Protein and Peptide Mass Spectrometry in Drug Discovery

The book that highlights mass spectrometry and its applicationin characterizing proteins and peptides in drug discovery

An instrumental analytical method for quantifying the mass andcharacterization of various samples from small molecules to largeproteins, mass spectrometry (MS) has become one of the most widelyused techniques for studying proteins and peptides over the lastdecade. Bringing together the work of experts in academia andindustry, Protein and Peptide Mass Spectrometry in DrugDiscovery highlights current analytical approaches, industrypractices, and modern strategies for the characterization of bothpeptides and proteins in drug discovery.

Illustrating the critical role MS technology plays incharacterizing target proteins and protein products, the methodsused, ion mobility, and the use of microwave radiation to speedproteolysis, the book also covers important emerging applicationsfor neuroproteomics and antigenic peptides. Placing an emphasis onthe pharmaceutical industry, the book stresses practice andapplications, presenting real–world examples covering the mostrecent advances in mass spectrometry, and providing an invaluableresource for pharmaceutical scientists in industry and academia,analytical and bioanalytical chemists, and researchers in proteinscience and proteomics.

PREFACE xv

CONTRIBUTORS xvii

PART I METHODOLOGY 1

1 Ionization Methods in Protein Mass Spectrometry 3
Ismael Cotte–Rodriguez, Yun Zhang, Zhixin Miao, and HaoChen

1.1 History of the Development of Protein Mass Spectrometry4

1.2 Laser–Based Ionization Methods for Proteins 5

1.3 Spray–Based Ionization Methods for Proteins 13

1.4 Ambient Ionization Methods 20

1.5 Conclusions 30

Acknowledgments 30

References 30

2 Ion Activation and Mass Analysis in Protein MassSpectrometry 43
Cheng Lin and Peter O Connor

2.1 Introduction 43

2.2 Ion Activation and Tandem MS Analysis 46

2.3 Mass Analyzers 59

References 81

3 Target Proteins: Bottom–up and Top–down Proteomics 89
Michael Boyne and Ron Bose

3.1 Mass Spectral Approaches to Targeted Protein Identification89

3.2 Bottom–up Proteomics 90

3.3 Top–down Approaches 96

3.4 Next–Generation Approaches 98

References 99

4 Quantitative Proteomics by Mass Spectrometry 101
Jacob Galan, Anton Iliuk, and W. Andy Tao

4.1 Introduction 101

4.2 In–Cell Labeling 105

4.3 Quantitation via Isotopic Labeling of Proteins 107

4.4 Quantitation via Isotopic Labeling on Peptides 112

4.5 Label–Free Quantitation 116

4.6 Conclusions 119

Acknowledgment 120

References 120

5 Comparative Proteomics by Direct Tissue Analysis UsingImaging Mass Spectrometry 129
Michelle L. Reyzer and Richard M. Caprioli

5.1 Introduction 129

5.2 Conventional Comparative Proteomics 130

5.3 Comparative Proteomics Using Imaging MS 131

5.4 Conclusions 136

Acknowledgments 137

References 137

6 Peptide and Protein Analysis Using Ion Mobility MassSpectrometry 139
Jeffrey R. Enders, Michal Kliman, SevugarajanSundarapandian, and John A. McLean

6.1 Ion Mobility Mass Spectrometry: Instrumentation andSeparation Selectivity 139

6.2 Characterizing and Interpreting Peptide and ProteinStructures 147

6.3 Applications of IM–MS to Peptide and ProteinCharacterizations 152

6.4 Future Directions 158

Acknowledgments 159

References 160

7 Chemical Footprinting for Determining Protein Propertiesand Interactions 175
Sandra A. Kerfoot and Michael L. Gross

7.1 Introduction to Hydrogen Deuterium Exchange 175

7.2 Experimental Procedures 178

7.3 Mass Spectrometry–Based HDX in Practice 182

7.4 Protein Footprinting via Free–Radical Oxidation 193

7.5 Chemical Crosslinking 198

7.6 Selective and Irreversible Chemical Modification 201

7.7 Conclusion 205

References 206

8 Microwave Technology to Accelerate Protein Analysis213
Urooj A. Mirza, Birendra N. Pramanik, and Ajay K.Bose

8.1 Introduction 213

8.2 Microwave Technology 215

8.3 Summary 224

Acknowledgments 224

References 224

9 Bioinformatics and Database Searching 231
Surendra Dasari and David L. Tabb

9.1 Overview 231

9.2 Introduction to Tandem Mass Spectrometry 231

9.3 Overview of Peptide Identification with Database Searching234

9.4 MyriMatch–IDPicker Protein Identification Pipeline 235

9.5 Results of a Shotgun Proteomics Study 246

9.6 Improvements to MyriMatch Database Search Engine 248

9.7 Applications of MyriMatch–IDPicker Pipeline 250

9.8 Conclusions 251

Acknowledgments 251

References 251

PART II Applications 253

10 Mass Spectrometry–Based Screening and Characterization ofProtein Ligand Complexes in Drug Discovery 255
Christine L. Andrews, Michael R. Ziebell, Elliott Nickbarg,and Xianshu Yang

10.1 Introduction 255

10.2 Affinity Selection Mass Spectrometry (AS–MS) 256

10.3 Solution–Based AS–MS as Screening Technologies 258

10.4 Gas–Phase Interactions 267

10.5 Enzyme Activity Assays Using MS for Screening or ConfirmingDrug Candidates 271

10.6 Conclusions and Future Directions 276

References 277

11 Utilization of Mass Spectrometry for the StructuralCharacterization of Biopharmaceutical Protein Products 287
Amareth Lim and Catherine A. Srebalus Barnes

11.1 Introduction 287

11.2 MS–Based Approach for the Characterization of RecombinantTherapeutic Proteins 288

11.3 Cell Culture Development 290

11.4 Purification Development 294

11.5 Formulation Development 300

11.6 Analytical Method Development 304

11.7 Confirmation of Structure/Product Comparability Assessment311

11.8 Conclusions 313

Acknowledgments 315

References 315

12 Post–translationally Modified Proteins: Glycosylation,Phosphorylation, and Disulfide Bond Formation 321
Anthony Tsarbopoulos and Fotini N. Bazoti

12.1 Introduction 321

12.2 Glycosylation 322

12.3 Phosphorylation 338

12.4 Disulfide Bond Detection and Mapping 347

12.5 Future Perspectives 350

Acknowledgments 352

Abbreviations 353

References 354

13 Mass Spectrometry of Antigenic Peptides 371
Henry Rohrs

13.1 Introduction 371

13.2 Analysis of Antigenic Peptides 374

13.3 Examples of the Application of Mass Spectrometry toAntigenic Peptide Study 381

13.4 Future Work 385

Acknowledgments 386

Abbreviations 387

References 387

14 Neuropeptidomics 393
Jonathan V. Sweedler, Fang Xie, and Adriana Bora

14.1 Introduction 393

14.2 Neuropeptidomics: Characterizing Peptides in the Brain394

14.3 Sample Preparation for Mass Spectrometry 395

14.4 Separations 405

14.5 Peptide Characterization via Mass Spectrometry 407

14.6 Conclusions 419

14.7 Future Perspectives 419

Acknowledgments 420

References 420

15 Mass Spectrometry for the Study of Peptide Drug Metabolism435
Patrick J. Rudewicz

15.1 Introduction 435

15.2 Peptide Drug Metabolism 436

15.3 LC–MS/MS for Metabolite Identification 437

15.4 Quantitative Analysis 439

15.5 Case Study: IL–1b Protease Inhibitors 440

15.6 Future Directions 445

References 445

INDEX 449

Michael L. Gross, PhD, is a Professor in the Departments ofChemistry, Medicine, and Immunology at Washington University in St.Louis. He is also Principal Investigator at the National Institutesof Health (NIH) Research Resource in Mass Spectrometry.

Guodong Chen, PhD, is Principal Scientist inBioanalytical and Discovery Analytical Sciences at Bristol–MyersSquibb in Princeton, New Jersey. He heads a mass spectrometry groupin support of drug discovery as well as development programs insmall molecule pharmaceuticals and biologics.

Birendra N. Pramanik, PhD, was a Distinguished Fellow atSchering–Plough Research Institute (SPRI), where he directed thespectroscopy, mass spectrometry, and NMR programs.

"This book will be a valuable reference as it contains plenty ofdepth and substance to be of interest to experienced practitionersof mass spectrometry and related techniques, but is stillaccessible to pharmaceutical researchers who want to learn moreabout MS technologies and its applications."  (AmericanSociety for Mass Spectrometry, 1 July 2012)