Westermeier R., Naven T., H?pker H.-R. Proteomics in Practice: A Guide to Successful Experimental Design

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Reiner Westermeier, Tom Naven,

and Hans-Rudolf Hçpker

Proteomics in Practice

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

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Reiner Westermeier, Tom Naven, and

Hans-Rudolf Hçpker

Proteomics in Practice

A Guide to Su ccessful Experimental Design

Second, Completely Revised Edition

The Authors

Dr. Reiner Westermeier

GE Healthcare Life Sciences

Oskar-Schlemmer-Str. 11

80807 Mnchen

Germany

Dr. Tom Naven

GE Healthcare UK Limited

Pollards Wood

Nightingales Lane

Chalfont St Giles

Bucks HP8 4SP

United Kingdom

Hans-Rudolf Hçpker

Weberweg 6

79183 Waldkirch

Germany

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ISBN 978-3-527-31941-1

Preface XI

Foreword XIII

Abbreviations, Symbols, Units XV

Introduction 1

1 History 1

2 Critical Points 8

2.1 Challenges of the Protein Samples 8

2.1 Challenges of the Analysis Systems 11

3 Proteomics Strategies 12

3.1 Proteome Mapping 12

3.2 Differential Analysis 12

3.3 Time Point Experiments 13

3.4 Verification of Targets or Biomarkers 13

3.5 Integration of Results into Biological Context 13

3.6 Systems Biology 13

4 Concept of Experimental Planning 14

4.1 Biological Replicates 14

4.2 Pooling of Samples: Yes or No? 14

4.3 Pre-fractionation of Samples: Yes or No? 14

4.4 Which is the Best Workflow to Start With? 15

Part I: Proteomics Technology

1 Electrophoretic Techniques

1.1 The Principle of Electrophoresis and Some Methodological

Background

1.1.1 Free Flow Electrophoretic Methods 20

1.1.2 Gels for Electrophoretic Techniques 21

1.1.3 Electroendosmosis Effects 21

1.2 Polyacrylamide Gel Electrophoresis 22

Contents

Proteomics in Practice. A Guide to Successful Experimental Design 2

Ed.

Reiner Westermeier, Tom Naven, and Hans-Rudolf Hçpker

ISBN: 978-3-527-31941-1

1.2.1 The Polyacrylamide Gel 22

1.2.2 SDS Polyacrylamide Gel Electrophoresis 27

1.2.3 Blue Native Electrophoresis 32

1.2.4 Cationic Detergent Electrophoresis 34

1.3 Blotting 35

1.3.1 Electrophoretic Transfer 36

1.3.2 Protein Detection on the Membrane 36

1.4 Isoelectric Focusing 38

1.4.1 Theoretical Background 39

1.4.2 Preparation of IEF Gels 44

1.4.3 Isoelectric Focusing in Proteomics 45

1.5 Two-dimensional Electrophoresis 53

1.5.1 Sample Preparation 53

1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis 68

1.5.3 First Dimension: Isoelectric Focusing in IPG Strips 77

1.5.4 Second Dimension: SDS Electrophoresis 100

1.5.5 Detection of Protein Spots 119

1.6 Image Analysis 125

1.6.1 Image Acquisition 125

1.6.2 Image Analysis and Evaluation 129

1.6.3 Use of 2-D Electrophoresis Data 137

1.7 Spot Handling 137

1.7.1 Spot Picking 139

1.7.2 Protein Cleavage 141

2 Liquid Chromatography Techniques 151

2.1 Basic Principles of Important Liquid Chromatography

Techniques

151

2.1.1 Ion Exchange Chromatography 153

2.1.2 Reversed Phase Chromatography 162

2.1.3 Affinity Chromatography 167

2.1.4 Gel Filtration 172

2.2 Strategic Approach and General Applicability 174

2.3 Liquid Chromatography Techniques and Applications in Proteome

Analysis

176

2.3.1 Peptide Separation 176

2.3.2 2DLC Peptide Separation 179

2.3.3 Affinity Chromatography and LC-MS/MS 187

2.3.4 Protein Pre-fractionation 189

2.4 Practical Considerations and Application of LC-based Protein

Pre-fractionation

194

2.4.1 Sample Extraction and Preparation 196

2.4.2 Experimental Setup 197

2.4.3 Ion Exchange Chromatography and

Protein Pre-fractionation

198

Contents

VII

2.4.4 Reversed Phase Chromatography and

Protein Pre-fractionation

205

2.4.5 Fraction Size and Number of Fractions 210

2.5 Critical Review and Outlook 211

3 Mass Spectrometry 215

3.1 Ionization 218

3.1.1 Matrix Assisted Laser Desorption Ionization 218

3.1.2 Electrospray Ionization 222

3.2 Ion Separation 225

3.2.1 Time-of-Flight Analyzer 225

3.2.2 Triple Quadrupole Analyzer 227

3.2.3 Quadrupole Ion Trap 228

3.2.4 Quadrupole Time-of-Flight 230

3.2.5 Hybrid Triple Quadrupole Linear Ion Trap 231

3.2.6 TOF/TOF Analyzer 231

3.2.7 Fourier Transform Ion Cyclotron 232

3.2.8 Orbitrap 233

3.3 Generating MS Data for Protein Identification 233

3.3.1 Peptide Mass Fingerprint 234

3.3.2 Peptide Mass Fingerprint Combined With Composition

Information

237

3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence

Information

238

3.3.4 Tandem Mass Spectrometry 242

3.4 Protein Characterization 258

3.4.1 Phosphorylation Analysis 259

3.4.2 Affinity Chromatography 260

3.4.3 Chemical Derivatization 261

3.4.4 Glycosylation 263

3.5 Protein Quantification Using Mass Spectrometry 264

3.5.1 Stable Isotope Labeling Approaches 264

3.5.2 Isotope-coded Affinity Tags 265

3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture 266

3.5.4 AQUA 267

3.5.5 iTRAQ 267

3.5.6 Non-labeling Software Approaches 268

3.6 MS Strategies 271

3.6.1 Bottom up Approach 271

3.6.2 Top down Approach 272

4 Functional Proteomics: Studies of Protein–Protein Interactions 273

4.1 Non-immunological Methods 273

4.1.1 Separation of Intact Multi-protein Complexes 273

4.1.2 Probing with Interaction Partners 273

Contents

4.1.3 Surface Plasmon Resonance 274

4.2 Antibody-based Techniques 275

4.2.1 Western Blotting and Dot Blots 275

4.2.2 Protein Microarrays 276

Part II: Practical Manual of Proteome Analysis 279

Equipment, Consumables, Reagents 281

Step 1: Sample Preparation 287

Step 2: Fluorescence Difference Gel Electrophoresis 299

Step 3: Isoelectric Focusing 309

Step 4: SDS Polyacrylamide Gel Electrophoresis 323

Step 5: Scanning of Gels Containing Pre-labeled Proteins 357

Step 6: Staining of Gels 361

Step 7: Image Analysis and Evaluation of DIGE Gels 373

Step 8: Spot Excision 383

Step 9: Sample Destaining 387

Step 10: Protein Digestion 389

Step 11: Microscale Desalting and Concentrating of Sample 393

Step 12: Chemical Derivatization of the Peptide Digest 397

Step 13: MS Analysis 399

Step 14: Calibration of MALDI-ToF MS 403

Step 15: Preparing for a Database Search 407

Part III: Trouble Shooting 411

1 Two-dimensional Electrophoresis 413

1.1 Sample Preparation 413

1.2 Isoelectric focusing in IGPG strips 414

1.3 SDS PAGE 416

1.4 Staining 417

ContentsVIII

1.5 DIGE Fluorescence Labeling 418

1.6 Results in 2-D Electrophoresis 421

2 Mass Spectrometry 429

References 433

Glossary of Terms 461

Index 473

Legal Statements 481

Contents IX