Proteomics in practice : a guide to successful experimental design / Reiner Westermeier, Tom Naven, and Hans-Rudolf Hopker. — 2nd completely rev. ed. — Weinheim : Wiley-VCH Verlag GmbH & Co. KGaA, c2008. – (58.17421/W527/2nd rev. ed.)

Contents

    Contents
    
    Preface XI
    Foreword XIH
    Abbreviations， Symbols， Units XV
    Introduction 1
    1 History 1
    2 Critical Points 8
    2.1 Challenges of the Protein Samples
    2.2 Challenges of the Analysis Systems
    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 19
    1.1 The Principle of Electrophoresis and Some Methodological Background 19
    1.2 Polyacrylamide Gel Electrophoresis 22
    1.3 Blotting 35
    1.4 Isoelectric Focusing 38
    1.5 Two-dimensional Electrophoresis 53
    1.6 Image Analysis 125
    1.7 Spot Handling 137
    2 Liquid Chromatography Techniques 151
    2.1 Basic Principles of Important Liquid Chromatography Techniques 151
    2.2 Strategic Approach and General Applicability 174
    2.3 Liquid Chromatography Techniques and Applications in Proteome Analysis 176
    2.4 Practical Considerations and Application of LC-based Protein Pre-fractionation 194
    2.5 Critical Review and Outlook 211
    3 Mass Spectrometry 215
    3.1 Ionization 218
    3.2 Ion Separation 225
    3.3 Generating MS Data for Protein Identification 233
    3.4 Protein Characterization 258
    3.5 Protein Quantification Using Mass Spectrometry 264
    3.6 MS Strategies 271
    4 Functional Proteomics: Studies of Protein-Protein Interactions 273
    4.1 Non-immunological Methods 273
    4.2 Antibody-based Techniques 275
    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
    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