216 6.3.9 Carbon Environment-Shared Phosphoproteome 217 6.3.9.

1 Two-Component His Kinases 217 6.3.9.2 Multiply Phosphorylated Kinases 217 6.3.9.3 pTPLAALpSAQSRRAR Peptide as Sensor His Kinase 217 6.3.

9.4 Aspartate Phosphorylated Tyr Kinase DivL 217 6.3.10 Carbon-Rich versus Carbon-Starved Class/Category 225 6.3.10.1 Localization of Phosphoproteome of C. crescentus 225 6.

3.10.2 Integral Membrane Proteins 225 6.3.10.3 Function of Phosphoproteome of C. crescentus 225 6.3.

11 Carbon-Rich versus Carbon-Starved Unique PhosphorylatedProteins 22.