Proteomics of Biological Systems: Protein Phosphorylation Using Mass Spectrometry Techniques

PROTEOMICS OF BIOLOGICAL SYSTEMS PROTEOMICS OF BIOLOGICAL SYSTEMS Protein Phosphorylation Using Mass Spectrometry Tec...

Author: Bryan M. Ham

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PROTEOMICS OF BIOLOGICAL SYSTEMS

PROTEOMICS OF BIOLOGICAL SYSTEMS Protein Phosphorylation Using Mass Spectrometry Techniques

Bryan M. Ham

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright © 2012 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permissions. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data: Ham, Bryan M. â•… Proteomics of biological systems : protein phosphorylation using mass spectrometry techniques / Bryan M Ham. â•…â•… p. cm. â•… Includes index. â•… ISBN 978-1-118-02896-4 (cloth) â•… 1.â•‡ Proteomics–Methodology.â•… 2.â•‡ Phosphorylation–Research–Methodology.â•… 3.â•‡ Phosphoproteins–Synthesis.â•… 4.â•‡ Mass spectrometry.â•… 5.â•‡ Biological systems–Research– Methodology.â•… I.â•‡ Title. â•… QP519.9.M3H367 2012 â•… 572'.62–dc23 2011019941 oBook ISBN: 9781118137048 ePDF ISBN: 9781118137017 ePub ISBN: 9781118137031 MOBI ISBN: 9781118137024 Printed in the United States of America. 10â•… 9â•… 8â•… 7â•… 6â•… 5â•… 4â•… 3â•… 2â•… 1

This Book Is Dedicated to the Most Important Person in My Life, My Ever Loving Wife.

CONTENTS Preface

xvii

Acknowledgments

xxi

About the Author

xxiii

1 Posttranslational Modification (PTM) of Proteins 1.1 1.2 1.3

1.4 1.5

1

Over 200 Forms of PTM of Proteinsâ•… 1 Three Main Types of PTM Studied by MSâ•… 2 Overview of Nano-Electrospray/Nanoflow LC-MSâ•… 2 1.3.1 Definition and Description of MSâ•… 2 1.3.2 Basic Design of Mass Analyzer Instrumentationâ•… 3 1.3.3 ESIâ•… 7 1.3.4 Nano-ESIâ•… 11 Overview of Nucleic Acidsâ•… 15 Proteins and Proteomicsâ•… 20 1.5.1 Introduction to Proteomicsâ•… 20 1.5.2 Protein Structure and Chemistryâ•… 22 1.5.3 Bottom-Up Proteomics: MS of Peptidesâ•… 27 1.5.3.1 History and Strategyâ•… 27 1.5.3.2 Protein Identification through Product Ion Spectraâ•… 30 1.5.3.3 High-Energy Product Ionsâ•… 36 1.5.3.4 De Novo Sequencingâ•… 37 1.5.3.5 Electron Capture Dissociation (ECD)â•… 40 1.5.4 Top-Down Proteomics: MS of Intact Proteinsâ•… 42 1.5.4.1 Backgroundâ•… 42 1.5.4.2 GP Basicity and Protein Chargingâ•… 42 1.5.4.3 Calculation of Charge State and Molecular Weightâ•… 44 1.5.4.4 Top-Down Protein Sequencingâ•… 46 vii

viiiâ•…â•… CONTENTS

1.5.5 Systems Biology and Bioinformaticsâ•… 48 1.5.6 Biomarkers in Cancerâ•… 52 Referenceâ•… 56 2 Glycosylation of Proteins

59

2.1 2.2 2.3 2.4

Production of a Glycoproteinâ•… 59 Biological Processes of Protein Glycosylationâ•… 59 N-Linked and O-Linked Glycosylationâ•… 60 Carbohydratesâ•… 60 2.4.1 Ionization of Oligosaccharidesâ•… 64 2.4.2 Carbohydrate Fragmentationâ•… 65 2.4.3 Complex Oligosaccharide Structural Elucidationâ•… 70 2.5 Three Objectives in Studying Glycoproteinsâ•… 72 2.6 Glycosylation Study Approachesâ•… 72 2.6.1 MS of Glycopeptidesâ•… 73 2.6.2 Mass Pattern Recognitionâ•… 75 2.6.2.1 High Galactose Glycosylation Patternâ•… 75 2.6.3 Charge State Determinationâ•… 76 2.6.4 Diagnostic Fragment Ionsâ•… 76 2.6.5 High-Resolution/High-Mass Accuracy Measurement and Identificationâ•… 76 2.6.6 Digested Bovine Fetuinâ•… 78 Referenceâ•… 79 3 Sulfation of Proteins as Posttranslational Modification 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8

Glycosaminoglycan Sulfationâ•… 81 Cellular Processes Involved in Sulfationâ•… 81 Brief Example of Phosphorylationâ•… 82 Sulfotransferase Class of Enzymesâ•… 82 Fragmentation Nomenclature for Carbohydratesâ•… 82 Sulfated Mucin Oligosaccharidesâ•… 83 Tyrosine Sulfationâ•… 84 Tyrosylprotein Sulfotransferases TPST1 and TPST2â•… 87 3.9 O-Sulfated Human Proteinsâ•… 89 3.10 Sulfated Peptide Product Ion Spectraâ•… 89 3.11 Use of Higher Energy Collisionsâ•… 93

81

CONTENTSâ•…â•… ix

3.12 Electron Capture Dissociation (ECD)â•… 94 3.13 Sulfation versus Phosphorylationâ•… 95 Referenceâ•… 97 4 Eukaryote PTM as Phosphorylation: Normal State Studies 4.1

4.2

99

Mass Spectral Measurement with Examples of HeLa Cell Phosphoproteomeâ•… 99 4.1.1 Introductionâ•… 99 4.1.2 Protein Phosphatase and Kinaseâ•… 99 4.1.3 Hydroxy-Amino Acid Phosphorylationâ•… 100 4.1.4 Traditional Phosphoproteomic Approachesâ•… 102 4.1.5 Current Approachesâ•… 103 4.1.5.1 Phosphoproteomic Enrichment Techniquesâ•… 103 4.1.5.2 IMACâ•… 104 4.1.5.3 MOACâ•… 105 4.1.5.4 Methylation of Peptides prior to IMAC or MOAC Enrichmentâ•… 107 4.1.6 The Ideal Approachâ•… 107 4.1.7 One-Dimensional (1-D) Sodium Dodecyl Sulfate (SDS) PAGEâ•… 108 4.1.8 Tandem MS Approachâ•… 108 4.1.8.1 pS Loss of Phosphate Groupâ•… 109 4.1.8.2 pT Loss of Phosphate Groupâ•… 112 4.1.8.3 pY Loss of Phosphate Groupâ•… 113 4.1.9 Alternative Methods: Infrared Multiphoton Dissociation (IRMPD) and Electron Capture Dissociation (ECD)â•… 115 4.1.10 Electron Transfer Dissociation (ETD)â•… 115 The HeLa Cell Phosphoproteomeâ•… 118 4.2.1 Introductionâ•… 118 4.2.2 Background of Studyâ•… 118 4.2.3 What is Coveredâ•… 119 4.2.4 Optimized Methods to Use for Phosphoproteomic Studiesâ•… 119 4.2.4.1 Cell Cultureâ•… 119 4.2.4.2 Extraction of HeLa Cell Proteinsâ•… 120 4.2.4.3 Trizol Extraction and Tryptic Digestionâ•… 120

xâ•…â•… CONTENTS

4.2.4.4

Solid-Phase Extraction (SPE) Desaltingâ•… 120 4.2.4.5 Converting Peptide Carboxyl Moieties to Methyl Estersâ•… 121 4.2.4.6 Roche Complete Lysis-M, EDTA-Free Extractionâ•… 122 4.2.4.7 1-D SDS-PAGE Cleanupâ•… 122 4.2.4.8 In-Gel Reduction, Alkylation, Digestion, and Extraction of Peptidesâ•… 122 4.2.4.9 Phosphopeptide Enrichment Using IMACâ•… 123 4.2.5 Description of Instrumental Analysesâ•… 123 4.2.5.1 RP/Nano-HPLC Separationâ•… 123 4.2.5.2 MS Analysisâ•… 125 4.2.6 Current Approaches for Peptide Identification and False Discovery Rate (FDR) Determinationâ•… 125 4.2.7 Results of the Protein Extraction and Preparationâ•… 126 4.2.7.1 Detergent Lysis, Trizol, and Ultracentrifugationâ•… 126 4.2.7.2 Nucleic Acid Removal with SDS-PAGEâ•… 127 4.2.8 HeLa Cell Phosphoproteome Methodology Comparisonâ•… 128 4.2.8.1 Roche In-Solution versus Trizol Extractionâ•… 129 4.2.8.2 In-Solution and In-Gel Digests Phosphoproteome Coverageâ•… 129 4.2.9 Overall Conclusionâ•… 134 4.3 Nonphosphoproteome HeLa Cell Analysisâ•… 135 4.3.1 IMAC Flow Through Peptide Analysisâ•… 135 4.3.2 IMAC NaCl Wash Peptide Analysisâ•… 136 4.3.3 IMAC Flow Through versus NaCl Wash Comparisonâ•… 138 4.3.4 Gene Ontology Comparisonâ•… 138 4.3.5 IMAC Bed Nonspecific Binding Studyâ•… 140 4.4 Reviewing Spectra Using the SpectrumLook Software Packageâ•… 143 Referenceâ•… 144

CONTENTSâ•…â•… xi

5 Eukaryote PTM as Phosphorylation: Perturbed State Studies 147 5.1

Study of the Phosphoproteome of HeLa Cells under Perturbed Conditions by Nano-High-Performance Liquid Chromatography HPLC Electrospray Ionization (ESI) Linear Ion Trap (LTQ)-FT/Mass Spectrometry (MS)â•… 147 5.1.1 Introductionâ•… 147 5.1.2 Ataxia Telangiectasia Mutated (ATM) and ATM and Rad3-Related (ATR)â•… 149 5.1.3 Background of Studyâ•… 149 5.1.3.1 PP5â•… 149 5.1.3.2 Functions of PP5â•… 151 5.1.3.3 DDR of PP5â•… 151 5.1.4 Review of Optimized Approach to Studyâ•… 151 5.1.4.1 Producing Cell Culturesâ•… 151 5.1.4.2 Protein Extractionâ•… 152 5.1.4.3 Phosphopeptide Enrichment by IMACâ•… 154 5.1.4.4 Reversed-Phase (RP)/Nano-HPLC Separationâ•… 155 5.1.4.5 LTQ-FT/MS/MSâ•… 156 5.1.4.6 Protein Identification and False Discovery Rate (FDR) Determinationâ•… 156 5.1.4.7 Phosphopeptide Quantitative Differential Comparisonâ•… 157 5.1.4.8 Data Set Peak Matching and Alignmentâ•… 157 5.1.4.9 Phosphopeptide Response Normalizationâ•… 160 5.1.5 Phosphoproteome Gene Ontology (GO) Comparisonâ•… 160 5.1.5.1 GO Cellular Componentâ•… 162 5.1.6 Potential Regulated Target Proteins of PP5â•… 162 5.1.6.1 Analysis of Variance (ANOVA)â•… 162 5.1.6.2 Four Potential Target Proteinsâ•… 166 5.1.7 GO Differential Comparisonâ•… 167 5.1.7.1 GO Cellular Componentâ•… 168 5.1.7.2 Influence of Classes or Categories of Proteinsâ•… 168 5.1.7.3 Molecular Function Interacting Modulesâ•… 168

xiiâ•…â•… CONTENTS

5.1.8 5.1.9

Conclusionâ•… 175 Reviewing Spectra Using the SpectrumLook Software Packageâ•… 175 Referenceâ•… 176 6 Prokaryotic Phosphorylation of Serine, Threonine, and Tyrosine 6.1

6.2

6.3

181

Introductionâ•… 181 6.1.1 Serine (Ser)/Threonine (Thr)/Tyrosine (Tyr) Phosphorylationâ•… 181 6.1.2 Histidine (His) Phosphorylationâ•… 181 6.1.3 Caulobacter crescentusâ•… 181 6.1.4 Ser/Thr/Tyr Phosphorylation of C. crescentusâ•… 183 6.1.5 Ser/Thr/Tyr Phosphorylation of Bacillus subtilis and Escherichia coliâ•… 184 6.1.6 C. crescentus as Cell Cycle Modelâ•… 185 6.1.7 Bacteria Starvation Responseâ•… 187 6.1.8 First Coverage of C. crescentus Phosphoproteomeâ•… 188 Optimized Methodology for Phospho Ser/Thr/Tyr Studiesâ•… 188 6.2.1 Bacterial Strain and Growth Conditionsâ•… 188 6.2.2 C. crescentus Cell Protein Extraction: Phosphoproteomicsâ•… 189 6.2.3 Solid-Phase Extraction (SPE) Desaltingâ•… 190 6.2.4 In Vitro Methylation of Peptidesâ•… 190 6.2.5 Phosphopeptide Enrichment by IMACâ•… 191 6.2.6 Normal Proteomicsâ•… 192 6.2.7 pY Enrichment by IPâ•… 192 6.2.8 RP/Nano-High-Performance Liquid Chromatography (HPLC) Separationâ•… 192 6.2.9 LC-Linear Ion Trap (LTQ)-Orbitrap MS/MSâ•… 193 6.2.10 LTQ-Fourier Transform (FT)/MS/MSâ•… 193 6.2.11 Peptide Identification and False Discovery Rate (FDR) Determinationâ•… 193 6.2.12 Peptide Quantitative Comparisonâ•… 194 Identification of the Components of the Ser/Thr/Tyr Phosphoproteome in C. crescentus Grown in the Presence and Absence of Glucoseâ•… 194 6.3.1 Total Phosphoprotein Identificationsâ•… 194 6.3.2 MSA Spectraâ•… 196

CONTENTSâ•…â•… xiii

6.3.3 6.3.4 6.3.5 6.3.6 6.3.7 6.3.8

6.3.9

6.3.10

6.3.11

6.3.12

6.3.13

Phosphorylation Sites Identifiedâ•… 196 Ser/Thr/Tyr Phosphoproteome of C. crescentusâ•… 205 Phosphorylated His and Aspartateâ•… 213 Cell Cycle His Kinase CckAâ•… 215 Phosphoglutamateâ•… 216 Enriched Tyr Phosphoproteome of C. crescentusâ•… 216 6.3.8.1 Sensor His Kinase KdpDâ•… 216 6.3.8.2 TonB-Dependent Receptor Proteinsâ•… 216 Carbon Environment-Shared Phosphoproteomeâ•… 217 6.3.9.1 Two-Component His Kinasesâ•… 217 6.3.9.2 Multiply Phosphorylated Kinasesâ•… 217 6.3.9.3 pTPLAALpSAQSRRAR Peptide as Sensor His Kinaseâ•… 217 6.3.9.4 Aspartate Phosphorylated Tyr Kinase DivLâ•… 217 Carbon-Rich versus Carbon-Starved Class/ Categoryâ•… 225 6.3.10.1 Localization of Phosphoproteome of C. crescentusâ•… 225 6.3.10.2 Integral Membrane Proteinsâ•… 225 6.3.10.3 Function of Phosphoproteome of C. crescentusâ•… 225 Carbon-Rich versus Carbon-Starved Unique Phosphorylated Proteinsâ•… 227 6.3.11.1 Carbon-Rich Environment Phosphorylated Proteinsâ•… 227 6.3.11.2 Carbon-Starved Environment Phosphorylated Proteinsâ•… 227 6.3.11.3 Decreased Normal Activityâ•… 232 Confirmation of Decreased Energy Pathwaysâ•… 232 6.3.12.1 Carbon-Rich Mitochondrial Localizationâ•… 232 6.3.12.2 Normal Proteome Glycolytic Pathwayâ•… 233 6.3.12.3 Starvation Survival Responseâ•… 233 Phosphopeptide Quantitative Differential Comparisonâ•… 233 6.3.13.1 Upregulation in Phosphorylationâ•… 234

xivâ•…â•… CONTENTS

6.3.13.2 Adaptive Response with Phosphorylationâ•… 234 6.3.13.3 Upregulation NAD-Dependent GDHâ•… 234 6.3.13.4 Downregulation of Flagellin Proteinâ•… 235 6.3.14 Carbon-Rich versus Carbon-Starved Normal Proteome Time Course Studyâ•… 235 6.3.14.1 Entire Proteome Localization and Functionâ•… 235 6.3.14.2 Regulated Proteinsâ•… 237 6.3.14.3 Localization of Regulated Proteinsâ•… 237 6.3.14.4 Function of Regulated Proteinsâ•… 238 6.3.14.5 Normal Proteome Energy Pathwaysâ•… 239 6.3.14.6 Overlap of Phosphorylated Proteins and Regulated Normal Proteomeâ•… 239 6.3.14.7 Differences of Phosphorylated Proteinsâ•… 240 6.3.14.8 Localization of Phosphorylated Proteinsâ•… 240 6.3.14.9 Direct Relationships Observedâ•… 240 6.3.15 Conclusionsâ•… 243 6.3.16 Supplementary Materialâ•… 243 6.3.16.1 Reviewing Spectra Using the SpectrumLook Software Packageâ•… 243 Referenceâ•… 244 7 Prokaryotic Phosphorylation of Histidine 7.1 7.2 7.3

7.4

249

Phosphohistidine as Posttranslational Modification (PTM)â•… 249 Bacterial Kinases and the Two-Component Systemâ•… 250 Measurement of Phosphorylated His (pH)â•… 251 7.3.1 Stabilities of Phosphorylated Amino Acidsâ•… 251 7.3.2 Immobilized Metal Affinity Chromatography (IMAC) and Mass Spectrometry (MS)â•… 252 In Vitro and In Vivo Study of pH-Containing Peptides by Nano-ESI Tandem MSâ•… 255 7.4.1 Introductionâ•… 255 7.4.2 Background of Studyâ•… 257 7.4.2.1 Bacteria Models of Ser/Thr/Tyr Phosphorylationâ•… 257

CONTENTSâ•…â•… xv

7.4.2.2 7.4.2.3 7.4.2.4 7.4.3

7.4.4 7.4.5 7.4.6

7.4.7 7.4.8

Prokaryotic Phosphorylation of Hisâ•… 258 C. crescentusâ•… 258 Mass Spectral Measurement of Phosphohistidineâ•… 258 Optimized Methodology for Phosphohistidine Studiesâ•… 259 7.4.3.1 In Vitro Selective pHis Phosphorylationâ•… 259 7.4.3.2 In Vitro Phosphorylation of Angio II (Sar1Thr8)â•… 261 7.4.3.3 In Vitro Methylation of Peptidesâ•… 262 7.4.3.4 C. crescentus Cell Protein Extraction with V-8 Protease Digestionâ•… 262 7.4.3.5 1-D SDS-Polyacrylamide Gel Electrophoresis (PAGE)â•… 263 7.4.3.6 Phosphohistidine Enrichment by Cu(II)Based IMACâ•… 264 7.4.3.7 Reversed-Phase (RP)/Nano-HPLC Separationâ•… 265 7.4.3.8 Nano-ESI Nano-HPLC MSâ•… 266 7.4.3.9 Peptide Identification and False Discovery Rate (FDR) Determinationâ•… 268 C18 RP LC Behaviorâ•… 268 Phosphohistidine Loses HPO3 and H3PO4â•… 270 7.4.5.1 Rational for H3PO4 Lossâ•… 272 Q-TOF/MS/MS Product Ion Spectraâ•… 277 7.4.6.1 pH-Containing Peptide INpHDLRâ•… 277 7.4.6.2 Doubly Charged (2+) Peptide INpHDLRâ•… 279 7.4.6.3 pH-Containing Peptide pHLGLARâ•… 279 7.4.6.4 Singly Charged (1+) Peptide pHLGLARâ•… 280 Behavior of Monophosphohistidine and Diphosphohistidine Peptideâ•… 281 7.4.7.1 Peptide Angio I as DRVYIHPFHLâ•… 281 Behavior of Phosphotyrosine and Phosphohistidine Peptideâ•… 285 7.4.8.1 Peptide Angio II as DRVpYIHPFâ•… 285 7.4.8.2 Phosphorylated Angio II as DRVpYIpHPFâ•… 285

xviâ•…â•… CONTENTS

7.4.9

Behavior of Phosphotyrosine-, Phosphothreonine-, and Phosphohistidine-Containing Peptideâ•… 287 7.4.9.1 Peptide Angio II (Sar1Thr8)â•… 287 7.4.10 Validation of Cu(II)-Based IMAC Phosphohistidine Enrichmentâ•… 291 7.4.10.1 Fe(III)-Based IMAC versus Cu(II) Basedâ•… 292 7.4.10.2 Cu(II)-Based IMAC of Angio Iâ•… 292 7.4.10.3 Cu(II)-Based IMAC of Angio IIâ•… 293 7.4.11 In Vivo Measurement of Phosphohistidineâ•… 293 7.4.11.1 Time-Based Digestion Studyâ•… 293 7.4.11.2 Phosphohistidine-Containing Peptidesâ•… 294 7.4.11.3 Phosphohistidine Product Ion Spectraâ•… 294 7.4.12 Gene Ontology of Phosphorylated Proteinsâ•… 296 7.4.12.1 Localization of Phosphorylated Proteinsâ•… 296 7.4.12.2 Function of Phosphorylated Proteinsâ•… 304 7.4.13 Predicted Regulatory Protein Motif Studyâ•… 307 7.4.14 Validation of Phosphohistidine-Containing Proteinsâ•… 308 7.4.14.1 Phosphorylation Motif Studyâ•… 308 7.4.14.2 Phosphohistidine Kinase Motifâ•… 309 7.4.15 The pDpH Motifâ•… 310 7.4.16 Conclusionsâ•… 311 7.5 Supplementary Materialâ•… 311 7.5.1 Reviewing Spectra Using the SpectrumLook Software Packageâ•… 311 Referenceâ•… 313 Appendix I Atomic Weights and Isotopic Compositions

317

Appendix II Periodic Table of the Elements

325

Appendix III Fundamental Physical Constants

327

Glossary

329

Index

345

PREFACE This book is a review of posttranslational modification (PTM) of proteins, including a special focus on a collection of mass spectral studies of phosphorylation as a PTM of both eukaryotic and prokaryotic proteomes utilizing the most recent advances and approaches in analytical chemistry. Protein PTM studies have now been studied for over 30 years in recognition of its importance in cellular processes. In particular, the study of protein phosphorylation as a PTM has received much attention in the last 5 years. The timing of this book is in accordance with the new advances in protein phosphorylation studies. A major focus of the book is in the first time reporting of the study of prokaryote phosphoproteomes, in particular the extensive study of the phosphorylation of the histidine residue that is extremely important in prokaryote signaling processes, with very little or no examples previously reported. The focus of the measurement of the phosphoproteomes is based on state-of-the-art mass spectrometry instrumentation and techniques. Also discussed are specific methodologies for performing PTM phosphoproteome studies, which include from cell cultures, through important steps during sample preparation, instrumental analysis using the most recent mass spectral approaches, the handling of the extensive data collected, and finally, recent tools available to the scientific community free of charge on the Internet currently being used to help understand and interpret the extensive and complex data collected during these studies. The data reduction approach utilizes the current “systems biology” viewpoint to rationalize the observations. The primary focus of the book is to teach the basic skills and methodologies needed for studies of phosphorylation as a PTM of proteins using real-life examples of actual phosphoproteome studies of both eukaryotic and prokaryotic systems. The book is a mixture of the fundamentals of sample preparation, nano-liquid chromatographic separation/ nano-electrospray ionization, tandem mass spectrometry instrumental analysis, followed by bioinformatic data interpretation concerning phosphoproteome studies. The book is presenting a number of first-time observations and studies involving the phosphoproteomes of prokaryotes. This is a xvii

xviiiâ•…â•… PREFACE

subject area that is timely and new with few previous examples in the literature. There is currently an overwhelming need and interest in the scientific community concerning phosphoproteomic studies of prokaryotic systems. This involves both the normal type of phosphoproteomes that are studied in eukaryotic systems and the novel areas of phosphorylation observed in prokaryotes. Researchers are currently applying the phosphoproteomic approaches that have now been well-optimized in eukaryotic systems to those of prokaryotic systems, and examples of these types of studies are presented in the book. However, the tremendously challenging area of phosphorylation of the histidine residue or “phosphohistidine proteomes” of prokaryotes has just begun to be studied. The book presents numerous first-time studies of this current topic of interest that has not been observed nor reported yet anywhere in the literature. The book describes the in vitro synthesis of phosphohistidine-containing peptides along with the mass spectral characterization of the peptides. Specifically, the book presents optimized methodologies for performing both eukaryotic phosphoproteome studies and prokaryotic phosphoproteome studies. The materials presented in the book are tried-and-tested sample preparation and analysis methods and approaches. While this can be collected in the literature for eukaryotic systems through exhaustive and time-consuming searches, the book has compiled the most recent approaches into one place. The methodologies that are presented for prokaryotic systems are, however, novel and new and not available in the literature in a systematic approach as presented in the book. In fact, the book is reporting prokaryotic study approaches that have not previously been reported before. This covers a new and quite challenging area of phosphohistidine prokaryote phosphoproteome studies. The book also presents methodologies for timebased studies of a prokaryote model that is undergoing a food-starved environment study. This is a first-ever reported quantitative differential study of a prokaryotic system under perturbed conditions. All studies reported in the book are actual laboratory experiments giving step-bystep sample preparation protocols using laboratory benchtop methodologies and the most recent vendor-optimized kits. For example, an immunoprecipitation study of phosphorylated tyrosine enrichment is described along with the associated mass spectral instrumental analysis results. The work and studies reported in the book can be an invaluable asset to the student and the researcher due to the combination of detailed step-by-step methodology for phosphoproteomic sample preparation, mass spectral instrumental analysis, and data interpretation approaches.

PREFACEâ•…â•… xix

The book also includes the use of some of the most recent systems biology bioinformatic internet tools such as the Blast2GO gene ontology (GO) tool. Also described are the most recent data processing approaches that have been developed by Dr. Richard Smith’s proteomics group at Pacific Northwest National Laboratory. The book is also an extensive reference concerning cell signaling studies associated with phosphorylation of proteins. Bryan M. Ham

ACKNOWLEDGMENTS I would like to acknowledge all those whose input, review, and criticisms helped enormously in the early structuring and final content of this book. I would like to include in the acknowledgement Pacific Northwest National Laboratory where much of the inspiration for this book was instilled within me while I was conducting research in Dr. Richard D. Smith’s proteomics group. Finally, and most important of all, is the acknowledgment of my wife, Dr. Aihui Ma Ham, whose consultations, support, reviewing, and invaluable encouragement saw through the entire process of this book from start to finish with an unending presence of which the project would most certainly not have completed to this level without. B. M. H.

xxi

ABOUT THE AUTHOR Bryan M. Ham, PhD, is a member of the American Society of Mass Spectrometry and the American Chemical Society. He has conducted proteomics and lipidomics research at The Ohio State University and Pacific Northwest National Laboratory in Richland, WA. He is currently working for the Department of Homeland Security at the U.S. Customs and Border Protection New York Laboratory. His research interests include the application of mass spectrometry for biomolecular analysis in the areas of proteomics, lipidomics, and metabolomics.

xxiii

1

Posttranslational Modification (PTM) of Proteins The study of posttranslational modification (PTM) of proteins using mass spectrometry (MS) approaches has now become a well-matured area of study. There are numerous approaches toward applying chromatography coupled with MS for PTM studies. The liquid chromatography (LC) front-end separation approach of choice is now nanoflow/ nano-electrospray, which allows increased sensitivity over previous LC methodology. This book looks at recent developments in PTM studies using MS and proteomic techniques with a focus upon a number of actual studies designed to instruct and highlight modern methodological approaches. A brief overview of nano-electrospray/nanoflow LC-MS is presented in Section 1.3. 1.1 OVER 200 FORMS OF PTM OF PROTEINS In the genomic sequencing field, the use of robotic gene sequencers allowed large-scale sequencing that was essentially automated. The robotic automation of determining gene sequences is possible because the sequences involved with genes involve only four bases (see “Overview of Nucleic Acids” in Section 1.4), and there are no variations induced in the form of postmodification. This has resulted in the wellpublicized entire sequencing of the human genome (Human Genome Project, Nature, February 2001). This is not the case with proteins where there is not only the observance of spliced variants from alternative splicing from the messenger ribonucleic acid (mRNA), there are also PTMs that can take place with the amino acids contained within the protein. There are over 200 PTMs that can take place with proteins as has been described by Wold.1 As examples, here are 22 different types

Proteomics of Biological Systems: Protein Phosphorylation Using Mass Spectrometry Techniques, First Edition. Bryan M. Ham. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc. 1

2â•…â•… POSTTRANSLATIONAL MODIFICATION (PTM) OF PROTEINS

of PTMs that can take place with proteins: acetylation, amidation, biotinylation, C-mannosylation, deamidation, farnesylation, formylation, flavinylation, gamma-carboxyglutamic acids, geranyl-geranylation, hydroxylation, lipoxylation, myristoylation, methylation, N-acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, phosphopantetheine, pyrrolidone carboxylic acid, pyridoxyl phosphate, and sulfation.2 There are also artifactual modifications such as oxidation of methionine (Met). A brief overview of proteins and an introduction to proteomics is presented in Section 1.5.

1.2 THREE MAIN TYPES OF PTM STUDIED BY MS Of these, the three types of PTM that are primarily observed and studied using mass spectrometric techniques are glycosylation, sulfation, and phosphorylation. The observance of PTM is increasingly being used in expression studies where a normal state proteome is being compared with a diseased state proteome. However, the PTM of a protein during a biological or physiological change within an organism may take place without any change in the abundance of the protein involved and often, is one piece of a complex puzzle. Methods that measure PTM using mass spectrometric methodologies often focus on the degree (increase or decrease, or alternatively, upregulation or downregulation) of PTM for any given protein or proteins. We shall briefly look at glycosylation and sulfation, which are less involved in cellular processes than phosphorylation, a major signaling cascade pathway for the response to a change in cellular condition(s).

1.3 OVERVIEW OF NANO-ELECTROSPRAY/ NANOFLOW LC-MS 1.3.1 Definition and Description of MS During the past decade, MS has experienced a tremendously large growth in its uses for extensive applications involved with complex biological sample analysis. MS is basically the science of the measurement of the mass-to-charge ratio (m/z) of ions in the gas phase (GP). Mass spectrometers are generally composed of three components: (1) an ionization source that ionizes the analyte of interest and effectively transfers it into the GP, (2) a mass analyzer that separates positively or negatively charged ionic species according to their mass-to-charge ratio

OVERVIEW OF NANO-ELECTROSPRAY/NANOFLOW LC-MSâ•…â•… 3

(m/z), and (3) a detector used to measure the subsequently separated GP ions. Mass spectrometers are computer controlled, which allows the collection of large amounts of data and the ability to perform various and complex experiments with the mass spectral instruments. Applications of MS include unknown compound identification, known compound quantitation, structural determination of molecules, GP thermochemistry studies, ion–ion and ion molecule studies, and molecule chemical property studies. MS is routinely used to determine elements such as Li+, Na+, Cl−, Mg,2+ inorganic compounds such as Li+(H2O)x or (TiO2 )+x , and organic compounds including lipids, proteins, peptides, carbohydrates, polymers, and oligonucleotides (deoxyribonucleic acid [DNA]/RNA). 1.3.2 Basic Design of Mass Analyzer Instrumentation Typical mass spectrometric instrumentation that is used in laboratories and research institutions is composed of six components: (1) an inlet, (2) an ionization source, (3) a mass analyzer, (4) a detector, (5) a data processing system, and (6) a vacuum system. Figure 1.1 illustrates the interrelationship of the six components that make up the fundamental construction of a mass spectrometer. The inlet is used to introduce a sample into the mass spectrometer and can be a solid probe, a manual syringe or syringe pump system, a gas chromatograph, or a liquid chromatograph. The inlet system can be either at atmospheric pressure as is shown in Figure 1.1 or at a reduced pressure under vacuum. The ionization source functions to convert neutral molecules into charged analyte ions, thus enabling their mass analysis. The ionization source can also be part of the inlet system. A typical inlet system and ionization source that is used with high-performance liquid chromatography (HPLC) is electrospray ionization (ESI). In an HPLC/ESI inlet system and ionization source, the effluent coming from the HPLC column is transferred into the ESI capillary that has a high voltage applied to it inducing the ESI process. In this configuration, the inlet system and ionization source are located at atmospheric pressure outside of the mass spectrometric instrumentation that is under vacuum. The spray that is produced passes through a tiny orifice that separates the internal portion of the mass spectrometer that is under vacuum from its ambient surroundings that are at atmospheric pressure. This orifice is also often called the inlet and/or the source. In the case of the coupling of a gas chromatograph to the mass spectrometer, the capillary column of the gas chromatograph is inserted through a heated transfer capillary directly into the internal portion of the mass spectrometer that is under


Data collection and processing station (PC) m/z Inlet orifice Electrospray ionization source (ESI) and spray

Atmospheric pressure region (760 torr)

Ion lens

Quadrupole mass analyzer

First-stage vacuum (10–2 to 10–4 torr)

Rotary-vane mechanical (rough) pump

Electron multiplier detector

Dynode converter

Second-stage vacuum (10–5 to 10–9 torr)

Turbo molecular pump

Figure 1.1.â•‡ The six components that make up the fundamental configuration of mass spectrometric instrumentation composed of (1) inlet and ionization system, (2) inlet orifice (source), (3) mass analyzer, (4) detector, (5) vacuum system, and (6) data collection and processing station (PC). [See Wikipedia, turbomolecular pump, http:// en.wikipedia.org/w/index.php?title=Turbomolecularpump&oldid=71160479 (as of August 24, 2006, 17:45 GMT)].

vacuum. This is possible due to the fact that the species eluting from the capillary column are already in the GP, making their introduction into the mass spectrometer more straightforward as compared with the liquid eluant from an HPLC where analytes must be transferred from the solution phase to the GP. An example of an ionization process that takes place under vacuum in the front end of the mass spectrometer is a process called matrix-assisted laser desorption ionization or MALDI. In this ionization technique, a laser pulse is directed toward a MALDI


target that contains a mixture of the neutral analytes and a strongly UV-absorbing molecule, often times a low-molecular-weight organic acid such as dihydroxybenzoic acid (DHB). The analytes are lifted off of the MALDI target plate directly into the GP in an ionized state. This is due to transference of the laser energy to the matrix and then to the analyte. The MALDI technique takes place within a compartment that is at the beginning of the mass spectrometer instrument and is under vacuum. The compartment that this takes place is often called the ionization source, thus combining the inlet system and the ionization source together into one compartment. As illustrated in Figure 1.1, the analyte molecules (small circles), in an ionized state, pass from atmospheric conditions to the first stage of vacuum in the mass spectrometer through an inlet orifice that separates the mass spectrometer that is under vacuum from ambient conditions. The analytes are guided through a series of ion lenses into the mass analyzer. The mass analyzer is the heart of the system, which is a separation device that separates positively or negatively charged ionic species in the GP according to their respective mass-to-charge ratios. The mass analyzer GP ionic species separation can be performed by an external field such as an electric field or a magnetic field or by a field-free region such as within a drift tube. For the detection of the GP-separated ionic species, electron multipliers are often used as the detector. Electron multipliers are mass impact detectors that convert the impact of the GP-separated ionic species into a cascade of electrons, thereby multiplying the signal of the impacted ion many times fold. The vacuum system ties into the inlet, the source, the mass analyzer, and the detector of the mass spectrometer at different stages of increasing vacuum as movement goes from the inlet to the detector (left to right in Fig. 1.1). It is very important for the mass analyzer and detector to be under high vacuum as this removes ambient gas, thereby reducing the amount of unwanted collisions between the mass-separated ionic species and gas molecules present. As illustrated in Figure 1.1, ambient, atmospheric conditions are generally at a pressure of 760â•¯torr. The firststage vacuum is typically at or near 10−3â•¯torr immediately following the inlet orifice and around the first ion transfer lenses. This stage of vacuum is obtained using two-stage rotary vane mechanical pumps that are able to handle high pressures such as atmospheric and large variation in pressures but are not able to obtain the lower pressures that are required further into the mass spectrometer instrument. The two-stage rotary vane mechanical pump has an internal configuration that utilizes a rotating cylinder that is off-axis within the pump’s hollow body. The off-axis-positioned rotor contains two vanes that are opposed and


directed radially and are spring controlled to make pump body contact. As the cylinder rotates, the volume between the pump’s body and the vanes changes; the volume increases behind each vane that passes a specially placed gas inlet port. This will cause the gas to expand behind the passing vane, while the trapped volume between the exhaust port and the forward portion of the vane will decrease. The exhaust gas is forced into a second stage and is then released by passing through the oil that is contained within the pump’s rear oil reservoir. This configuration is conducive for starting up at atmospheric pressure and working toward pressures usually in the range of 10−3 to 10−4â•¯torr. The lower stages of vacuum are obtained most often using turbo molecular pumps as illustrated in Figure 1.1. Turbo molecular pumps are not as rugged as the mechanical pumps described previously and need to be started in a reduced pressure environment. Typically, a mechanical pump will perform the initial evacuation of an area. When a certain level of vacuum is obtained, the turbo molecular pumps will then turn on and bring the pressure to higher vacuum. Using a mechanical vane pump to provide a suitable forepump pressure for the turbo molecular pump is known as roughing or “rough out” the chamber. Therefore, two-stage rotary vane mechanical pumps are often referred to as rough pumps. As illustrated in Figure 1.1, the turbo molecular pump contains a series of rotor/stator pairs that are mounted in multiple stages. The principle of turbomolecular pumps is to transfer energy from the fast rotating rotor (turbo molecular pumps operate at very high speeds) to the molecules that make up the gas. After colliding with the blades of the rotor, the gas molecules gain momentum and move to the next lower stage of the pump and repeat the process with the next rotor. Eventually, the gas molecules enter the bottom of the pump and exit through an exhaust port. As gas molecules are removed from the head or beginning of the pump, the pressure before the pump is continually reduced as the gas is removed through the pump, thus achieving higher and higher levels of vacuum. Turbo molecular pumps can obtain much higher levels of vacuum (up to 10−9â•¯torr) as compared with the rotary vane mechanical pumps (up to 10−4â•¯torr). The final component of the mass spectrometer is a data processing system. This is typically a personal computer (PC) allowing the mass spectrometric instrumentation to be software controlled, enabling precise measurements of carefully designed experiments and the collection of large amounts of data. Commercially bought mass spectrometers will come with its own software that is used to set the operating parameters of the mass spectrometer and to collect and interpret the data, which is in the form of mass spectra.


1.3.3 ESI ESI is a process that enables the transfer of compounds in solution phase to the GP in an ionized state, thus allowing their measurement by MS. The use of ESI coupled with MS was pioneered by Whitehouse et al.3 and Fenn4 in 1985 and 1993 by extending the work of Dole et al.5 in 1968, who demonstrated the production of GP ions by spraying macromolecules through a steel capillary that was electrically charged and subsequently monitoring the ions with an ion-drift spectrometer. The process by which ESI works has received much theorization, study, and debate,6–12 in the scientific community, especially the formation of the ions from the Taylor13 cone droplets and offspring droplets. Figure 1.2

HPLC column

HPLC pump

Data collection and processing station (PC) m/z N2 Curtin gas Electrospray capillary

Electron multiplier detector

N2 Electrospray sleave (3–5 kV)

Electrospray counter electrode (600 V)

Mass spectrometer at 10–5 torr

Electrospray Taylor cone at 760 torr Figure 1.2.â•‡ General setup for ESI when measuring biomolecules by electrospray mass spectrometry.


Taylor Cone

ESI Droplets

Spray Needle 2–5 kV

Metal Plate ~100 V Mass Spectrometer

ESI Solution

Oxidation

Excess Charge on Surface

Reduction

Solvent and Neutralized Ions

Spray Current (i)

+

2–5 kV Power Supply

_

Figure 1.3.â•‡ Electrospray ionization process illustrated in positive ion mode. (Reprinted with permission of John Wiley & Sons, Inc. Cech, N.B., and Enke, C.G. Practical implications of some recent studies in electrospray ionization fundamentals. Mass Spectrometry Reviews, 2001, 20, 362–387. Copyright 2001.)

shows the general setup for ESI when measuring biomolecules by electrospray MS. The electrospray process is achieved by placing a potential difference between the capillary and a flat counter electrode. This is illustrated in Figure 1.3 where the “spray needle” is the capillary and the “metal plate” is the flat counter electrode. The generated electric field will penetrate into the liquid meniscus and create an excess abundance of charge at the surface. The meniscus becomes unstable and protrudes out, forming a Taylor cone. At the end of the Taylor cone, a jet of emitting droplets (number of drops estimated at 51,250 with radius of 1.5â•¯µm) will form that contains an excess of charge. Pictures of jets of offspring droplets are illustrated in Figure 1.4. As the droplets move toward the counter electrode, a few processes take place. The drop shrinks due to evaporation, thus increasing the surface charge until columbic repulsion is great enough that offspring droplets are produced. This is known as the Rayleigh limit, producing a columbic explosion. The produced offspring droplets have 2% of the parent droplets’ mass and 15% of the parent droplets’ charge. This process will continue until the drop contains one molecule of analyte and charges that are associated with basic sites (positive ion mode). This is referred to as the


(a)

(b)

Figure 1.4.â•‡ Pictures illustrating the jet production of offspring droplets. (Reprinted with permission from Alessandro Gomez, Physics of Fluids, 6, 404 (1994). Copyright 1994, American Institute of Physics.)

“charged residue model” that is most important for large molecules such as proteins. This process is illustrated in Figure 1.5. As the droplets move toward the counter electrode, a second process also takes place known as the “ion evaporation model.” In this process, the offspring droplet will allow evaporation of an analyte molecule from its surface along with charge when the charge repulsion of the analyte with the solution is great enough to allow it to leave the surface of the drop. This usually takes place for droplets with a radius that is less than 10â•¯nm. This type of ion formation is most important for small molecules.


Taylor cone “budding” sample in solution

+HV

+ + +

+ + + + + +

+

+

+

+

+ + +

+ + +

+ + +

+ + +

+ +

+ + +

+

+ +

+

+

+

droplet fission at Rayleigh limit

+ +

+

+

solvent evaporation

+

+ +

[M+nH]n+

+

+ +

+

+

formation of desolvated ions by further droplet fission and/or ion evaporation

Figure 1.5.â•‡ Gas-phase ion formation process from electrospray droplets. (Reprinted from Gaskell, S.J. Electrospray: principles and practice. J. Mass Spectrom. 1997, 32, 677–688. Copyright John Wiley & Sons Limited 1997. Reproduced with permission.)

In the ensuring years since its introduction, electrospray MS has been used for structural elucidation and fragment information,14–16 and noncovalent complex studies,17,18 just to name a few recent examples of its overwhelmingly wide range of applications. Electrospray3,4,7,8 is an ionization method that is now well known to produce intact GP ions with very minimal, if any, fragmentation being produced during the ionization process. In the transfer process of the ions from the condensed phase to the GP, several types of “cooling” processes of the ions are taking place in the source: (1) cooling during the desolvation process through vibrational energy transfer from the ion to the departing solvent molecules, (2) adiabatic expansion of the electrospray as it enters the first vacuum stage, (3) evaporative cooling, and (4) cooling due to low-energy dampening collisions with ambient gas molecules. The combination of these effects, and the fact that


electrospray can effectively transfer a solution phase complex to the GP with minimal interruption of the complex, makes the study of noncovalent complexes from solution by ESI-MS attractive. 1.3.4 Nano-ESI A major application of biomolecule analysis using MS has been the ability to allow liquid flows to be introduced into the source of the mass spectrometer. This has enabled the coupling of HPLC to MS where HPLC is used for a wide variety of biomolecule analysis. Normal ESI, introduced in the preceding section, typically has flow rates in the order of microliters per minute (∼1–500â•¯µL/min). Traditional analytical HPLC systems designed with UV/Vis detectors generally employ flow rates in the range of milliliters per minute (∼0.1–1â•¯mL/min). A recent advancement in the ESI technique has been the development of nanoelectrospray where the flows employed are typically in the range of nanoliters per minute (∼1–500â•¯nL/min). Following the progression of the development of electrospray from Dole’s original reporting in 1968 through Fenn’s work reported in 1984 and 1988, a more efficient electrospray process was reported by Wilm et al.19 employing flows in the range of 25â•¯nL/min. This early reporting of low flow rate electrospray was initially termed as microelectrospray by Wilm et al. but was later changed to nano-electrospray.20 At the same time that Wilm et al.19 reported the microelectrospray, Caprioli et al.21 also reported a miniaturized ion source that they had named microelectrospray. The name “nanoelectrospray” for Wilm’s source is actually more descriptive due to flow rates used in the nanoliter per minute range and the droplet sizes that are produced in the nanometer range. Conventional electrospray sources before the introduction of nano-electrospray produced droplets on the order of 1–2â•¯µm. The nano-electrospray source produces droplets in the size range of 100–200â•¯nm, which is 100–1000 times smaller in volume. When spraying standard solutions at concentrations of 1â•¯pmol/µL, it is estimated that droplets of the nanometer size contain only one analyte molecule per droplet. The original nano-electrospray sources that were used were composed of pulled fused-silica capillary tips 3–5â•¯cm long with orifices of 1–2â•¯µm in diameter. The tips also have thin gold plating that allows current flow. The tips are loaded with 1–5â•¯µL of sample directly using a pipette22 and coupled to the electrospray source, completing the closed circuit required for the production of the applied voltage electrospray Taylor cone generation. This is illustrated in Figure 1.6 where in the top portion of the figure a sample is being loaded into the


(1) Load sample directly into nanospray tip

High-voltage (HV) power supply Current meter

m/z (2) Place loaded nanospray tip into electrospray HV source

Resistor

(3) Apply HV to produce electrospray process

(4) Measure m/z values with mass spectrometer

Figure 1.6.â•‡ Top of figure illustrates the loading of a nano-electrospray tip. Bottom of figure illustrates the coupling of the nano-electrospray tip to the closed-circuit system.

nanospray tip using a pipette. The tip is then placed into the closedcircuit system for the electrospray to take place. The sample flow rate is very low using the nanospray tips allowing the measurement of a very small sample size over an extended period of time. It has also been observed that nanospray requires a lower applied voltage for the production of the electrospray that helps to reduce problems with corona electrical discharges that will interrupt the electrospray. In nanoelectrospray, the flow rate is lower than in conventional electrospray and is felt to have a direct impact on the production of the droplets within the spray and the efficiency of ion production. The lower flow rate produces charged droplets that are reduced in size as compared with conventional electrospray. This has been described in detail by Wilm et al.,19 by Fernandez de la Mora et al.,23 and by Pfeifer and Hendricks.24 There are fewer droplet fission events required with smaller


(a)

(b)

1 µm i.d.

(c)

2 µm i.d.

5 µm i.d.

Figure 1.7.â•‡ Illustration of different nano-electrospray tip orifice diameters. Scanning electron microscopy images of employed nanospray emitters: (a) 1-, (b) 2-, and (c) 5-µm tip. Images were obtained after 2 hours of use. (Reprinted with permission from Li, Y.; Cole, R.B. Shifts in Peptide and Protein Charge State Distributions with Varying Spray Tip Orifice Diameter in Nano-Electrospray Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Anal. Chem. 2003, 75, 5739–5746. Copyright 2003 American Chemical Society.)

(a)

(b)

>1 µm 50 µm

(c)

500 µm (d)

12.5 µm 50 µm

500 µm

Figure 1.8.â•‡ Examples of nanospray tip sizes and the influence upon the ESI Taylor cone. The cone is not observed in (b) at a diameter of >1â•¯µm. The cone is observed in (d) for a diameter of 12.5â•¯µm. (Reprinted with permission. This article was published in J Am Soc Mass Spectrom, Schmidt, A., Karas, M. Effect of different solution flow rates on analyte ion signals in nano-ESI MS, or: when does ESI turn into nano-ESI?, 2003, 14, 492–500. Copyright Elsevier 2003.)

initial droplets in conjunction with less solvent evaporation taking place before ion release into the GP.25,26 A result of this is that a larger amount of the analyte molecule is transferred into the mass spectrometer for analysis. Though the efficiency of ionization is increased with nano-electrospray, the process is also influenced by the size and shape of the orifice tip.27,28 Pictures of nano-electrospray orifice tips are illustrated in Figure 1.7. Figure 1.8 shows an example of the production and observance of and ESI Taylor core.


Figure 1.9.â•‡ Photograph of nine stable electrosprays generated from the nine-spray emitter array. (Reprinted with permission from Tang, K.; Lin, Y.; Matson, D.W.; Kim, T.; Smith, R.D. Anal. Chem. 2001, 73, 1658–1663. Copyright 2001 American Chemical Society.)

While Figure 1.8d does show a Taylor cone formed, Figure 1.9 gives a good picture of an array of Taylor cones formed from a microelectrospray emitter. In the figure, multiple cones can be seen along with their associated spray produced from the electrospray process. As mentioned previously, nano-HPLC is increasingly being coupled to nano-electrospray for biomolecule analysis. A nano-HPLC-ESI system is illustrated in Figure 1.10. The flow involved in nano-HPLCESI often ranges between 10 and 100â•¯nL/min. The fused-silica capillary columns that are used in nano-HPLC have very small diameters, often around 50â•¯µm. These small-diameter columns can often create high back pressures in the HPLC system. One way to achieve the very low flow rate through the fused-silica nano-HPLC column is to use a flow splitter that is located in-stream between the column and the HPLC pump as illustrated in Figure 1.10. The tubing from the splitter to waste is called a restrictor and is used to regulate the flow through the nanocolumn. A smaller-diameter restrictor used will increase the back pressure, forcing more mobile phase through the nanocolumn. If a larger diameter restrictor is used, the back pressure will be lower, resulting is less flow being directed through the column. The nanocolumns have a nano-ESI tip coupled to them (diameters can range from 1â•¯µm up to 100â•¯µm) to produce the electrospray. Another difference observed here as compared with the atmospheric pressure source is the absence of a nebulizing gas or a drying gas. These are not needed or used in nano-ESI.

OVERVIEW OF NUCLEIC ACIDSâ•…â•… 15

Restrictor split to waste

Flow splitter

m/z

Fused-silica (packed) capillary column

Figure 1.10.â•‡ Design of a nano-HPLC nano-ESI system for mass spectrometric analysis of biomolecules.

1.4 OVERVIEW OF NUCLEIC ACIDS Nucleic acids are an important consideration in PTM study; they are present in cellular protein extracts and must be separated. In traditional studies, the Trizol precipitation method was used to isolate a nucleic acid fraction and a protein fraction. This afforded the opportunity to study both from an extraction. Due to their close relationship and importance, we will look at a brief overview of nucleic acids and their measurement by MS. Nucleic acids are also analyzed using mass spectrometric techniques, and we will start with a background look at the makeup of nucleic acids before looking at the MS. In contrast to polysaccharides, and similar to proteins, nucleic acids are specifically directional in their makeup and contain nonidentical monomers that have a distinct sequence that produce informational macromolecules. The nucleic acids reside in the nucleus of the cell and are the storage, expression, and transmission of genetic information of living species. The two types of nucleic acids are


DNA and RNA. There are two distinct parts of their chemical structure and makeup that differentiate the two. First, DNA contains the fivecarbon sugar deoxyribose, while RNA contains ribose; and second, DNA contains the base thymine (T), while RNA contains uracil (U). The molecules that make up the DNA and RNA structures are illustrated in Figure 1.11. This constitutes the purine bases adenine (A) and guanine (G), the pyrimidine bases cytosine (C), uracil (U), and thymine. Also illustrated in Figure 1.11 are the two sugars d-deoxyribose and d-ribose and finally, the phosphate group that acts as the backbone of the nucleic acids linking the nucleotides together. Nucleotides are the monomeric units that make up the nucleic acids. There are actually only four nucleotides that make up DNA and RNA, a much smaller number than the 20 amino acids found in proteins. Examples of nucleotides found in DNA and RNA are illustrated in Figure 1.12. Figure 1.12a is a DNA nucleotide where the number 2′ carbon in the sugar ring contains a hydrogen atom for d-deoxyribose. One of the bases will be attached to the 1′ carbon of the sugar through an aromatic nitrogen, and the phosphate will be attached to the number 5′ sugar carbon with a phosphoester bond. The RNA nucleotide illustrated in Figure 1.12b has the same types of bonding as illustrated for the DNA nucleotide but to a d-ribose sugar. In the case that the phosphate group is removed from the nucleotide, the remaining base sugar structure is called a nucleoside. The nucleotides are linked to each other through the phosphate group, forming a linear polymer. The nucleotides undergo a condensation reaction through the linking of the phosphate group on the 5′ carbon to the 3′ carbon of the next nucleotide known as a 3′,5′ phosphodiester bond. The resulting polynucleotide therefore has a 5′ hydroxyl group at the start (by convention) and a 3′ hydroxyl group at the end (by convention). Representative linear nucleotide structures are illustrated for RNA and DNA in Figure 1.13. A similar naming scheme that is used for the fragmentation ions generated by collision-induced dissociation (CID) of peptides was proposed by Glish et al.29 for nucleic acids and is illustrated in Figure 1.14. There are four cleavage sites producing fragmentation along the phosphate backbone from CID. When the product ion contains the 3′-OH portion of the nucleic acid, the naming includes the letters w, x, y, and z, where the numeral subscript is the number of bases from the associated terminal group. When the product ion contains the 5′-OH portion of the nucleic acid, the naming includes the letters a, b, c, and d. Losses are also more complicated than that shown in Figure 1.14 due to the neutral loss of base moieties. Figure 1.15 illustrates an actual structural

OVERVIEW OF NUCLEIC ACIDSâ•…â•… 17

Pyrimidines

Purines NH2

N

N

N H

O

NH2

NH

N

N

N H

Adenine (A)

N H

O

Uracil (U) (in RNA)

Cytosine (C)

O

O

N

NH

NH

N H

O

N

N H

NH2

O

Thymine (T) (in DNA)

Guanine (G)

Sugars

HO

OH O H

H

OH

OH

H

O H

D-ribose

O

P

O

(in RNA) O

HO

OH

Phosphate group

O H

H

OH

H

H

H

D-deoxyribose

(in DNA)

Figure 1.11.â•‡ Structures of the molecules that make up the nucleic acids DNA and RNA. The purine bases adenine (A) and guanine (G), the pyrimidine bases cytosine (C), uracil (U), and thymine, the sugars d-deoxyribose and d-ribose, and the phosphate group.

18â•…â•… POSTTRANSLATIONAL MODIFICATION (PTM) OF PROTEINS (a)

NH2

N

Phosphodiester bond

N

O -O

N

P

N

5′

O

O

4′

O-

H

2′H

3′

H

1′

H

OH

H

(b)

NH2

N

N

O -O

P

O

O

O

O-

H

H

OH

OH

H

H

Figure 1.12.â•‡ (a) DNA nucleotide and (b) RNA nucleotide.

cleavage at the w2/a2 site of a 4-mer nucleic acid’s phosphate backbone according to the naming scheme of Figure 1.14. Numerous mechanisms have also been reported for the fragmentation pathways leading to charged base loss and also neutral base loss. These are losses that are observed in product ion spectra other than the cleavage along the phosphate backbone that is illustrated in Figures 1.14 and 1.15. Neutral and charged base losses add to the complexity of the product ion spectra but also add information concerning the makeup of the oligonucleotide. Figure 1.16 illustrates a couple examples of proposed fragmentation pathway mechanisms for neutral and charged base losses. In Figure 1.16a, a simple nucleophilic attack on the C-1′ carbon atom by the phosphodiester group results in the elimination of a charged base.30 Figure 1.16b illustrates a two-step reaction where in the first step there is neutral base loss followed by breakage of the 3′-phosphoester bond.31 There are other proposed fragmentation pathways for a number of other possible mechanisms for the production of the product ions observed in tandem mass spectra of the nucleic acids.

OVERVIEW OF NUCLEIC ACIDSâ•…â•… 19 O

(a)

N

Guanine

NH

N

NH2

N O

HO

O H

H

H

H

N

O O

Thymine

NH H O

NH2 O

P

O

H

N

N

N NH2

P

O

O H

O-

H

H

N H

H

N

O O

O

P

H

O

(b) NH

N

N

Cytosine O

O

O-

N

Adenine

H

H

O O

N

H

H

O-

H

H

OH

H

H

Guanine NH2

NH2

HO

O H

H

N

O O

Cytosine

N

H OH

H

P

O

O NH2

O H

H

O-

N

N

H OH

H

N

O

Adenine

N O

O

P

O

O H

H

OH

N

O O

P

Uracil

NH

H OH

O

O

O

OH

H

H

OH

H OH

Figure 1.13.â•‡ Linear nucleic acid structures for (a) DNA and (b) RNA.

20â•…â•… POSTTRANSLATIONAL MODIFICATION (PTM) OF PROTEINS w3 x 3

y3

z3

B1

w2 x 2

B2

O

O

P

1

b1

c1

O

O O

P

O

O-

d1

y1 z 1 B4

O

O-

a

w1 x 1

B3

O

5′ HO

y2 z 2

a2 b 2

c 2 d2

OH 3′

O

P O-

a

3 b3

c3

d3

Figure 1.14.â•‡ Naming scheme for nucleic acid product ions. When the product ion contains the 3′-OH portion of the nucleic acid, the naming includes the letters w, x, y, and z. When the product ion contains the 5′-OH portion of the nucleic acid, the naming includes the letters a, b, c, and d. In both 3′-OH and 5′-OH containing product ions, the numeral subscript is the number of bases from the associated terminal group.

1.5 PROTEINS AND PROTEOMICS 1.5.1 Introduction to Proteomics Proteomics, the study of a biological system’s compliment of proteins (e.g., from cell, tissue, or a whole organism) at any given state in time, has become a major area of focus for research and study in many different fields and applications. In proteomic studies, MS can be employed to analyze either the intact, whole protein or the resultant peptides obtained from enzyme-digested proteins. The mass spectrometric analysis of whole, intact proteins is often called top-down proteomics where the measurement study starts with the analysis of the intact protein in the GP and subsequently investigating its identification and any possible modifications through CID measurements. The mass spectrometric analysis of enzyme-digested proteins that have been converted to peptides is known as bottom-up proteomics. Finally, MS is also used to study PTMs that have taken place with the proteins such as glycosylation, sulfation, and phosphorylation. We shall begin with a look at bottom-up proteomics, the most common approach, followed by topdown proteomics, which is seeing more applications and study lately, and finally, the PTMs of glycosylation, sulfation, and phosphorylation. Bioinformatics has become an important tool used in the interpretation of results obtained from MS studies. In the last part of this chapter, we will briefly look at what bioinformatics is and what it can be used for in relation to MS and proteomic studies. Due to the enormous impact

PROTEINS AND PROTEOMICSâ•…â•… 21 O N

NH

N

N

NH2 O

HO

O H

H

H

H

NH H N

O

O NH2

O

O

P

O H

OH

a2

N

H H

H

P

N

w2

O O

N

O

N NH2

O H

O-

H

N

H

H

H

N

O O

P

O

O

OH

O N

O

H

H

OH

H

H

NH NH2

N

NH2

N

N

O HO

N

O H

H

H

H

NH

P O-

N NH2

N

O O

N

O

H O

HO

P

O

O H

O-

O

O H

H H

a2

H

H

H

N H N

O

O

H O

P

O

O

OH

H

H

OH

H

H

w2 Figure 1.15.â•‡ Example of structural cleavage at the w2/a2 site of a 4-mer nucleic acid’s phosphate backbone according to the naming scheme of Figure 1.14.

22â•…â•… POSTTRANSLATIONAL MODIFICATION (PTM) OF PROTEINS (a)

O

O N

N

O O

P

N

NH

N

N

NH 2 O

O

O

O

O O

O

O

O

O

(b) N

N

O

P

O

O H O

O

O P O

H

H

P

O

+ O

+ O

O

H

O H

O

H

O O

P

O

P

NH2

N

O

H

H

O

O

N H

NH 2

OH

NH

NH

N

O

O

O

N

O P

O O

O

NH2

N

+

O

P

P O

NH

H

O

P O

O

O

O H H

H

O

Figure 1.16.â•‡ Proposed fragmentation pathways associated with the base substituent groups. (a) Nucleophilic attack on the C-1′ carbon atom by the phosphodiester group results in the elimination of a charged base. (b) Two-step reaction mechanism where in the first step there is neutral base loss followed by breakage of the 3′-phosphoester bond.

of proteomics on research into biological processes, organisms, diseased states, tissues, and so on, we will begin this section starting with a brief overview of proteins including their structure and makeup. 1.5.2 Protein Structure and Chemistry Of all biological molecules, proteins are one of the most important, next only to the nucleic acids. All living cells contain proteins, and their name is derived from the Greek word proteios, which has the meaning of “first.”32 There are two broad classifications for proteins related to their structure and functionality: water-insoluble fibrous proteins and water-soluble globular proteins. The three-dimensional configuration of a protein is described by its primary, secondary,

PROTEINS AND PROTEOMICSâ•…â•… 23

Figure 1.17.â•‡ Ribbon structure representation of the RNase protein illustrating substructures of alpha helices and beta sheets.

tertiary, and quaternary structures. Figure 1.17 is a three-dimensional ribbon representation of the protein RNase. The primary structures of proteins are made up of a sequence of amino acids forming a polypeptide chain. Typically, if the chain is less than 10,000â•¯ Da, the compound is called a polypeptide; if greater than 10,000â•¯Da, the compound is called a protein. There are 20 amino acids that make up the protein chains through carbon to nitrogen peptide bonds. Figure 1.18 illustrates the 20 amino acid structures that make up the polypeptide backbone chain of proteins. Amino acids possess an amino group (NH2) and a carboxyl group (COOH) that are bonded to the same carbon atom that is alpha to both groups; therefore, amino acids are called alpha amino acids (α-amino acid). At physiological pH (∼7.36), the amino acids can be subdivided into four classes according to their structure, polarity, and charge state: (1) negatively charged composed of aspartic acid (Asp) and glutamic acid (Glu); (2) positively charged composed of lysine (Lys), arginine (Arg), and histidine (His); (3) polar composed of serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine (Cys), glutamine (Gln), and asparagine (Asn); and (4) nonpolar composed of glycine (Gly), leucine (Leu), isoleucine (Ile), alanine (Ala), valine (Val), proline (Pro), Met, tryptophan (Trp), and phenylalanine (Phe). The carbon to nitrogen peptide bonds are formed through condensation reactions between the carboxyl and amino groups. An example condensation reaction between the amino acids Leu and Tyr is illustrated in Figure 1.19. The peptide C-N bonds are found to be

24â•…â•… POSTTRANSLATIONAL MODIFICATION (PTM) OF PROTEINS Positively charged

Negatively charged O O H3N

CH

H3N

O

C

CH2 C

O

CH

O

O

C

O

H3N

CH

CH2

CH2

CH2

CH2

C

H3N

O

CH

O

C

O

H3N

CH

CH2

C

O

CH2

CH2 HN

C

O

CH2

CH2

O

NH O

NH

CH2

Aspartic acid (Asp) Glutamic acid (Glu)

C

NH3

NH2

NH2

Lysine (Lys)

Arginine (Arg)

Histidine (His)

Polar O H3N

CH

C

O

H3N

CH2 OH

CH

C

CH

OH

H3N

O

CH

C

H3N

O

CH

CH2

CH3

C

H3 N

O

CH

CH2

CH2

SH

CH2

Cysteine (Cys)

Threonine (Thr)

Serine (Ser)

O

O

O

O

C

C

O

O

O OH H3N

CH

C

Glutamine (Gln)

O

OH

Tyrosine (Tyr)

CH2 C

O

OH

Asparagine (Asn)

Nonpolar O

O H3N

CH

C

H3N

O

CH

C

O

H3 N

CH2

H

Glycine (Gly)

CH

CH3

C

CH

CH3

H3 N

CH3

CH

CH3

Alanine (Ala)

CH3

C

O

CH

O

Valine (Val)

C

O

O O

H3 N

CH

CH2 H2N

C

CH

Isoleucine (Ile) O

O O

H3N

CH

H3N

O

CH3

Leucine (Leu)

C

CH

CH2

CH3

O

O

O

C

O

CH2

H3N

CH

C

O

CH2

CH2 S

Proline (Pro)

HN CH3

Methionine (Met)

Tryptophan (Trp)

Phenylalanine (Phe)

Figure 1.18.â•‡ Structures of the 20 amino acids that make up the polypeptide backbone of proteins. Divisions include negatively charged, positively charged, polar, and nonpolar.

PROTEINS AND PROTEOMICSâ•…â•… 25 O

O

O

O H3 N

CH

C

CH2 CH

H3 N

O

+

CH CH2

C

O

H3 N

CH

N H

CH2 CH

CH3

C

CH3

CH

C

O

CH2

+

H2O

CH3

CH3

OH OH

Leucine

Tyrosine

Figure 1.19.â•‡ Condensation reaction between the amino acids leucine and tyrosine forming a peptide bond.

shorter than most amine C–N bonds due to a double-bond nature that contributes to 40% of the peptide bond.33 This double-bond character lessons the free rotation of the bond, thus affecting the overall structure of the protein.34 The secondary structure of the protein is described by two different configurations and turns. The two configurations are α-helices (first proposed by Linus Pauling and Robert B. Corey in 1951) and β-sheets (parallel and antiparallel) and are illustrated in Figure 1.20. The α-helix is described as a right-hand-turned spiral that has hydrogen bonding between oxygen and the hydrogen of the nitrogen atoms of the chain backbone. This hydrogen bonding stabilizes the helical structure. The R-group side chains that make up the amino acid residues extrude out from the helix. The β-sheet is a flat structure that also has hydrogen bonding between oxygen and the hydrogen of the nitrogen atoms but from different β-sheets (parallel and antiparallel) that run along side each other. These hydrogen bonds also work to stabilize the structure. The R-group side chains alternatively extrude out flat with the sheet from the sides of the sheet. The third secondary structure, the turn, basically changes the direction of the polypeptide strand. The tertiary structure, which includes the disulfide bonds, is composed of the ordering of the secondary structure, which is stabilized through side chain interactions. The quaternary structure is the arrangement of the polypeptide chains into the final working protein. All four structures describe what is actually a folded protein, where the apolar regions of the protein are tucked away inside the structure, away from the aqueous medium they are found in naturally, and more polar regions are on the surface.

26â•…â•… POSTTRANSLATIONAL MODIFICATION (PTM) OF PROTEINS (a) α-Helix O

R

O

O

CH R

CH

NH2

H

O N H

C

O

N

CH

C

O

N

CH

C

R

R

O

H

C

O

H N

O

CH R

C

O

H N

O R CH

C

O

O O O

HO

H N

CH

C

C

O

H N

CH

O

C

CH

N H

R R

R

NH2

(b) Parallel β-sheets

R NH2 R

C

CH O

O

R

R

H R

R

H R

R

O

H R

R

O

N CH

N

C

O

O C

HO C

H

O

CH

N CH

N

O C

O

O

CH C

O C

H

CH

CH

HO

O

N

N

H

C

O

O

N

O C

O

O

CH

CH

CH

H

R N

N

C

H

O

O

O

R

C O

C

O

CH

CH

CH

H

R N

N

C

R

H

NH2

O

O C

H

(c) Antiparallel β-sheets

CH

H

O R

N CH

O

C

O

HO

Figure 1.20.â•‡ (a) α-Helices. β-Sheets: (b) parallel and (c) antiparallel.


1.5.3 Bottom-Up Proteomics: MS of Peptides 1.5.3.1 History and Strategy.â•‡ The proteomic approach was composed of measuring the enzymatic products of the protein digestion (after protein extraction from the biological sample), namely the peptides, using MS is known as bottom-up proteomics. In the bottom-up approach using nano-ESI-HPLC/MS, the peptides are chromatographically separated and subjected to collision-induced dissociation in the GP. The product ion spectra thus obtained of the separated peptides are then used to identify the proteins present in the biological system being studied. Prior to the use of nano-ESI-HPLC/MS for peptide measurement, Edman degradation was used to sequence unknown proteins. The method of Edman sequencing involves the removal of each amino acid residue one by one from the polypeptide chain starting from the N-terminus of the peptide or protein.35 The method worked well for highly purified protein samples that contained a free amino N-terminus, but the analysis was slow, usually taking a day to analyze the sequence of one protein. MS was first coupled with Edman sequencing in 1980 by Shimonishi et al.,36 where the products of the Edman degradation were measured using field desorption (FD) MS. FD, introduced in 1969 by Beckey, is an ionization technique not commonly in use today. FD consists of depositing the sample, either solid or dissolved in solvent, onto a needle and applying a high voltage. The process of desorption and ionization are obtained simultaneously. The analyte ions produced from FD are then introduced into the mass spectrometer for mass analysis. Fast atom bombardment was also used as an ionization technique to measure peptides obtained from the Edman sequencing approach.37 Another early approach to proteomics using MS was the application of MALDI time-of-flight (TOF) MS (MALDI-TOF/MS) to the measurement of peptides obtained from in-gel digestions of proteins separated by gel electrophoresis. This technique was reported by several groups and was called peptide mass fingerprinting (PMF).38–40 In the PMF approach, proteins are first separated using two-dimensional gel electrophoresis (2-DE), a protein separation technique first introduced in the 1970s.41 The gel used in electrophoresis is a rectangular gel composed of polyacrylamide. The protein sample is loaded onto the gel and the proteins are separated according to their isoelectric point (pH where the protein has a zero charge). This is the first dimension of the separation. The second dimension is a linear separation of the proteins according to their molecular weights. In preparation for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins


are first denatured (usually with 8â•¯M urea and boiling) and sulfide bonds are cleaved effectively unraveling the tertiary and secondary structure of the protein. SDS, which is negatively charged, is then used to coat the protein in a fashion that is proportional to the proteins’ molecular weight. The proteins are then separated within a polyacrylamide gel by placing a potential difference across the gel. Due to the potential difference across the gel, the proteins will experience an electrophoretic movement through the gel, thus separating them according to their molecular weight with the lower-molecular-weight proteins having a greater mobility through the gel and the higher molecular weight proteins having a lower mobility through the gel. The resultant 2-DE separation is a collection of spots on the gel that can be up to a few thousand in number. In 2-D SDS-PAGE, the proteins have been essentially separated into single protein spots. This allows the digestion of the protein within the spot (excised from the gel) using a protease with known cleavage specificity into subsequent peptides that are unique to that particular protein. The peptides extracted from the in-gel digested proteins separated by 2-D SDS-PAGE are then measured by MALDI-TOF/MS, creating a spectrum of peaks that represent the molecular weight of the protein’s enzymatic generated peptides. This list of measured peptides can be compared with a theoretical list according to the specificity of the enzyme used for digestion. There is an extensive list of references and searching software that has been introduced for the PMF approach to proteomics that has been reviewed.42 The 2-D SDS-PAGE and peptide mass fingerprint approach to proteomics is illustrated in Figure 1.21. In bottom-up proteomics, the proteins are generally extracted from the sample of interest, which can include a sample of cultured cells, bacterium, tissue, or a whole organism. A general scheme for the extraction and peptide mass fingerprint mass spectrometric analysis typically followed in early proteomic studies is illustrated in Figure 1.21. The initial sample is lysed and the proteins are extracted and solubilized. The proteins can then be separated using one-dimensional (1-D) or 2-D SDS-PAGE. Proteins can be digested in the gels, or the proteins in solution are digested using a protease such as trypsin. Trypsin is an endopeptidase that cleaves within the polypeptide chain of the protein at the carboxyl side of the basic amino acids Arg and Lys (the trypsin enzyme has optimal activity at a pH range of 7–10 and requires the presence of Ca+2). It has been observed though that trypsin does not efficiently cleave between the residues Lys–Pro and Arg–Pro. Tryptic peptides are predominantly observed as doubly or triply charged when using electrospray as the ionization source. This is due to the amino


Eukaryote cell

Prokaryote

Whole organism

Extraction of proteins

1-D and 2-D SDS-PAGE

In-gel or in-solution digestion

SCX fractionation

IMAC/TiO 2

Nano-LC-MS MS/MS spectra Database searching Swiss-Prot

NCBI

Bioinformatics Figure 1.21.â•‡ General strategy and sample flow involved in proteomics. IMAC, immobilized metal affinity chromatography.

terminal residue being basic in each peptide, except for the C-terminal peptide. There exist a number of proteases that are available to the mass spectrometrist when designing a digestion of proteins into peptides. These can be used to target cleavage at specific amino acid residues within the polypeptide chain. Examples of available proteases and their cleavage specificity are listed in Table 1.1. The enzymes will cleave the proteins into smaller chains of amino acids (typically from five

30â•…â•… POSTTRANSLATIONAL MODIFICATION (PTM) OF PROTEINS TABLE 1.1.â•‡ Examples of Proteases Available for Polypeptide Chain Cleavage Protease

Trypsin Chymotrypsin Proteinase K Factor Xa Carboxypeptidase Y Submaxillary Arg-C protease Staphylococcus aureus V-8 protease Aminopeptidase M Pepsin Ficin Papain

Polypeptide Cleavage Specificity

At carboxyl side of arginine and lysine residues At carboxyl side of tryptophan, tyrosine, phenylalanine, leucine, and methionine residues At carboxyl side of aromatic, aliphatic, and hydrophobic residues At carboxyl side of Glu-Gly-Arg sequence Sequentially cleaves residues from the carboxy (C) terminus At carboxy side of arginine residues At carboxy side of glutamate and aspartate residues Sequentially cleaves residues from the amino (N) terminus Nonspecifically cleaves at exposed residues favoring the aromatic residues Nonspecifically cleaves at exposed residues favoring the aromatic residues Nonspecifically cleaves at exposed residues

amino acid residues up to 100 or so). These short-chain amino acids are mostly water soluble and can be directly analyzed by MS. However, often, a lysis and extract from a biological system will constitute a very complex mixture of proteins that requires some form of separation to decrease the complexity prior to mass spectral measurement. 1.5.3.2 Protein Identification through Product Ion Spectra.â•‡ More recently, nano-ESI-HPLC-MS/MS has been employed using reversedphase (RP) C18 columns to initially separate the peptides prior to introduction into the mass spectrometer. If a highly complex compliment of digested proteins are being analyzed such as those obtained from eukaryotic cells or tissue, a greater degree of complexity reduction is employed such as strong cation exchange (SCX) fractionation, which can separate the complex peptide mixture up to 25 fractions or more. The coupling of online SCX with nano-ESI C18 RP HPLC-MS/ MS has also been employed and is called 2-D HPLC and multidimensional protein identification technology (MudPIT).43 This is a gel-free approach that utilizes multiple HPLC-MS analysis of in-solution digestions of protein fractions. The separated peptides are introduced into the mass spectrometer, and product ion spectra are obtained. The


product ions within the spectra are assigned to amino acid sequences. A complete coverage of the amino acid sequence within a peptide from the product ion spectrum is known as de novo sequencing. This can unambiguously identify a protein (except for a few anomalies that will be covered shortly) according to standard spectra stored in protein databases. Two examples of protein databases are NCBInr, a protein database composed of a combination of most public databases compiled by the National Center for Biotechnology Information (NCBI), and Swiss-Prot, a database that includes an extensive description of proteins including their functions, PTMs, and domain structures. The correlation of peptide product ion spectra with theoretical peptides was introduced by Yates et al. in 1994.44 At the same time, Mann et al.45 proposed a partial sequence error-tolerant database searching for protein identifications from peptide product ion spectra. There exists now a rather large choice of searching algorithms that are available for protein identifications from peptide product ion spectra. A list of identification algorithms and their associated uniform resource locators (URLs) is illustrated in Table 1.2. The final step in the proteomic analysis of a biological system is the interpretation of the identified proteins, which has been called bioinformatics. Bioinformatics attempts to map and decipher interrelationships between observed proteins and the genetic description. Valuable information can be obtained in this way concerning biomarkers for diseased states, the descriptive workings of a biological system, biological interactions, and so on. In the identification of proteins from peptide collision-induced dissociation using MS is performed to fragment the peptide and identify its amino acid residue sequence. In most mass spectrometers used in proteomic studies such as the ion trap, the quadrupole TOF, the triple quadrupole, and the Fourier transform ion cyclotron resonance (FTICR), the collision energy is considered low (5–50â•¯eV) and the product ions are generally formed through cleavages of the peptide bonds. According to the widely accepted nomenclature of Roepstorff and Fohlman,46 when the charge is retained on the N-terminal portion of the fragmented peptide, the ions are depicted as a, b, and c. When the charge is retained on the C-terminal portion, the ions are denoted as x, y, and z. The description of the dissociation associated with the peptide chain backbone and the nomenclature of the produced ions is illustrated in Figure 1.22. The ion subscript, for example, the “2” in y2, indicates the number of residues contained within the ion, two amino acid residues in this case. The weakest bond is between the carboxyl carbon and the nitrogen located directly to the left in the peptide chain. At low-energy collision-induced dissociation of the peptide in MS, the

TABLE 1.2.â•‡ List of Identification Algorithms

MS identification algorithms and URLs PMF â•… Aldente http://www.expasy.org/tools/aldente/ â•… Mascot http://www.matrixscience.com/search_form_select.html â•… MOWSE http://srs.hgmp.mrc.ac.uk/cgi-bin/mowse â•… MS-Fit http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm â•… PeptIdent http://www.expasy.org/tools/peptident.html â•… ProFound http://65.219.84.5/service/prowl/profound.html MS/MS identification algorithms and URLs PFF â•… Phenyx http://www.phenyx-ms.com/ â•… Sequest http://fields.scripps.edu/sequest/index.html â•… Mascot http://www.matrixscience.com/search_form_select.html â•… PepFrag http://prowl.rockefeller.edu/prowl/pepfragch.html â•… MS-Tag http://prospector.ucsf.edu/ucsfhtml4.0/mstagfd.htm â•… ProbID http://projects.systemsbiology.net/probid/ â•… Sonar http://65.219.84.5/service/prowl/sonar.html â•… TANDEM http://www.proteome.ca/opensource.html â•… SCOPE N/A â•… PEP_PROBE N/A â•… VEMS http://www.bio.aau.dk/en/biotechnology/vems.htm â•… PEDANTA N/A De novo sequencing â•… SeqMS http://www.protein.osaka-u.ac.jp/rcsfp/profiling/SeqMS.html â•… Lutefisk http://www.hairyfatguy.com/Lutefisk â•… Sherenga N/A â•… PEAKS http://www.bioinformaticssolutions.com/products/ peaksoverview.php Sequence similarity search â•… PeptideSearch http://www.narrador.embl-heidelberg.de/GroupPages/ Homepage.html â•… PepSea http://www.unb.br/cbsp/paginiciais/pepseaseqtag.htm â•… MS-Seq http://prospector.ucsf.edu/ucsfhtml4.0/msseq.htm â•… MS-Pattern http://prospector.ucsf.edu/ucsfhtml4.0/mspattern.htm â•… Mascot http://www.matrixscience.com/search_form_select.html â•… FASTS http://www.hgmp.mrc.ac.uk/Registered/Webapp/fasts/ â•… MS-Blast http://dove.embl-heidelberg.de/Blast2/msblast.html â•… OpenSea N/A â•… CIDentify http://ftp.virginia.edu/pub/fasta/CIDentify/ Congruence analysis â•… MS-Shotgun N/A â•… MultiTag N/A Tag approach â•… Popitam http://www.expasy.org/tools/popitam/ â•… GutenTag http://fields.scripps.edu/GutenTag/index.html Reprinted with permission of John Wiley & Sons, Hernandez, P., Muller, M., Appel, R.D. Automated protein identification by tandem mass spectrometry: Issues and strategies. Mass Spectrom. Rev. 2006, 25, 235–254.

32


primary breakage will take place at the weakest bond, generally along the peptide backbone chain, and produce a, b, and y fragments. Notice that the c ions and the y ions contain an extra proton that they have abstracted from the precursor peptide ion. There has also been a proposed third structure for the b ion that is formed as a protonated oxazolone, which is suggested to be more stable through cyclization47 (see b2 ion in Fig. 1.23). The stability of the y ion can be attributed to the N-terminus x3

y3

R1

O

C H

C

H 2N

a1

H 2N

z3

N H b1

R1

O

C H

C

x2

y2

R2

O

C H

C

c1

a2

z2

N H b2

x1 R3

O

C H

C

c2

a3

R2 N H

O

CH

C

y1

N H b3

O

C H

C

a2

H 2N

R1

O

C H

C

R4

O

C H

C

OH

c3

R3 N H

C-terminus

z1

R4

O

C H

C

N H

OH

x2 R2 N H

C H

C

O

H 3N

R3

O

C H

C

N H

R4

O

C H

C

OH

y2 R1

O

C H

C

R2 R3

H 2N

N H

C H

C

HC

b2

H 2N

R1

O

C H

C

O

R4

O

C H

C

O C

N H

OH

z2

N H

R2

O

C H

C

NH 3

c2 Figure 1.22.â•‡ Dissociation associated with the peptide chain backbone and the nomenclature of the produced ions. Charge retained on the N-terminal portion of the fragmented peptide the ions are depicted as a, b, and c. Charge retained on the C-terminal portion the ions are denoted as x, y, and z. Ion subscript, for example, “2” in y2, indicates the number of residues (two) contained within the ion.


H 3N

R1

O

C H

C

N H

R2

O

C H

C

N H

R3

O

C H

C

N H

R4

O

C H

C

OH

Mobile proton

H 2N

R1

O

C H

C

R2 N H

C H

C

H

R3

O

N H

C H

C

N H

R4

O

C H

C

OH

O

R1 CH

H 2N

O

H 2N

C

O

C H

C

N H

R4

O

C H

C

R4

O

C H

C

OH

O

C HN

R3

CH R2 O C

O R1

CH

C CH

R2

N

H 2N

H H 2N

R3

O

C H

C

N H

OH

O C

O R1

CH

C CH H 2N

N

R3

O

C H

C

R4

O

C H

C

R2 H 3N

N H

OH

H

b2 ion

y2 ion

Figure 1.23.â•‡ Fragmentation pathway leading to the production of the b and y ions from collision-induced dissociation from the polypeptide backbone chain.


transfer of the proton that is producing the charge state to the terminal nitrogen, thus inducing new bond formation and a lower energy state. The model that describes the dissociation of protonated peptides during low-energy collision-induced excitation is called the “mobile proton” model.48 Peptides fragment primarily from charge-directed reactions where protonation of the peptide can take place at side chain groups, amide oxygen and nitrogen, and at the terminal amino acid group. On the peptide chain backbone, protonation of the amide nitrogen will lead to a weakening of the amide bond inducing fragmentation at that point. However, it is more thermodynamically favored, as determined by molecular orbital calculations,48,49 for protonation to take place on the amide oxygen, which also has the effect of strengthening the amide bond. Inspection of peptide product ion fragmentation spectra has demonstrated though that the protonating of the amide nitrogen is taking place over the protonating of the amide oxygen. This is in contrast to the expected site of protonation from a thermodynamic point of view that indicates the amide oxygen protonation and not the amide nitrogen. This discrepancy has been explained by the “mobile proton model,” introduced by Wysocki et al.,48,50 which describes that the proton(s) added to a peptide, upon excitation from CID, will migrate to various protonation sites provided they are not sequestered by a basic amino acid side chain prior to fragmentation. The fragmentation pathway leading to the production of the b and y ions is illustrated in Figure 1.23. The protonation takes place first on the N-terminus of the peptide. The next step is the mobilization of the proton to the amide nitrogen of the peptide chain backbone where cleavage is to take place. The protonated oxazolone derivative is formed from nucleophilic attack by the oxygen of the adjacent amide bond on the carbon center of the protonated amide bond. Depending on the location of the retention of the charge, either a b ion or a y ion will be produced. Besides the amide bond cleavage producing the b and y ions that are observed in low-energy collision product ion spectra, there are also a number of other product ions that are quite useful in peptide sequence determination. Ions that have lost ammonia (−17â•¯Da) in low-energy collision product ion spectra are denoted as a*, b*, and y*. Ions that have lost water (−18â•¯Da) are denoted as ao, bo, and yo. The a ion illustrated in Figure 1.22 is produced through loss of CO from a b ion (−28â•¯Da). Upon careful inspection of the structures in Figure 1.22 for the product ions, it can be seen that the a ion is missing CO as compared with the structure of the b ion. When a difference of 28 is observed in product ion spectra between two m/z values, an a–b ion pair is suggested and can be useful in ion series identification. Internal cleavage


H2N

R2

O

C H

C

R3 N H

C H

C

Amino-acylium ion b-type and y-type cleavage

O

H2N

R2

O

C H

C

R3 N H

CH

Amino-immonium ion a-type and y-type cleavage

Figure 1.24.â•‡ Structure of (left) an amino-acylium ion produced through a combination of b- and y-type cleavage and (right) an amino-immonium ion through a combination of a- and y-type internal cleavage.

ions are produced by double backbone cleavage, usually by a combination of b- and y-type cleavage. When a combination of b- and y-type cleavage takes place, an amino-acylium ion is produced. When a combination of a- and y-type internal cleavage takes place, an aminoimmonium ion is produced. The structures of an amino-acylium ion and an amino-immonium ion are illustrated in Figure 1.24. These types of product ions that are produced from internal fragmentation are denoted with their one-letter amino acid code. Though not often observed, xtype ions can be produced using photodissociation. 1.5.3.3 High-Energy Product Ions.â•‡ Thus far, the product ions that have been discussed, the a-, b-, and y-type ions, are produced through low-energy collisions such as those observed in ion traps. The collisioninduced activation in ion traps is a slow heating mechanism, produced through multiple collisions with the trap bath gas, which favors lowerenergy fragmentation pathways. High-energy collisions that are in the kiloelectron volt range such as those produced in MALDI TOF-TOF MS produce other product ions in addition to the types that have been discussed so far. Side-chain cleavage ions that are produced by a combination of backbone cleavage and a side-chain bond are observed in high-energy collisions and are denoted as d, v, and w ions. Figure 1.25 contains some illustrative structures of d-, v-, and w-type ions. Immonium ions are produced through a combination of a-type and y-type cleavage that results in an internal fragment that contains a single side chain. These ions are designated by the one-letter code that corresponds to the amino acid. Immonium ions are not generally observed in ion trap product ion mass spectra but are in MALDI TOFTOF product ion mass spectra. The structure of a general immonium ion is illustrated in Figure 1.26. Immonium ions are useful in acting as confirmation of residues suspected to be contained within the peptide backbone. Table 1.3 is a compilation of the amino acid residue


H

H

R'

H2N

R1

O

C H

C

O

HC N H

HN

CH

C H

C

N H

R3

O

C H

C

OH

v2

d2

R'

H CH HC

O C

N H

R4

O

C H

C

OH

w2 Figure 1.25.â•‡ Structures of d-, v-, and w-type ions produced by a combination of backbone cleavage and a side chain bond observed in high-energy collision product ion spectra.

R H2N

CH

Figure 1.26.â•‡ Structure of a general immonium ion.

information that is used in MS analysis of peptides. The table includes the amino acid residue’s name, associated codes, residue mass, and immonium ion mass. 1.5.3.4 De Novo Sequencing.â•‡ An example of de novo sequencing is illustrated in Figure 1.27. The product ion spectrum in Figure 1.27a is for a peptide composed of seven amino acid residues. The peptide product ion spectrum in Figure 1.27b is also composed of seven amino acid residues; however, the Ser residue (Ser, C3H5NO2, 87.0320â•¯amu) in Figure 1.27a has been replaced by a Thr residue (Thr, C4H7NO2, 101.0477â•¯amu) in Figure 1.27b. The product ion spectra are very similar, but a difference can be discerned with the b5 ion and the y3 ions where a shift of 14â•¯Da is observed due to the difference in amino acid residue composition associated with Ser and Thr. Though the sequencing of the amino acids contained within a peptide chain can be discerned by de novo MS as just illustrated, there is a

38â•…â•… POSTTRANSLATIONAL MODIFICATION (PTM) OF PROTEINS TABLE 1.3.â•‡ Amino Acid Residue Names, Codes, Masses, and Immonium Ion m/z Values Residue

Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

One-Letter Code

Three-Letter Code

Residue Mass

Immonium Ion (m/z)

A R N D C E Q G H I L K M F P S T W Y V

Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

71.04 156.10 114.04 115.03 103.01 129.04 128.06 57.02 137.06 113.08 113.08 128.09 131.04 147.07 97.05 87.03 101.05 186.08 163.06 99.07

129 87.09 88.04 76 102.06 101.11 30 110.07 86.1 86.1 101.11 104.05 120.08 70.07 60.04 74.06 159.09 136.08 72.08

problem associated with isomers and isobars. Isomers are species that have the same molecular formula but differ in their structural arrangement, while isobars are species with different molecular formulas that possess similar (or the same) molecular weights. For example, it is not possible to determine whether a particular peptide contains Leu (Leu, C6H11NO, 113.0841â•¯amu) or its isomer Ile (Ile, C6H11NO, 113.0841â•¯amu) both at a residue mass of 113.0841â•¯amu. Furthermore, even though the remaining 18 amino acid residues each contain distinctive elemental compositions and thus distinct molecular masses, some combinations of residues will actually equate to identical elemental compositions. This produces an isobaric situation where different peptides will possess either very similar or identical sequence masses. If every single peptide amide bond cleavage is not represented within the product ion spectrum, then it is not possible to discern some of these possible combinations. The use of high-resolution/high-mass accuracy instrumentation such as the FTICR mass spectrometer or the Orbitrap can be used to

PROTEINS AND PROTEOMICSâ•…â•… 39 y6

y4

y3

y2

y1

Cys-Gln-Ile-Ala-Ser-Pro-Cys

(a)

b1

100

y5

Cys+Gln

b2

b3

b4

Ile

b5

b6

Ala

Ser

Pro

Cys

b2 b3

y2

%

y2

b5

b4 y2

b6

+

[M+H]

0 100

150

200

250

300

y6

450

400

y4

y3

y2

500

550

600

650

700 800

y1

Cys-Gln-Ile-Ala-Thr-Pro-Cys

(b)

b1 100

y5

350

Cys+Gln

b2

b3

b4

Ile

b5

Ala

b6

Thr

Pro

Cys

b2 b3

y2

%

y2

b5

b4

+

b6 [M+H]

y2 0 100

150

200

250

300

350

400

450

500

550

600

650

700 800

Figure 1.27.â•‡ Example of de novo sequencing using product ion spectra collected by collision-induced dissociation mass spectrometry. (a) Peptide composed of seven amino acid residues. (b) Peptide composed of seven amino acid residues with the serine residue (Ser, C3H5NO2, 87.0320â•¯amu) replaced by a threonine residue (Thr, C4H7NO2, 101.0477â•¯amu). The product ion spectra are very similar, but a difference can be discerned with the b5 ion and the y3 ions where a shift of 14â•¯Da is observed due to the difference in amino acid residue composition associated with serine and threonine.

help reduce this problem when complete de novo sequencing is not possible. Table 1.4 is a listing of some of the amino acid combinations that may arise that can contribute to unknown sequence determination when complete de novo sequencing is not being obtained. For a peptide with a mass of 800â•¯Da, the differences in the table for, for example, the Gln versus Lys difference at 0.03638â•¯Da would take a mass accuracy of better than 44â•¯ppm to distinguish the two. For the Arg versus Glyâ•¯+â•¯Val at 0.01124â•¯Da, it would require a mass accuracy of 14â•¯ppm to distinguish the two. For the FTICR mass spectrometers and the hybrid mass spectrometers such as the linear ion trap-Fourier transform (LTQ-FT)

40â•…â•… POSTTRANSLATIONAL MODIFICATION (PTM) OF PROTEINS TABLE 1.4.â•‡ Examples of Combinations of Amino Acid Residues Where Isobaric Peptides Can Be Observed Amino Acid Residue

Leucine Isoleucine Glutamine Glycineâ•¯+â•¯alanine Asparagine 2â•¯×â•¯glycine Oxidized methionine Phenylalanine Glutamine Lysine Arginine Glycineâ•¯+â•¯valine Asparagine Ornithine Leucine/isoleucine Hydroxyproline 2â•¯×â•¯valine Prolineâ•¯+â•¯threonine

Residue Mass (Da)

113.08406 113.08406 128.05858 128.05858 (57.02146â•¯+â•¯71.03711) 114.04293 114.04293 (2â•¯×â•¯57.02146) 147.03540 147.06841 128.05858 128.09496 156.10111 156.08987 (57.02146â•¯+â•¯99.06841) 114.04293 114.07931 113.08406 113.04768 198.13682 (2â•¯×â•¯99.06841) 198.10044 (97.05276â•¯+â•¯101.04768)

Δ Mass (Da)

0 0 0 0.03301 0.03638 0.01124 0.03638 0.03638 0.03638

or the LTQ-Orbitrap, this is readily achievable, but often, for ion traps this is not always achievable. 1.5.3.5 Electron Capture Dissociation (ECD).â•‡ Other techniques such as ECD51 and electron transfer dissociation (ETD) have also been used to alleviate the problem of isobaric amino acid combinations by giving complimentary product ions (such as c and z ions) that help to obtain complete sequence coverage. The technique of ECD tends to promote extensive fragmentation along the polypeptide backbone, producing c- and z-type ions while also preserving modifications such as glycosylation and phosphorylation. The general z-type ion that is shown in Figure 1.22 is different though from the z-type ion that is produced in ECD, which is a radical cation. Peptide cation-radicals are produced by passing or exposing the peptides, which are already multiplyprotonated by ESI through low-energy electrons. The mixing of the protonated peptides with the low-energy electrons will result in exothermic ion–electron recombinations. There are a number of dissociations that can take place after the initial peptide cation-radical is formed. These include loss of ammonia, loss of H atoms, loss of side chain fragments, cleavage of disulfide bonds, and most importantly, peptide backbone cleavages. The c-type ion is produced through homolytic cleavage


at the N–C peptide bond, and charges are present in the amino-terminal fragment. The z-type ion is produced when charges are present in the carboxy-terminal fragment. The mechanism that has been given for the promotion of fragmentation of the peptides is due to electron attachment to the protonated sites of the peptide. The now cation radical intermediate that has formed will release a hydrogen atom. A nearby carbonyl group will capture the released hydrogen atom and the peptide will dissociate by cleavage of the adjacent N–C peptide bond. The mechanism for the production of an α-amide radical of the peptide C-terminus, a z-type ion, and the enolamine of the N-terminus portion of the peptide, a c-type ion, is illustrated in Figure 1.28.

H

H

H3N

R1

O

C H

C

N H

R2

O

C H

C

R3

O

C H

C

N H

Electron capture

N H

R4

O

C H

C

e H

H

H3N

R1

O

C H

C

N H

R2

O

C H

C

.

N H

R3

O

C H

C

N H

R4

O

C H

C

H

H

H3N

O

C H

C

N H

R2

O

C H

C H

.

OH

Loss and recapture

H transfer

R1

OH

N H

R3

O

C H

C

N H

R4

O

C H

C

H

H R1 H3N

C H

O C

R2 N H

C H

c-Type ion

O C

OH

R3 NH

+

HC

.

O C

N H

R4

O

C H

C

OH

z-Type ion

Figure 1.28.â•‡ Mechanism for the production of c- and z-type ions observed in ECD.


1.5.4 Top-Down Proteomics: MS of Intact Proteins 1.5.4.1 Background.â•‡ Measuring the whole, intact protein in the GP using mass spectrometric methodologies is known as “top-down” proteomics. Top-down proteomics measures the intact protein’s mass followed by collision-induced dissociation of the whole protein, breaking it into smaller parts. A vital component of top-down proteomics is the accuracy in which the masses are measured. Often, high-resolution mass spectrometers such as the FTICR mass spectrometer are used to accurately measure the intact protein’s mass and the product ions produced during collision-induced dissociation experiments. In early topdown experiments, though, this was not the case. Mass spectrometers such as the triple quadrupole coupled with electrospray were first used to measure intact proteins in the GP.52,53 However, the triple quadrupole mass spectrometer does not allow the resolving of the isotopic distribution of the product ions being generated in the top-down approach. The use of FT-MS/MS was later reported with high enough resolution to resolve isotopic peaks.54,55 An example of these early topdown experiments utilizing FT-MS is illustrated in Figure 1.29. Extensive initial, premass analysis sample preparation, such as cleanup, digestion, desalting, and enriching, all often incorporated in “bottomup” proteomics, is not necessarily required in top-down approaches. The dynamic range in top-down proteomics can be limited by the number of analytes that can be present during analysis, but this is usually overcome by using some type of separation prior to introduction into the mass spectrometer. The separation of complex protein mixtures can be obtained using techniques such as RP-HPLC, gel electrophoresis, anion exchange chromatography, and capillary electrophoresis. Typically in bottom-up analysis, the digested protein peptides are 2000 genes).1 In eukaryotic cells, the protein phosphorylation takes place with the serine (Ser), threonine (Thr), and tyrosine (Tyr) residues. The reversible protein phosphorylation of the Ser, Thr, and Tyr residues is an integral part of cellular processes involving signal transduction.2,3 The identification of the phosphorylation sites is important in understanding cellular signal transduction. 4.1.2 Protein Phosphatase and Kinase Protein phosphatases are enzymes responsible for the removal of phosphate groups from a target (i.e., reversible protein

Proteomics of Biological Systems: Protein Phosphorylation Using Mass Spectrometry Techniques, First Edition. Bryan M. Ham. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc. 99

100â•…â•… EUKARYOTE PTM AS PHOSPHORYLATION: NORMAL STATE STUDIES

phosphorylation), and protein kinases are enzymes responsible for the addition of phosphate groups to a target. These two enzymes work together to control cellular processes and signaling pathways. Greater attention has been given in the literature to the study of signaling pathways primarily involved with protein kinase as compared with specific types of phosphatase.4–9 However, the importance of studying protein phosphatase enzymes and their targets has been demonstrated in recently reported disease state studies where the abnormal condition has been attributed at least in part to malfunctioning protein phosphatase enzymes.10–12 In covalent modification of proteins such as phosphorylation, the activity of the modified enzyme has been altered in the form of activated, inactivated, or to otherwise regulate its activity upwardly or downwardly. The most common mechanism for phosphorylation is the transfer of a phosphate group from adenosine triphosphate (ATP) to the hydroxyl group of Ser, Thr, or Tyr within the protein. Figure 4.1 illustrates the general cellular mechanism involving protein kinase phosphorylation and protein phosphatase dephosphorylation. In the top portion of Figure 4.1, a cellular signal is received in a kinase/phosphatase cycle, often in the form of a messenger biomolecule such as lipid (diacylglycerol shown), which initiates the phosphorylation of the target protein by the protein kinase–ATP action. The enzyme has been phosphorylated resulting in its participation in the signaling cycle. Usually, the phosphorylated enzyme does not permanently stay in its modified form but will undergo dephosphorylation through interaction with protein phosphatase. Figure 4.2 illustrates the nonphosphorylated structure of the Ser, Thr, and Tyr residues, each having a hydroxyl moiety and the phosphorylated form of the amino acid residue. 4.1.3 Hydroxy-Amino Acid Phosphorylation While the Ser, Thr, and Tyr residues all have a side group hydroxyl moiety available for PTM, over 99% of the phosphorylated modification takes place with the Ser and Thr amino acid residues in eukaryotic cells.13 Recently, Olsen et al.3 have reported a slight variation to the widely referred study of Hunter and Sefton,14 where the relative abundances of amino acid residue phosphorylation were assigned as 0.05% for phosphotyrosine (pY), 10% for phosphothreonine (pT), and 90% for phosphoserine (pS). In Olsen’s recent study, this has been adjusted to 1.8% pY, 11.8% pT, and 86.4% pS where the larger percentage value allocated to pY was attributed to more sensitive methodology being employed, thus allowing the characterization of lower

MASS SPECTRAL MEASUREMENT WITH EXAMPLES â•…â•… 101 O H2 C HO

C CH O

H2C C

Incoming signal from messenger

Inactive form of enzyme OH O

P

N H2N

R

OH

O

N

O

O

P

N

O

O

N

HO

OH HO

OH

OH O

P

O

P O

OH

Adenosine triphosphate

ON

OFF

Protein kinase N H2N

O

N

Protein phosphatase

OH P

N

O

O

N

O

OH O

P OH

HO

OH

Adenosine diphosphate Active form of enzyme

O HO

OH

P O

Signal out

Figure 4.1.â•‡ Example of the general cellular mechanism involving protein kinase phosphorylation and protein phosphatase dephosphorylation. (Top) A cellular signal is received in the form of the messenger biomolecule diacylglycerol lipid that initiates the phosphorylation of the target protein by the protein kinase–ATP action. The phosphorylated enzyme propagates the signaling cycle and is then recycled through dephosphorylation by a phosphatase.

abundant phosphorylated proteins. However, the stoichiometry of the phosphorylated proteome (in the form of tryptic peptides) is small (≤1) in relation to the nonphosphorylated proteome (in the form of tryptic peptides), thus requiring that the sensitivity of the phosphorylated proteome analysis to be as optimal as possible.

102â•…â•… EUKARYOTE PTM AS PHOSPHORYLATION: NORMAL STATE STUDIES O H2N

CH

C

OH

O

O

CH2 H2N

H2N

CH

C

CH2

CH

OH

OH

CH3

CH

C

OH

OH

Threonine (Thr)

Serine (Ser)

OH

Tyrosine (Tyr) O O

O H2N

CH

C

CH2

OH

H2N

CH

H3C

CH

P

OH

CH

C

OH

CH2

O

O O

C

H2N

O

O

Phosphorylated serine (pS)

O

P

O

O

Phosphorylated threonine (pT)

O O

P

O

O

Phosphorylated tyrosine (pY) Figure 4.2.â•‡ Structures of the nonphosphorylated (top) and phosphorylated (bottom) states of the amino acids serine, threonine, and tyrosine.

4.1.4 Traditional Phosphoproteomic Approaches Mass spectrometry (MS) is often used to identify the sites of phosphorylation in the protein backbone when studying cellular signaling pathways.15–18 Prior to the introduction of mass spectrometric methodology for phosphorylated protein analysis, researchers studied protein phosphorylation using 32P labeling followed by two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) and finally, Edman

MASS SPECTRAL MEASUREMENT WITH EXAMPLES â•…â•… 103

sequencing. This methodology was time-consuming and involved the handling of radioactive isotopes. This has prompted a considerable amount of methodology development using mass spectrometric techniques that are able to measure whole proteomes from complex biological systems and not just single proteins that the Edman sequencing approach is most suited for. There are numerous studies reported in the literature of signaling pathways primarily involved with protein kinase.12,19–23 The study of protein phosphatase enzymes and their targets has also gained importance where there are recently reported numerous disease states that have been attributed at least in part to malfunctioning protein phosphatase enzymes.5,24–26 4.1.5 Current Approaches The mass spectrometric instrumentation in use today possesses the sensitivity needed for PTM analysis; therefore, often, the limiting factor in phosphorylated proteome analyses lies in the sample treatment prior to mass spectral measurement. Immobilized metal affinity chromatography (IMAC) is the methodology of choice for phosphorylated proteome cleanup and enrichment.27–29 Labeling is also the most widely used approach for relative quantitation of the phosphorylated proteome when comparing a normal state with a perturbed state. Labeling typically involves using stable isotopes with either 16O/18O, 2D-methanol or stable isrotope labeling by amino acids in cell culture (13C-SILAC), or isobaric peptide tags for relative and absolute quantification (iTRAQ) reagent.30–32 However, often, labeling procedures can cause an increase in sample complexity, can be cumbersome or incomplete, and can ultimately result in sample losses. 4.1.5.1 Phosphoproteomic Enrichment Techniques.â•‡ The two phosphoproteome enrichment methods of choice in proteomics laboratories are IMAC and metal oxide affinity chromatography (MOAC). These two approaches are based on capturing and enriching phosphorylated species on the peptide level after the proteins have been digested by an enzyme such as trypsin. There are a number of approaches that are based on the capture and enrichment of the phosphoproteome on the protein level such as the Pierce Phosphoprotein Enrichment Kit that uses affinity spin columns33 but typically is not as commonly used when nano-liquid chromatography (LC) nano-electrospray ionization (ESI) MS is the instrumental analyses performed. A second protein level approach is to perform immunoprecipitation of Tyr-phosphorylated proteins, but this is a highly specific methodology for Tyr phosphorylation studies and not the entire phosphoproteome.34,35

104â•…â•… EUKARYOTE PTM AS PHOSPHORYLATION: NORMAL STATE STUDIES IDA-Fe(III)-phospho complex O P

protein digest

phosphopeptide

O C

O

O

Fe N O

O Ti

IMAC

rinse

O

O

C O C O

load

O

O

P

O

O Ti

MOAC

elute

IMAC or TiO2 resin nonphosphopeptide

digest reagent

enriched phosphopeptides

Figure 4.3.â•‡ Schematic diagram of phosphopeptide isolation by immobilized metal affinity chromatography (IMAC) through binding of the phosphate group with a metal–ligand complex such as the iron(III)-iminodiacetate (IDA) or iron(III)nitrilotriacetate (NTA) complexes where the Fe(III)-IDA forms a tridentate complex and the Fe(III)-NTA forms a tetradentate complex. (Reprinted with permission from Dunn, J.D., Reid, G.E., Bruening, M.L., Mass Spectrometry Reviews 2010, 29, 29–54. Copyright 2009 by Wiley Periodicals, Inc.)

4.1.5.2 IMAC.â•‡ IMAC is able to enrich phosphorylated peptides utilizing a binding of the phosphate group with a metal–ligand complex such as the iron(III)-iminodiacetate (IDA) or iron(III)-nitrilotriacetate (NTA) complexes. These complexes are illustrated in Figure 4.3 where the Fe(III)-IDA forms a tridentate complex and the Fe(III)-NTA forms a tetradentate complex. The middle column in Figure 4.3 illustrates the IMAC binding complex. A typical IMAC enrichment protocol entails washing of the IMAC column bed with organic solvents (to remove organics), water (to remove metals and water soluble compounds), and ethylenediaminetetraacetic acid (EDTA) to strip the column of all metals and remove them, activation of the column bed with Fe(III), loading of the sample, acidic washing, and a final basic elution of the


phosphorylated peptides. To stabilize the peptide phosphate moieties and to prepare them for carbon 18 (C18) reversed-phase (RP) LC, the eluant is usually acidified to approximately pH 4 with formic, acetic, or trifluoracetic acid (TFA). A schematic diagram of an IMAC column and the ensuing enrichment steps is illustrated in Figure 4.3. A detailed step-by-step optimized protocol for IMAC phosphopeptide enrichment can be found in Section 4.2.4.9. A recent optimization step added to the IMAC phosphopeptide enrichment methodology includes the conversion of the acidic peptide residues aspartic acid (Asp) and glutamic acid (Glu) to methyl esters. This is performed in order to decrease the amount of nonspecific binding taking place between nonphosphorylated peptides containing Asp or Glu residues. Nonspecific binding can compete for the binding sites in the IMAC column bed and thus decrease the enrichment of the phosphorylated peptides and can also contribute to the presence of nonphosphorylated peptides in the final analysis of the enriched fraction. Removing nonspecific binding peptides from the enrichment fraction is thought to decrease ionization suppression of the phosphorylated peptides by nonphosphorylated peptides that are coeluting from the high-performance liquid chromatography (HPLC) column. The most common reagents used for methylating the acidic residues are acetyl chloride and/or thionyl chloride in methanol (methanolic HCl). Later in the chapter, in Section 4.3, we will look at an IMAC column bed study where the normal nonphosphorylated proteome of a HeLa cell protein extract is measured by collecting the flow through and NaCl salt wash of the IMAC column. Included in the study is a description of the influence of peptide molecular weight and charge state (CS) that appears to be dictating the functionality of the nonspecific binding of peptides not containing a phosphoryl group. 4.1.5.3 MOAC.â•‡ MOAC has recently found greater use in a number of proteomic laboratories that have incorporated different metal oxides including titanium dioxide (TiO2), aluminum oxide (Al2O3), niobium oxide (Nb2O5), and zirconium dioxide (ZrO2). The MOAC approaches have shown to be stable at both large pH ranges and elevated temperatures. It was first thought that methyl esterification was not necessary for MOAC with good recoveries and low nonspecific binding, but some studies have indicated otherwise. For example, in a study by Pinkse et al.36 using TiO2 MOAC, it was demonstrated that the nonmethylated form of a synthetic peptide containing a phosphorylation was recovered at 90% in the presence of its nonphosphorylated form. However, a

106â•…â•… EUKARYOTE PTM AS PHOSPHORYLATION: NORMAL STATE STUDIES TiO2-phospho complex O P

protein digest

phosphopeptide

O C

O

O

Fe N O

O Ti

IMAC

rinse

O

O

C O C O

load

O

O

P

O

O Ti

MOAC

elute

IMAC or TiO2 resin nonphosphopeptide

digest reagent

enriched phosphopeptides

Figure 4.4.â•‡ Schematic diagram of phosphopeptide isolation by metal oxide affinity chromatography (MOAC). Titanium dioxide has a positively charged surface at acidic pH that has excellent selectivity in absorbing and enriching phosphorylated peptides. A bridging bidentate complex is formed between the phosphate group and the TiO2. (Reprinted with permission from Dunn, J.D., Reid, G.E., Bruening, M.L., Mass Spectrometry Reviews 2010, 29, 29–54. Copyright 2009 by Wiley Periodicals, Inc.)

simple tryptic digestion mixture from cyclic guanosine monophosphate (cGMP)-dependent protein kinase resulted in 11 phosphorylated peptides recovered along with a number of nonphosphorylated peptides. They determined that 98% of the nonmethylated form of the [Glu1]fibrinopeptide B was retained where the methylated form was not. For phosphopeptide enrichment, TiO2 has turned out to be the most widely used metal oxide. Titanium dioxide has a positively charged surface at acidic pH that has excellent selectivity in absorbing and enriching phosphorylated peptides. A bridging bidentate complex is formed between the phosphate group and the TiO2 as illustrated in Figure 4.4. The right end column in Figure 4.4 illustrates the MOAC binding complex. Titania has a pKa value of 4.4, and a pKb value of 7.7. Thus, at low pH values, the titania is positively charged, and at high pH,


the titania is negatively charged. At a pH of ∼10 of an NH4OH eluent, the phosphorylated peptides dissociate from the TiO2 bed. As a reference, the first and second pKa values of pS and pT are ∼1.7 and 6. The typical TiO2 MOAC loading solutions are composed of 0.1% TFA with a pH of 1.9 and 0.1â•¯M acetic acid that has a pH of 2.7. These pH values are low enough to effectively protonate the acidic amino acid residues, thus preventing adsorption of nonphosphorylated peptides to the TiO2. Typically, in TiO2 MOAC, the peptides are loaded and washed under acidic conditions and then eluted under basic conditions. 4.1.5.4 Methylation of Peptides prior to IMAC or MOAC Enrichment.â•‡ Thionyl chloride (Sigma-Aldrich St. Louis, MO) was used for methyl esterification of the peptides.37 The steps involved in methylating the peptides are as follows: 1. Only use dry peptide material that has been previously extensively dried in a SpeedVac (Eppendorf, Hamburg, Germany). 2. Carefully add dropwise 40â•¯µL of thionyl chloride to 1â•¯mL methanol (both anhydrous). 3. Add thionyl chloride/methanol mixture to dry peptide at a ratio of 75â•¯µL thionyl chloride/methanol solution per 100â•¯µg peptide. 4. Vortex the reaction mixture for 5–10 minutes to ensure dissolution of the dry peptide material. 5. Sonicate the reaction mixture for 10 minutes. 6. Let the mixture react at room temperature for 1 hour. 7. Bring the methylated peptide to dryness in a SpeedVac (Eppendorf) and store at −80°C until further processed. For the methylated form of peptide, 730â•¯µL of a solution composed of 40â•¯µL of thionyl chloride in 1â•¯mL anhydrous methanol was added to 1â•¯mg of peptide. The mixture was sonicated for 10 minutes at 37°C for 10 minutes and allowed to react at room temperature for 1 hour. The solvent was removed in the SpeedVac and the peptide was reconstituted in IMAC loading solutions that were composed of 1:1:1 methanol/ acetonitrile/0.01% acetic acid at a ratio of 100â•¯µL solution to 100– 200â•¯µg peptides. 4.1.6 The Ideal Approach The ideal methodology for PTM studies would entail both high sample recovery and sample specificity while avoiding any additional


modifications to the proteins being studied. The first critical step in sample preparation is the lysis and solubilization of the sample’s compliment of proteins. This is followed by additional steps for sample cleanup and treatment that is often accompanied by protein/peptide loss at each step. Another limiting factor is the presence of nucleic acids during the IMAC enrichment step, which are known to poison or compete with the phosphorylated peptides for binding sites on the IMAC bed. Alternative approaches exist for the removal of ribonucleic acid (RNA) and DNA such as the addition of RNase and DNase to the whole cell lysate buffer,38 passing the lysate repeatedly through a tuberculin syringe fitted with a 21-gauge needle to shear the RNA and DNA mechanically,39 or using the QIAShredder (QIAGEN Inc., Valencia, CA).40 The use of RNase and DNase is often avoided in an effort to reduce the amount of additives and the production of low-molecularweight nucleic acid lysed products, which are difficult to remove and also poison the IMAC bedding. The choice of ultracentrifugation can be made over mechanical shearing for similar reasons that are associated with the production of low-molecular-weight nucleic acid species. 4.1.7 One-Dimensional (1-D) Sodium Dodecyl Sulfate (SDS) PAGE The use of 1-D SDS-PAGE is another alternative approach for whole cell lysate intact protein cleanup that includes removal of the nucleic acids. Current examples include a study by Gygi et al.5 who used preparatory gels for HeLa cell nuclear protein separation followed by whole gel digestion (cut into 10 regions) where 967 proteins revealed 2002 phosphorylation sites. A recent study by Mann et al.41 reported that five times more proteins from tear fluid were identified after gel electrophoresis as compared with in-solution digest. Other potential advantages for the use of 1-D SDS-PAGE gel electrophoresis is the ability to target specific molecular weight ranges that can be identified and excised from various band regions within the gels and a more efficient tryptic digest due to the enhanced accessibility of the protein backbone denatured into a linear orientation locked within the gel. Recoveries from gels can be an added concern though as all steps in protein/peptide preparation can contribute to an overall loss of protein. 4.1.8 Tandem MS Approach Mass spectrometric studies of phosphorylation PTMs typically employ tandem MS to generate product ion spectra of phosphorylated


peptides. This usually entails a premass spectrometric separation of a complex mixture of phosphorylated peptides by RP C18 stationary phase HPLC with an ESI source (RP C18 HPLC ESI-MS/MS). Fragmentation pathway studies using ion trap MS of the phosphorylation of the three possible sites in peptides, Ser, Thr, and Tyr, have been studied and characterized, revealing different mechanisms. 4.1.8.1 pS Loss of Phosphate Group.â•‡ Peptides that contain pS generally will lose the phosphate group as the predominant product ion in the spectrum. This often results in limited information about the sequence of the peptide where other peptide backbone fragmentation is not well observed. Neutral loss of the phosphate group for the plus one charge state (+1 CS) is observed at 98.0â•¯Da, [Mâ•¯+â•¯Hâ•¯−â•¯H3PO4]+; for the plus two CS (+2 CS), neutral loss of the phosphate group is observed at 49.0â•¯Da, [Mâ•¯+â•¯2Hâ•¯−â•¯H3PO4]2+; for the plus 3 CS (+3 CS), neutral loss of the phosphate group is observed at 32.7â•¯Da, [Mâ•¯+â•¯3Hâ•¯−â•¯H3PO4]3+. 4.1.8.1.1â•… pS Collision-Induced Dissociation (CID) Spectra.â•‡ Figure 4.5 illustrates product ion spectra for a pS-containing peptide at m/z 987.5 (monoisotopic mass of 1972.98â•¯Da). The spectrum in Figure 4.5a is the MS/MS spectrum of the doubly charged phosphorylated peptide, [Mâ•¯+â•¯2H]2+, at m/z 987.5 where the predominant product ion is observed at m/z 938.7 for the neutral loss of the phosphate group for the doubly charged precursor (−49.0â•¯Da), [Mâ•¯+â•¯2Hâ•¯−â•¯H3PO4]2+. There is coverage in the spectrum of the peptide backbone sequence as illustrated by the b- and y-type ions; however, their response is quite low and often are not observed in product ion spectra of phosphorylated peptides when using tandem ion trap MS. The loss of the phosphate modification from the peptide is the preferred fragmentation pathway and is usually the predominant one observed. The phosphorylated peptide’s sequence is shown in the spectrum as RApSVVGTTYWMAPEVVK, where the phosphorylation is on the Ser amino acid residue that is third from the left. In the collision-induced fragmentation of the phosphorylated peptide, all of the y-type ions observed in Figure 4.5a (y5â•¯−â•¯y14) do not contain the phospho group. Only one of the b-type ions contains the phospho group at b12. This is due to a preferential cleavage taking place on the peptide backbone at the proline residue, while all of the other b-type ions include neutral loss of the phospho group from the Ser residue and are denoted as bn∆ , which equals bnâ•¯−â•¯H3PO4. The loss of the phosphorylation that is associated with Ser is through a β (beta)-elimination mechanism producing dehydroalanine. The mechanism for dehydroalanine production through β-elimination is

Relative Abundance

(a) 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

+2 –H3PO4 938.7

y

Relative Abundance

9

7 6 5

RApS VVGTTYWMAPE VVK

b

4 5

10 1112 14 15 16

b∆n = bn-H3PO4

y12 1381.6 y5 ∆ 571.4 y6 396.3 b 5 642.5 495.3 b∆4

400

500

600

773.5

700

800

b∆14 1531.7

y9 b∆12 b 12 ∆ 1122.5 b∆111305.5 1403.4y13 y14 ∆ b∆16 b 15 b 10 1234.6 1480.6 1579.6 1103.3 1630.7 1729.6

y7

900

1000 1100 1200 1300 1400 1500 1600 1700 1800

m/z

(b) 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

1413 12

+2 –H2O

b12 1305.6

930.0

y 15 14 1312

98

7 6 5

3

RABVVGTTYWMA PEVVK

b

4 5 67

9 10 1112 141516

B = dehydroalanine y5 571.3

y7

y6 b4 y3 397.0 b6 345.3 b5552.3

642.4

b7

773.5

b11 1234.4

b9

917.4

653.3

y8 959.4

b10

y9 1122.5

1103.6

495.3

400

500

600

700

800

900

b14

b15 1630.6

1531.6

y12 y y14 13 1381.4 1480.6 1579.5

b16 1729.7 y15 1648.8

1000 1100 1200 1300 1400 1500 1600 1700 1800

m/z Figure 4.5.â•‡ Phosphoserine loses predominantly H3PO4 through β-elimination to produce dehydroalanine in tandem ion trap mass spectrometry. (a) MS2 spectrum of a doubly charged phosphopeptide ion (m/z 987.5). A loss of 98â•¯Da (H3PO4) is observed. The bn∆ label denotes a loss of 98â•¯Da (i.e., bn-H3PO4). (b) MS3 spectrum of the ion arising from loss of 98â•¯Da (m/z 938.7 of the doubly charged ion in [a]). The y14 and y15 fragments have a mass difference of 69â•¯Da that corresponds to the mass of dehydroalanine, identifying the product of phosphoserine after losing 98â•¯Da as dehydroalanine. The “B” label denotes the dehydroalanine residue. (Reprinted with permission. This article was published in J Am Soc Mass Spectrom, DeGnore, J.P., Qin, J. Fragmentation of phosphopeptides in an ion trap mass spectrometer, 1998, 9, 1175–1188. Copyright Elsevier 1998.) 110


R

N H

H

O

C

C

CH2

P

R1

OH

O R

O O

O

N H

C

C

O

CH2 OH

Dehydroalanine

R1

+

O

P

OH

OH

Phosphate

OH

Phosphoserine β-elimination Figure 4.6.â•‡ β-Elimination mechanism for the fragmentation pathway producing dehydroalanine for the phosphorylated serine amino acid residue.

illustrated in Figure 4.6 for pS. This would represent the structure of the Ser in the peptide that is represented by the m/z 938.7 product ion in Figure 4.5, [Mâ•¯+â•¯2Hâ•¯−â•¯H3PO4]2+. Figure 4.5b is an example of the application of MS3 product ion spectral collection for the enhanced fragmentation of phosphorylated peptides. In this approach, the main product ion collected from MS2, m/z 938.7, is isolated and subjected to a third stage of fragmentation. In the MS3 product ion spectrum of Figure 4.5, there is a predominant peak for water loss from the m/z 938.7 precursor peak. This also does not afford much information for the sequence of the peptide; however, there are also observed in the spectrum numerous product ion peaks of the b and y types that have a much greater response than that observed in the MS2 product ion spectrum of Figure 4.5a. Notice that in the fragmentation of the phosphorylated peptide in Figure 4.5a, product ions derived through cleavage from both sides directly adjacent to the Ser residue did not take place. This means that a positive identification of the residue that contains the phosphate modification, namely the Ser residue, is not possible. Notice in the third-stage product ion spectrum of Figure 4.5b that fragmentation on both sides of the Ser residue did in fact take place. Because the precursor ion that was subjected to CID in the third-stage product ion spectrum of Figure 4.5b was the m/z 938.7 product ion from the second-stage fragmentation in Figure 4.5a, the Ser residue has been replaced in the sequence by a “B” ion, which stands for dehydroalanine. Due to the absence of the phosphate modification on the peptide chain, there is observed substantial sequence coverage in the product ions observed in Figure 4.5b, thus allowing specific identification of the peptide sequence and the location of the phosphorylation. Further studies concerning phosphorylated Ser


(pS) have demonstrated that loss of the phosphate H3PO4 group in product ion spectra is not dependent on the CS of the precursor ion. All three commonly observed CSs, +1, +2, and +3 for ESI CID product ion spectral collection resulted in the loss of the phosphate group as the predominant product ion peak with other minor losses as shown in Figure 4.5a. 4.1.8.2 pT Loss of Phosphate Group.â•‡ When the phosphorylation modification of a peptide takes place on the Thr amino acid residue, the predominant product ion spectral peak derived from CID is also observed to be through β-elimination of the phosphate group. The neutral loss of the phosphate group, −98â•¯ Da as [Mâ•¯+â•¯Hâ•¯−â•¯H3PO4]+, from the Thr residue produces dehydroaminobutyric acid. A product ion spectrum illustrating the major species produced from a peptide containing a phosphorylated Thr is illustrated in Figure 4.7. The sequence of the phosphorylated peptide illustrated in Figure 4.7 is RASVVGTpTYWMAPEVVK, which has a monoisotopic mass of 1972.98â•¯Da. The phosphorylation of the peptide has taken place on the Thr amino acid residue (pT). The CS of the peptide in the product ion spectrum of Figure 4.7 is +2 at m/z 987 as [Mâ•¯ +â•¯ 2H]2+. Loss of

Relative Abundance

100 90 80

MS2

939.0

×5

b5 513.5

y5

571.5

400

b∆8

y7

754.5 773.5

y6 642.5

600

b∆9 917.1

b9 1015.3

b∆10

b8 852.3

800

b

5

8 9 1011 12 1415 17

1122.3

b∆12 1305.5

b10

b12 1403.3

b11 1332.4

1201.4

1200

b15

b∆14 1531.5

1103.5

1000

7 6 5

RASWGTpT YWMAPEVVK

978.4

947.5

9

y

b17+2 +2 y –HPO3 9

70 60 50 40 30 20 10 0

+2 –H3PO4

1400

b14

1728.5

1629.5

1600

1800

m/z Figure 4.7.â•‡ Product ion spectrum of phosphorylated threonine illustrating the major species produced. The sequence of the phosphorylated peptide is RASVVGTpTYWMAPEVVK (monoisotopic mass of 1972.98â•¯Da). Charge state of the peptide is +2 at m/z 987 as [Mâ•¯+â•¯2H]2+. Loss of the H3PO4 phosphate group is the primary fragmentation pathway at m/z 939.0 as [Mâ•¯+â•¯2Hâ•¯−â•¯H3PO4]2+. (Reprinted with permission. This article was published in J Am Soc Mass Spectrom, DeGnore, J.P., Qin, J. Fragmentation of phosphopeptides in an ion trap mass spectrometer, 1998, 9, 1175–1188. Copyright Elsevier 1998.)

MASS SPECTRAL MEASUREMENT WITH EXAMPLES â•…â•… 113 H

O

H2N

C

C

H3C

CH

OH O H2N

C

H3C

CH

C

OH OH

+

O

P

OH

O O

P

OH

OH

Dehydoaminobutyric acid

Phosphate

OH

Phosphorylated threonine (pT) β-elimination Figure 4.8.â•‡ Mechanism for the production of the dehydroaminobutyric acid from phosphorylated threonine through neutral loss, β-elimination of the phosphate group.

the H3PO4 phosphate group is the primary fragmentation pathway that is observed in the product ion spectrum at m/z 939.0 as [Mâ•¯ +â•¯2Hâ•¯−â•¯ H3PO4]2+. In the product ions produced from CID of the m/z 987 species, there are a number of b-type ions that both contain the phosphate modification (b8, b9, b10, b11, b12, b14, b15) and that do not ∆ ∆ ∆ , b12 , b14 ). Figure 4.8 illustrates the fragmentation pathway (b8∆ , b9∆, b10 mechanism for the production of the dehydroaminobutyric acid from the neutral loss, β-elimination of the phosphate group from phosphorylated Thr amino acid. One difference in the product ion spectrum for fragmentation of phosphorylated Thr as compared with phosphorylated Ser, Figure 4.5a, is the product ion peak observed at m/z 947.5 for the neutral loss of 80â•¯ Da. The product ion at m/z 947.5 represents dephosphorylation through neutral loss of HPO3 from the precursor peak as [Mâ•¯+â•¯2Hâ•¯ −â•¯HPO3]2+. This particular loss is not observed in the product ion spectrum of phosphorylated Ser. The mechanism for dephosphorylation of the Thr amino acid is illustrated in Figure 4.9. The dephosphorylation results in the structure of the original amino acid residue of Thr. 4.1.8.3 pY Loss of Phosphate Group.â•‡ Product ion spectra of phosphorylated Tyr (pY) containing peptides also illustrate losses associated with 80 and 98â•¯Da, which would appear to be similar to the losses observed with phosphorylated Thr peptides. The 80-Da loss is due to dephosphorylation of the Tyr residue resulting in the original structure of the Tyr residue. The mechanism is illustrated in Figure 4.10. However, due to the structure of Tyr, a similar mechanism of β-elimination for

114â•…â•… EUKARYOTE PTM AS PHOSPHORYLATION: NORMAL STATE STUDIES O H2N H3C

H C

C

O

OH

CH

H2N

H C

O

H3C

CH

O C

OH

+

P O

OH

H O

P

OH

O

OH

Threonine

HPO 3

Dephosphorylation of threonine Figure 4.9.â•‡ Mechanism for dephosphorylation of the threonine amino acid resulting in the structure of the original amino acid residue of threonine. O H2N

CH

O

C

OH

CH2

H2N

CH

C

OH

CH2

HO

+

O P O

O O

P

H O

OH

OH

Dephosphorylated tyrosine

HPO 3

Phosphorylated tyrosine (pY) Figure 4.10.â•‡ Mechanism for the dephosphorylation of tyrosine.

loss of the phosphate (−98â•¯Da, H3PO4) group is most probably not likely. The neutral loss of phosphate at 98â•¯Da has also been proposed to not happen through a two-step mechanism involving both water (H2O) loss of 18â•¯Da and HPO3 loss of 80â•¯Da, in any order. The neutral loss of 98â•¯Da is more than likely associated with some form of rearrangement in the fragmentation pathway mechanism.


4.1.9 Alternative Methods: Infrared Multiphoton Dissociation (IRMPD) and Electron Capture Dissociation (ECD) Other mass spectrometric methods are used in phosphorylated peptide analysis that has been investigated to help increase the efficiency of the fragmentation that takes place. This can allow more direct approaches for modification location without the need of third-stage fragmentation experiments that require the interpretation of two individual spectra. One example of an alternative mass spectrometric approach has been the use of the Fourier transform ion cyclotron resonance (FTICR) mass spectrometer using IRMPD and ECD. Figure 4.11 illustrates a comparison of these two dissociation approaches. In the top figure, IRMPD was used to excite and dissociate the phosphorylated peptide. The phosphorylation is located on a Ser (pS) residue within the middle of the peptide chain having the sequence AKRRRL(pS)SLRASTS. In the product ion spectrum collected using IRMPD, the major product ions that were observed were all associated with the neutral loss of the phosphate group as [Mâ•¯+â•¯3Hâ•¯−â•¯H3PO4]3+ and also, phosphate and water loss as [Mâ•¯+â•¯3Hâ•¯−â•¯H3PO4â•¯−â•¯H2O]3+. As can also be observed in the top product ion spectrum of Figure 4.11, very little information is given concerning the peptide chain’s sequence. The bottom product ion spectrum of Figure 4.11 illustrates the effectiveness of using ECD for phosphorylated peptide sequence determinations. In this spectrum, there is an appreciable amount of peptide backbone fragmentation ions in the form of c-type and z-type ions. The major peak in the spectrum is the triply protonated precursor ion as [Mâ•¯+â•¯3H]3+. Also, notice that the phosphorylation was maintained within the structure of the product ions. The two product ion spectra are complimentary where the top spectrum is diagnostic for the determination of a phosphorylation of the peptide, while the bottom spectrum gives very good sequence coverage of the peptide. 4.1.10 Electron Transfer Dissociation (ETD) More recently, the ETD capability has been incorporated into linear ion traps that also allow significant peptide backbone cleavages similar to the ECD capability usually associated with FTICR mass spectrometers. Figure 4.12 illustrates the extensive fragmentation that can be achieved with ETD for a quite long sequence of peptide. The top spectrum is the dissociation spectrum for a 35-residue phosphopeptide that has a molecular weight of 4093â•¯Da. The phosphopeptide is in the plus six CS (+6) at m/z 683.3 for [Mâ•¯+â•¯6H]6+. As can be seen in the figure,

116â•…â•… EUKARYOTE PTM AS PHOSPHORYLATION: NORMAL STATE STUDIES [M - H3PO4 - H2O + 3H]3+

[M - H3PO4 + 3H]3+

Off-Axis IRMPD

(y13 - H3PO4)3+

AKRRRL(pS)SLRASTS (y8 - H3PO4 -17)+

(b7 - H3PO4)2+

(b7 - H3PO4)+ 3+

[M + 3H]

(y9 - H3PO4)+

y6+

+

y7

400

500 [M + 3H]3+

600

700

800

900

m/z

AKRRRL(pS)SLRASTS

~ x 10

ECD

[M + 3H - 17]2+• 2+

[M + 2H] and [M + 3H]2+•

(z12 + 3H)2+• c112+ +

c3+

400

4

500

c102+

600

c122+ c132+

700

800

c7+

c8+

c9+ z10+•

900 1000 1100 1200

z11+•

m/z

Figure 4.11.â•‡ (Top) Product ion spectrum obtained from off-axis IRMPD FTICR MS/MS of a population of quadrupole- and SWIFT-isolated [AKRRRL(pS) SLRASTSâ•¯+â•¯3H]3+ phosphopeptide ions. The spectrum is dominated by ions resulting from neutral losses of H3PO4, NH3, and H2O. Five (out of 13) peptide backbone bonds are broken, and the location of the phosphorylation site is identified only by observation of the (y8–H3PO4–NH3) ion (present at very low abundance) and the singly and doubly charged (b7–H3PO4) ions. Irradiation was for 500â•¯ms at ∼36-W laser power, and the data represent a sum of 10 scans. (Bottom) Product ion spectrum obtained from ECD (20-ms irradiation) FTICR MS/MS of the same quadrupole- and SWIFT-isolated phosphopeptide as in the top figure. Twelve out of 13 peptide backbone bonds are cleaved, and the location of the phosphate is readily assigned by observation of the abundant c7 ions. (Reprinted with permission. This article was published in J Chromatogr B, Chalmers, M.J., Kolch, W., Emmett, M.R., Marshall, A.G., Mischak, H. Identification and analysis of phosphopeptides, 2004, 803, 111–120. Copyright Elsevier 2004.)


(a)

• • • • • • • • • •• • • • • • • ••• • • • • • • H S G F F H S S KKE E Q Q N N Q ATA G E H D A S I T R S pS L D R K * * * ** ** * * ** * * * * * * * * * * * * *** * c4 c3 c5 • c6 c7 • z2 • • z c z7 6 8 •c9 c2 z • 5 * z4 * * • y1 * * * •* * z3 * * 100 400 700 1000 z12 c 13 z c12 * z c14 * c15* z16 c16 • c10 z10* c11 11 z8 * * z9 • 13 • • • •* * z17* • * •* • •* • • * *• * z14 •z15 • m/z 1400 1100 1700 2000

(b)

z1 100

z9 c10 z10

1100

• E N A N S R S S A pH M S S N A I Q R * * z4 z5 z2 * z8 c9 c6 z c c 8 z 5 c c 7 6 7 c3 z3 4 • * 400 700 1000 z17 z12 z16 c13 c14 z15 c16 c18 c12 z13 z 14 c15 z11c 11 1400

1700

2000

Figure 4.12.â•‡ Phosphopeptide mass spectra. ETD mass spectra recorded on [Mâ•¯+â•¯6H]+6 ions at m/z 683.3 for a 35-residue phosphopeptide of molecular weight 4093 (a), and [Mâ•¯+â•¯3H]+3 ions from a phosphorylated His-containing peptide at the C-terminus of the septin protein, Cdc10 (b). Observed c and z· ions are indicated on the peptide sequence by ⎤ and ⎣, respectively. Observed doubly charged c and z· ions are indicated by an additional label, circle and asterisk, respectively. (Reprinted with permission from 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. PNAS 2007, 104, 2193–2198. Copyright 2007 National Academy of Sciences, U.S.A.)

for this very long sequence phosphorylated peptide, essentially complete coverage of the peptide chain has been achieved along with the determination of the phosphorylation site. The ETD product ion spectrum illustrated in Figure 4.12b is for a phosphorylated histidine (His) (pH) peptide. Phosphorylation on the His residue is an important type of PTM that is observed in prokaryotic proteomes and will be discussed in the next chapter. The rest of this chapter is an illustrative example of applying mass spectral techniques for the study of phosphorylation of a eukaryotic cell system as a PTM. The studies described utilize HeLa cells that are both normal and that have sustained induced DNA damage using the nonlinear peptide bleomycin to study the signaling cascade effect of


cellular perturbation. The discussion brings the reader through specific methodological approaches utilizing the most recent advances in proteomic sample preparation, phosphopeptide enrichment, mass spectral measurement, and treatment of the collected data sets.

4.2 THE HELA CELL PHOSPHOPROTEOME 4.2.1 Introduction To improve both coverage and confidence in protein identifications, ongoing optimization of proteomic methodologies are currently being investigated. In order to avoid the pitfalls associated with single-point analysis and undersampling, the optimization of sample preparation, inclusion of technical replicates (repeated instrumental analysis of the same sample), and biological replicates (multiple individual samples) are crucial steps in proteomic studies. The following work illustrates results where phosphopeptides were isolated from HeLa cells and analyzed by nano-RP-LC-MS/MS. A detergent-based protein extraction approach, followed with additional steps for nucleic acid removal, is shown to provide a simple alternative to the broadly used Trizol extraction. The measurement reproducibility from the evaluation of four technical replicates demonstrated low percent variance in peptide responses at approximately 3%, where additional peptide identifications were made with each added technical replicate. The inclusion of six technical replicates affords the optimal collection of peptide information for moderately complex protein extracts (approximately 4000 uniquely identified peptides per data set). 4.2.2 Background of Study For regulating cellular processes, such as in HeLa cells, involving signal transduction reversible phosphorylation of Ser, Thr, and Tyr residues in proteins represent an important mechanism in eukaryotes.2 To identify protein phosphorylation sites in eukaryotic systems such as HeLa cells, analytical approaches utilizing MS have been applied extensively.15–18 An enrichment step is a necessary requirement due to the often low stoichiometry of the phosphoproteome within a cell (≤1%) to identify low abundance phosphopeptides from complex mixtures. One current and widely used enrichment technique in phosphoproteomics is immobilized metal affinity chromatography,27–29 which has been optimized over the years for high specificity enrichment and

THE HELA CELL PHOSPHOPROTEOMEâ•…â•… 119

recovery of phosphopeptides.17,24,30 It is possible that the presence of nucleic acids in proteomic samples may interfere with IMAC enrichment steps by competing with phosphopeptides for available binding sites in the IMAC stationary phase. Because of this sample preparation, methods upstream of IMAC enrichment are highly important. Next, if the reader is unaware, Trizol extraction has been the most commonly used protein extraction method in IMAC applications.9,22,24,42 The Trizol approach removes nucleic acids that reduce phosphopeptide recovery and provides an increase in method sensitivity during IMAC enrichment; however, the Trizol method involves multiple steps, including phase separation and precipitation to remove RNA and DNA and collect protein. As a precipitation-based approach, the Trizol methodology may result in selective protein loss, and minor variations in sample handling during this procedure can undermine both uniform protein recovery among samples and sample quality.41 4.2.3 What is Covered In the following sections, we will look closely at a study in which samples were prepared using a detergent-based cell lysis method followed by in-solution or in-gel digestion and IMAC enrichment to explore the phosphoprotein coverage of HeLa cell lysates. As a comparison with existing techniques, samples were also prepared using Trizol extraction. It was observed that as compared with Trizol extraction, detergent-based extraction such as with the Roche Complete lysis approach is fast, requires a single, easily reproduced step, and gives a good protein yield but requires additional steps to remove nucleic acids prior to IMAC enrichment. To facilitate nucleic acid removal, several simple steps were taken during phosphopeptide sample preparation. The inclusion of both technical replicates (repeated instrumental analysis of the same sample) and biological replicates (multiple individual samples), as well as the effect of these sample preparation steps on coverage, was evaluated. We will see that these experimental design parameters are crucial to avoid the pitfalls associated with single-point analysis and undersampling. 4.2.4 Optimized Methods to Use for Phosphoproteomic Studies 4.2.4.1 Cell Culture.â•‡ A very common system used in proteomic studies are HeLa cells. The cultures are often grown in Dulbecco’s modified Eagle medium (DMEM) with high glucose (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Clontech,


Mountain View, CA) and 100 units/mL penicillin and 100â•¯µg/mL streptomycin (Invitrogen) at 37°C in 5% CO2. This is a generalized approach for preparing cell cultures for proteomic studies. Manufacturer guidelines are also very helpful in preparing a system for study. 4.2.4.2 Extraction of HeLa Cell Proteins.â•‡ In the example illustrated here, two sets of samples are prepared for analysis. In the first set (biological replicate 1), five nearly confluent 100-mm plates of cells were extracted with Trizol, and five matched plates of cells were solubilized with Roche lysis buffer. The sample solubilized with Roche Complete Lysis-M was then split into two equal portions. One portion was subjected to in-solution tryptic digestion, while the other portion was subjected to SDS-PAGE and in-gel tryptic digestion. For the second sample (biological replicate 2), five nearly confluent 100-mm plates of cells were again solubilized with Roche lysis buffer and divided into two portions for in-solution digest or in-gel digest. 4.2.4.3 Trizol Extraction and Tryptic Digestion.â•‡ Trizol reagent (Invitrogen), according to the manufacturer’s suggested protocol with the exception that the initial Trizol volume was doubled (∼2â•¯mL Trizol reagent/5â•¯×â•¯106 cells), was used to extract protein11: 1. The protein pellet was resuspended in 8â•¯M urea. 2. The proteins were then reduced in a 5â•¯mM solution of dithiothrietol (DTT). 3. The proteins were then alkylated in a 15â•¯mM solution of iodoacetamide. 4. The denatured and alkylated proteins were digested with modified trypsin at a 1:20 ratio for 4 hours at 37°C after twofold dilution with 50â•¯mM NH4HCO3 (pH 7.4). 5. After fivefold further dilution, a second trypsin digestion at a 1:20 ratio was performed overnight at 37°C. 6. The digestion was stopped by adding acetic acid to a final pH of ∼3.5–4. 4.2.4.4 Solid-Phase Extraction (SPE) Desalting.â•‡ A C18 RP peptide SPE cartridge is used to desalt the tryptic digests. A 1â•¯mL/100â•¯mg tube is usually sufficient to use for up to 5â•¯mg of protein extract: 1. Condition the SPE column with 3â•¯mL of methanol on an SPE vacuum chamber.


2. Rinse the column with 2â•¯mL of acidified water (0.1% TFA). 3. Slowly put the protein extract sample through the column using minimal vacuum (∼0.5–1â•¯mL per minute flow rate). 4. Wash the column containing the sample with 4â•¯mL of 95:5 H2Oâ•›:â•›ACN, 0.1% TFA. 5. Allow the column to go to dryness and whip the needles below the columns dry. 6. Place appropriate collection tubes under the columns. 7. Close off the tubes from the vacuum and add 1â•¯mL of 80:20 ACNâ•›:â•›H2O, 0.1% TFA. 8. Allow the elution buffer to slowly flow through the tube until the column is dry (∼0.5 to 1â•¯mL per minute flow rate). 9. When completed, remove the sample from the SPE vacuum chamber. 4.2.4.5 Converting Peptide Carboxyl Moieties to Methyl Esters.â•‡ To remove the possibility of nonspecific binding of peptides to the IMAC column bed, peptide residue carboxyl moieties are converted to methyl esters. The tryptic peptides are converted to peptide methyl esters according to the general procedure of White et al.22 except that a second methyl esterification step was performed to ensure complete esterification. Samples were reconstituted in IMAC loading solutions that were composed of 1:1:1 methanol/acetonitrile/0.01% acetic acid at a ratio of 100â•¯µL solution to 100–200â•¯µg peptides: 1. Only use dry peptide material that has been previously extensively dried in a SpeedVac. 2. Carefully add dropwise 40â•¯µL of thionyl chloride to 1â•¯mL methanol (both anhydrous). 3. Add thionyl chloride/methanol mixture to dry peptide at a ratio of 75â•¯µL thionyl chloride/methanol solution per 100â•¯µg peptide. 4. Vortex the reaction mixture for 5–10 minutes to ensure dissolution of the dry peptide material. 5. Sonicate the reaction mixture for 10 minutes. 6. Let the mixture react at room temperature for 1 hour. 7. Bring the methylated peptide to dryness in a SpeedVac (Eppendorf). 8. Reconstitute in IMAC loading solution composed of 1:1:1 methanol/acetonitrile/0.01% acetic acid at a ratio of 100â•¯µL solution to 100–200â•¯µg peptides.


4.2.4.6 Roche Complete Lysis-M, EDTA-Free Extraction.â•‡ As an alternative to the Trizol extraction approach, eukaryotic systems can be treated with a detergent-based lysis and extraction buffer. For this detergent-based approach, HeLa cells were lysed and extracted using the Roche Complete Lysis-M, EDTA-free kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s guidelines. Phosphatase Inhibitor Cocktail Sets I and Set II (EMD Biosciences, San Diego, CA) were added to the extracts, following the manufacturer’s protocol. For the first portion split (described earlier), urea was added to the extract to a final concentration of 8â•¯M, and the proteins were reduced, alkylated, and digested as described earlier. Following tryptic digestion, ultracentrifugation (166,000â•¯×â•¯g for 30 minutes at 4°C) was used to deplete nucleic acids from the sample prior to SPE desalting. Following SPE desalting, extracts were methyl esterified and the phosphopeptides enriched using IMAC, as described later. Biological replicate 1 was analyzed using a liquid chromato-graphy linear ion trap-Fourier transform mass spectrometer (LC-LTQ-FT MS), while biological replicate 2 was analyzed using LC-LTQ-Orbitrap MS (Thermo Fisher Scientific, Bremen, Germany). 4.2.4.7 1-D SDS-PAGE Cleanup.â•‡ To remove nucleic acids from the second portion split, as well as to investigate sample cleanup and recovery, samples of the total cell lysates prepared with the Roche Complete Lysis-M, EDTA-free kit were separated using 1-D SDS-PAGE as a preparatory stage as described elsewhere.42 Briefly, the separations were performed according to the manufacturer’s guidelines using a Mini-PROTEAN 3 Cell (Bio-Rad, Hercules, CA) and 1-mm-thick Ready Gel Tris-HCl gels with a 4%–20% gradient acrylamide composition (Bio-Rad). Precision Plus Protein Standards (Bio-Rad) ranged from 10 to 250. Prior to gel loading, the protein samples were mixed with a dye solution that contained the reducing agent Bond-Breaker TCEP (Pierce, Rockford, IL) and heated at 95°C for 4 minutes. Approximately 3â•¯mg of extracted protein determined by the bicinchoninic acid (BCA) protein assay (Pierce) were subjected to SDS-PAGE on two gels (1.5â•¯mg per gel) at a constant voltage of 200â•¯V. The gels were fixed, stained, destained, and then stored until analyzed.42 4.2.4.8 In-Gel Reduction, Alkylation, Digestion, and Extraction of Peptides.â•‡ Multiple identical lanes were pooled for each of the two gels, and the resulting two gel samples were digested. Details of in-gel reduction, alkylation, digestion, and peptide extraction have been described elsewhere.42 A C18 RP peptide Macrotrap SPE cartridge


(Michrom BioResources, Inc., Auburn, CA) was used to desalt the in-gel tryptic digests. Peptides were converted to methyl esters as described earlier, and the samples were then reconstituted in IMAC loading solution (1:1:1 methanol/acetonitrile/0.01% acetic acid) at a ratio of 100â•¯µL to 100–200â•¯µg of peptide. The first biological replicate Roche extract sample was analyzed using LC-LTQ-FT MS, while the second biological replicate sample was analyzed using LC-LTQ-Orbitrap MS. 4.2.4.9 Phosphopeptide Enrichment Using IMAC.â•‡ Phosphopeptides are enriched using an IMAC protocol that includes advances and optimizations recently summarized by Ross et al.37 with the exception of using thionyl chloride during the methyl esterification process. Also suggested is using custom-packed IMAC Macrotrap cartridges with a 50-µL bed volume (Michrom BioResources, Inc.) for phosphopeptide enrichment. The procedure consists of the following: 1. The column is stripped with 500â•¯µL 50â•¯mM EDTA (adjusted to pH 9–10 with ammonium hydroxide) at a flow rate of 50â•¯µL/min. 2. The column is washed with 1000â•¯µL nanopure water at 100â•¯µL/min. 3. The column is activated with 375â•¯µL 100â•¯mM FeCl3 at 25â•¯µL/min. 4. The column is washed to remove excess metal ions with 400â•¯µL 0.1% acetic acid at 50â•¯µL/min. 5. The column is loaded with approximately 1.5â•¯mg sample reconstituted in IMAC loading solution (1:1:1 methanol/ acetonitrile/0.01% acetic acid) at a ratio of 100â•¯µL to 100–200â•¯µg of peptide at 4â•¯µL/min. 6. The column is washed with 400â•¯µL wash buffer (100â•¯mM NaCl, 1% acetic acid, and 25% acetonitrile) at 25â•¯µL/min. 7. The column is re-equilibrated with 300â•¯µL of 0.01% acetic acid. 8. The column is eluted with 250â•¯µL of 50â•¯mM Na2HPO4 (pH ∼8.5). 9. The eluate is immediately acidified with acetic acid to a pH of ∼4. 4.2.5 Description of Instrumental Analyses 4.2.5.1 RP/Nano-HPLC Separation.â•‡ Peptide mixtures from HeLa cell extracts were separated using an automated dual-column phosphoproteome nano-HPLC platform assembled in-house and has now been reported in the literature.43 All portions of the separation system that come in contact with peptide mixtures with the exception of the autosampler syringe (but including the valve apparatus and transfer lines)


are nonmetal to minimize the loss of phosphopeptides. The platform includes two 103-mL syringe pumps (Model 100DM, Teledyne Isco, Inc., Lincoln, NE) controlled using a single series D controller and a 1.5-mL mobile phase mixer, which was built in-house. One pump is dedicated to mobile phase A and is operated at 1000â•¯psi, and the other is dedicated to mobile phase B and is operated at 1500â•¯psi. Eight, twoposition Valco valves (Valco Instruments Co., Houston, TX) are used, including a six-port injection valve with a 10-µL sample loop, two fourport valves for mobile phase and mixer purge selection, and a 10-port and two four-port valves for directing the sample to either of two pairs of SPE and analytical columns. Two four-port valves are used to connect the pump to either the fluidic system or to a pair of refill reservoirs. With the two-column design, samples can be loaded, desalted, and analyzed using one pair of SPE and analytical columns while the other pair is being re-equilibrated, which allows for continuous sample analysis. The SPE precolumns are prepared from 150-µm i.d., ∼10-cm-long fused-silica capillaries packed in-house with 5-µm octadecylsilane (ODS-AQ) C18 material (YMC Co., Ltd., Kyoto, Japan) to a bed length of 4â•¯cm. The SPE precolumns are double fritted (one Kasil® potassium silicate, PQ Corporation, Valley Forge, PA, chemical frit at each end) due to the procedural backwashing of the SPE columns directly after sample loading and prior to analytical column separation. The two analytical separation columns are composed of 50-µm i.d. fused silica (Polymicron Technologies Inc., Phoenix, AZ), 40-cm-long capillaries packed in-house with 5-µm ODS-AQ C18 RP material. The tips coupled to the columns for electrospray are 10-µm i.d. open tubular fused silica that have been etched with hydrofluoric acid (HF) for uniform tip bevel and opening.44 The SPE precolumn and tips are connected to the analytical column using PicoClear unions (New Objective, Inc., Woburn, MA). An in-house constructed rack assembly supports the valve and column system and was fitted to a PAL autosampler (Leap Technologies, Carrboro, NC) for automated sample loading and analysis. Peptide samples were loaded onto the SPE precolumn and backwashed with 0.1â•¯M acetic acid in nanopure water. A voltage of 2.3â•¯kV is applied at the split “tee” at the head of the column instead of at the union between column and the ESI tip to minimize loss of phosphopeptides. The ESI tips are positioned at the MS inlet, using a set of encoding translation stages (Newport, Irvine, CA). All components of the LC system are controlled by custom software that runs on a laptop computer that communicates with the various hardware components via a 16-port USB hub and that triggers MS data acquisition using a contact closure connection.


The HPLC mobile phases were composed of 0.1â•¯M acetic acid in nanopure water (A), and 70% acetonitrile/0.1â•¯M acetic acid in nanopure water (B). The system was equilibrated at 1000â•¯psi for 20 minutes with 100% mobile phase A. Next, an exponential gradient was created by valve switching from pump A to B, which displaced mobile phase A in the mixer with mobile phase B. The gradient was controlled by the split flow (∼9â•¯µL/min) under constant pressure conditions. The final composition of mobile phase B was approximately 70% by the end of the HPLC run (180 minutes). 4.2.5.2 MS Analysis.â•‡ A linear ion trap/Fourier transform hybrid MS was used for some of the product ion spectral data set collection, where data-dependent analysis (DDA) data sets were collected for the 10 most abundant species after each high-resolution MS scan by the LTQ-FT (100,000 resolution and mass scan range of 400–2000 m/z). A linear ion trap/Orbitrap hybrid MS was also used for some of the product ion spectral data set collection. Data-dependent data sets were collected for the 10 most abundant species after each high-resolution MS scan by the LTQ-Orbitrap (100,000 resolution and mass scan range of 300–2000 m/z). Data sets were also collected with high mass accuracy precursor scans by the LTQ-Orbitrap, data-dependent MS/MS of the top five peptides, followed by MS3 of the neutral loss peak in the MS2 scan that was associated with a precursor peak loss corresponding to phosphate loss (i.e., a neutral loss of 32.7â•¯Da [3+], 49.0 [2+], 65.4, and 98.0 [1+]). To enhance identification of phosphopeptides, data sets were collected following an additional gas-phase (GP) separation45 within the MS, which entails scanning for shorter, predefined m/z ranges, which are 300–850 and 750–1575, both with the precursor scan at 100,000 resolution. 4.2.6 Current Approaches for Peptide Identification and False Discovery Rate (FDR) Determination To identify peptides, all data collected from LC-MS/MS analyses (LCLTQ-FT MS/MS and LC-LTQ-Orbitrap MS/MS) were analyzed using SEQUEST and the following search criteria for phosphorylated peptides: static methyl esterification on D-, E-, and C-termini of the peptides in conjunction with dynamic phosphorylation of S, T, and Y residues, all searched as full tryptic cleavage products. As the precursor masses were collected with high mass accuracy, the SEQUEST parameter file also contained a search criteria cutoff of ±1.5â•¯Da for the precursor masses. A no-enzyme search was performed for the standard


extract. Data were searched against the human International Protein Index (IPI) database (version 3.20 containing 61,225 protein entries; available at www.ebi.ac.uk/IPI). To determine the FDR, the IPI database was searched as a decoy database; that is, the reversed human IPI was appended to the forward database and included in the SEQUEST search. The FDR was estimated from the forward and reverse (decoy) filtered matches and was calculated as a ratio of two times the number of false positive peptide identifications to the total number of identified peptides.46 For phosphorylated peptide search results (fully tryptic only), the following filtering criteria were applied for an FDRâ•¯≤â•¯5%: 1+ CS, XCorrâ•¯≥â•¯1.4; 2+ CS, XCorrâ•¯≥â•¯2.4; 3+ and 4+ CS, XCorrâ•¯≥â•¯3.3, all CSs with DelCn2â•¯≥â•¯0.13. All phosphopeptide filtering criteria included a mass error cutoff within ±6.5â•¯ppm. For the standard extract, the following filtering criteria were applied for an FDRâ•¯≤â•¯5%: 1+ CS, DelCn2â•¯≥â•¯0.1, XCorrâ•¯≥â•¯1.5, both partially and fully tryptic ends; 2+ CS, DelCn2â•¯≥â•¯0.1, XCorrâ•¯≥â•¯2.2, fully tryptic ends; 2+ CS, DelCn2â•¯≥â•¯0.1, XCorrâ•¯≥â•¯4.0, partially tryptic ends; 3+ CS, DelCn2â•¯≥â•¯0.1, XCorrâ•¯≥â•¯2.9, fully tryptic ends; 3+ CS, DelCn2â•¯≥â•¯0.1, XCorrâ•¯≥â•¯4.6, partially tryptic ends. High-confidence identifications were obtained using the accurate mass and time tag approach and in-house developed programs Viper and MultiAlign that have been described elsewhere.47 4.2.7 Results of the Protein Extraction and Preparation This section looks at a comparison of three types of methodology approaches for performing eukaryote phosphoproteomic analyses. Figure 4.13 illustrates an overview of the steps and methodologies used in this study. 4.2.7.1 Detergent Lysis, Trizol, and Ultracentrifugation.â•‡ In this study, normal HeLa cells (i.e., unperturbed cells) were lysed prior to protein extraction and solubilization. The associated differences in the study include the use of the detergent-based Roche Complete lysis kit versus Trizol lysis and extraction. Also, the use of 1-D SDS-PAGE to separate extracted proteins was incorporated, and ultracentrifugation was used to facilitate removal of nucleic acids from the protein digest in the detergent extraction approach prior to SPE cleanup (i.e., desalting and further removal of nucleic acids). A clear gelatinous substance was observed after centrifugation and decanting as a pellet on the bottom of the centrifuge tubes thought to be composed of nucleic acids. High recovery of peptides (98%) was observed following


Normal HeLa cells Biological replicate 1 & Biological replicate 2

Trizol lysis

Roche lysis

In-solution digest

In-solution digest

Ultracentrifugation

1-D gel digest

SPE Methylation IMAC Phosphopeptides nano-RP-LC ESI-MS/MS

Figure 4.13.â•‡ Overview of the methodology studied in the analysis of HeLa cell total proteome coverage. The initial step of the study is composed of the lysis of the normal HeLa cells and subsequent protein extraction and solubilization. Key differences include the use of the Roche Complete lysis kit versus Trizol lysis and extraction, and incorporation of 1-D SDS-PAGE separation of extracted proteins. (Reprinted with permission from Ham et al. J. Proteome Res. 2008, 7, 2215–2221. Copyright 2008 American Chemical Society.)

ultracentrifugation. When ultracentrifugation was performed on the undigested extract, protein loss was greater and ranged from 15% to as high as 48%. 4.2.7.2 Nucleic Acid Removal with SDS-PAGE.â•‡ Finally, SDS-PAGE was also used as an alternative approach for removing nucleic acids prior to IMAC enrichment of the phosphopeptides.5,41 Advantages of a gel-based approach is the ability to target specific molecular weight ranges of proteins for more comprehensive phosphopeptide identification without additional fractionation prior to digestion and IMAC enrichment. Other advantages include a more efficient tryptic digest


due to the enhanced accessibility of the protein backbone denatured into a linear orientation locked within the gel. Low throughput can be a disadvantage due to the labor intensive aspect of in-gel digestions and generally low recoveries, that is, 40.0â•¯±â•¯16.8%, (nâ•¯=â•¯4). The overall recovery of this approach is comparable with other approaches for comprehensive phosphopeptide identification. Additional losses are usually expected to occur during fractionation steps such as strong cation exchange (SCX) that are often required when applying nongelbased approaches. As an example, recoveries from SPE used for desalting/detergent cleanup steps were approximately 51.4â•¯±â•¯15.6% (nâ•¯=â•¯9, includes data from all three approaches). 4.2.8 HeLa Cell Phosphoproteome Methodology Comparison The results from the two biological replicates for the Roche Complete in-solution digest method and the Roche Complete in-gel digest method phosphoproteomic analyses are listed in Table 4.1. The complementary nature of the two extraction methodologies are illustrated in these results. For the normal HeLa cell phosphoproteome, a combined total of 651 phosphorylation sites and 597 unique phosphoproteins were identified. Spectra for the 597 phosphopeptides along with SEQUEST identification information are included in the SpectrumLook Software Package (see Section 4.4). Table 4.1 shows the three types of sample processing procedure (Roche Complete in-solution digest, Roche Complete in-gel digest, and Trizol) results that allow an assignment of the efficiencies. The Roche Complete in-solution digest approach yielded the greatest number of phosphorylated protein identifications followed by the in-gel digest approach.

TABLE 4.1.â•‡ Phosphoproteomic Comparison of Total HeLa Cell Lysate Methodology Biological Replicate 1

Number of Unique

Phosphopeptides Phosphorylated sites Phosphoproteins

Roche

Roche

InSolution

In-Gel

172 337 311

Biological Replicate 2 Rep1

Roche

Roche

Rep2

Trizol

Total

InSolution

In-Gel

Total

Total

143 195

116 222

302 521

153 267

135 179

248 397

380 651

294

260

498

313

301

459

597


4.2.8.1 Roche In-Solution versus Trizol Extraction.â•‡ Figure 4.14 shows the overlap in unique phosphopeptides and phosphoproteins between the two extraction methods (namely, Roche in-solution digest and the Trizol extraction) for sample 1 in the form of Venn diagrams. The phosphoproteins identified in the Roche solution digest sample contained approximately 57% of the unique phosphorylated peptides (74% of the phosphoproteins) identified in the Trizol sample. We can conclude from this that the complement of proteins within the two extracts is similar, which is consistent with our observations for other samples prepared using the two methodologies. In contrast to the Trizol extraction, the Roche lysis approach does not require numerous protein precipitation steps that can result in poor recovery of precipitated proteins. We can also conclude from this that the good overlap in protein with the Roche lysis affords a good alternative to Trizol extraction. 4.2.8.2 In-Solution and In-Gel Digests Phosphoproteome Coverage.â•‡ To compare the reproducibility of phosphorylated protein identifications as a function of the method used for extraction and digestion, the sample workup and analysis of biological replicates was performed. 4.2.8.2.1â•… Biological Replicates.â•‡ The extent of overlap in unique phosphopeptide and phosphorylated protein identifications measured Roche in-solution

106

Trizol

66

57% overlap in unique phosphopeptides

50

Trizol

Roche in-solution

119

192

68

89% overlap in unique phosphopeptides

Figure 4.14.â•‡ Venn diagrams comparing the overlap in unique phosphorylated peptides and proteins for the samples from the Roche in-solution digest and the Trizol extraction. There was a 57% overlap in unique phosphorylated peptides (left) and an 89% overlap in unique phosphorylated proteins between the two extraction methodologies. (Reprinted with permission from Ham et al. J. Proteome Res. 2008, 7, 2215–2221. Copyright 2008 American Chemical Society)


between the two biological replicates is illustrated in a set of Venn diagrams in Figure 4.15. For the in-solution digest samples (Fig. 4.15a), an overlap of 53% in phosphopeptides and 64% in phosphoproteins identified between biological replicates is seen in the results. There is also a similar observation for the overlap between biological replicates for the in-gel digest where it is 55% for phosphopeptides and 70% for phosphorylated proteins (Fig. 4.15b). However, there is a decrease to 28% for unique phosphopeptides and 40% for phosphorylated proteins in the overlap between the in-solution digest of one biological replicate with that of the in-gel digest of the second replicate as illustrated in Figure 4.15c. A 64% overlap in phosphopeptides and a 72% overlap in phosphorylated proteins between the two biological replicates are obtained (Fig. 4.15d) when the unique phosphorylated peptides and proteins identified in the in-solution and the in-gel digestion samples are combined. This indicates that the increase in the number of overlapped phosphopeptides/proteins between these two sample preparation methods provides complementary coverage and, when combined, will offer more comprehensive coverage of the HeLa cell phosphoproteome. 4.2.8.2.2â•… The Effect of Performing Technical Replicates on Phosphoproteome Coverage.â•‡ In the following analyses, four technical replicates were obtained by repeatedly collecting nano-RP-LC-MS/MS data sets for each extraction methodology (the Trizol sample was measured and reported for biological replicate 1 only) to investigate the influence of technical replicates on the phosphoproteome coverage. The total number of unique phosphopeptide identifications was determined from the individual data set identifications (FDRâ•¯

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