Proteomics in Practice: A Guide to Successful Experimental Design

✍ Scribed by Reiner Westermeier, Tom Naven, Hans-Rudolf Höpker

• Publisher: Wiley-VCH
• Year: 2008
• Tongue: English
• Leaves: 505
• Edition: 2
• Category: Library

✦ Synopsis

This is a really great resource for people starting out in proteomics research. I'm a graduate student, and I have not yet seen such a well laid out book. The protocols are really well formatted. The importance of steps is explained (to the side, so you can skip over them if you already know), there are hints similar to what you would get if you were training from a real person, and the diagrams are very clear. I keep checking this out at the library (grad student salary), but if I had the money, I would definitely buy this book.

✦ Table of Contents

Proteomics in Practice......Page 4
Contents......Page 8
Preface......Page 14
Foreword......Page 16
Abbreviations, Symbols, Units......Page 18
1 History......Page 24
2.1 Challenges of the Protein Samples......Page 31
2.2 Challenges of the Analysis Systems......Page 34
3.2 Differential Analysis......Page 35
3.6 Systems Biology......Page 36
4.3 Pre-fractionation of Samples: Yes or No?......Page 37
4.4 Which is the Best Workflow to Start With?......Page 38
Part I: Proteomics Technology......Page 40
1.1 The Principle of Electrophoresis and Some Methodological Background......Page 42
1.1.1 Free Flow Electrophoretic Methods......Page 43
1.1.3 Electroendosmosis Effects......Page 44
1.2.1 The Polyacrylamide Gel......Page 45
1.2.2 SDS Polyacrylamide Gel Electrophoresis......Page 50
1.2.3 Blue Native Electrophoresis......Page 55
1.2.4 Cationic Detergent Electrophoresis......Page 57
1.3 Blotting......Page 58
1.3.2 Protein Detection on the Membrane......Page 59
1.4 Isoelectric Focusing......Page 61
1.4.1 Theoretical Background......Page 62
1.4.2 Preparation of IEF Gels......Page 67
1.4.3 Isoelectric Focusing in Proteomics......Page 68
1.5.1 Sample Preparation......Page 76
1.5.2 Pre-labeling of Proteins for Difference Gel Electrophoresis......Page 91
1.5.3 First Dimension: Isoelectric Focusing in IPG Strips......Page 100
1.5.4 Second Dimension: SDS Electrophoresis......Page 123
1.5.5 Detection of Protein Spots......Page 142
1.6.1 Image Acquisition......Page 148
1.6.2 Image Analysis and Evaluation......Page 152
1.7 Spot Handling......Page 160
1.7.1 Spot Picking......Page 162
1.7.2 Protein Cleavage......Page 164
2.1 Basic Principles of Important Liquid Chromatography Techniques......Page 174
2.1.1 Ion Exchange Chromatography......Page 176
2.1.2 Reversed Phase Chromatography......Page 185
2.1.3 Affinity Chromatography......Page 190
2.1.4 Gel Filtration......Page 195
2.2 Strategic Approach and General Applicability......Page 197
2.3.1 Peptide Separation......Page 199
2.3.2 2DLC Peptide Separation......Page 202
2.3.3 Affinity Chromatography and LC-MS/MS......Page 210
2.3.4 Protein Pre-fractionation......Page 212
2.4 Practical Considerations and Application of LC-based Protein Pre-fractionation......Page 217
2.4.1 Sample Extraction and Preparation......Page 219
2.4.2 Experimental Setup......Page 220
2.4.3 Ion Exchange Chromatography and Protein Pre-fractionation......Page 221
2.4.4 Reversed Phase Chromatography and Protein Pre-fractionation......Page 228
2.4.5 Fraction Size and Number of Fractions......Page 233
2.5 Critical Review and Outlook......Page 234
3 Mass Spectrometry......Page 238
3.1.1 Matrix Assisted Laser Desorption Ionization......Page 241
3.1.2 Electrospray Ionization......Page 245
3.2.1 Time-of-Flight Analyzer......Page 248
3.2.2 Triple Quadrupole Analyzer......Page 250
3.2.3 Quadrupole Ion Trap......Page 251
3.2.4 Quadrupole Time-of-Flight......Page 253
3.2.6 TOF/TOF Analyzer......Page 254
3.2.7 Fourier Transform Ion Cyclotron......Page 255
3.3 Generating MS Data for Protein Identification......Page 256
3.3.1 Peptide Mass Fingerprint......Page 257
3.3.2 Peptide Mass Fingerprint Combined With Composition Information......Page 260
3.3.3 Peptide Mass Fingerprint Combined With Partial Sequence Information......Page 261
3.3.4 Tandem Mass Spectrometry......Page 265
3.4 Protein Characterization......Page 281
3.4.1 Phosphorylation Analysis......Page 282
3.4.2 Affinity Chromatography......Page 283
3.4.3 Chemical Derivatization......Page 284
3.4.4 Glycosylation......Page 286
3.5.1 Stable Isotope Labeling Approaches......Page 287
3.5.2 Isotope-coded Affinity Tags......Page 288
3.5.3 Stable Isotope Labeling with Amino Acids in Cell Culture......Page 289
3.5.5 iTRAQ......Page 290
3.5.6 Non-labeling Software Approaches......Page 291
3.6.1 Bottom up Approach......Page 294
3.6.2 Top down Approach......Page 295
4.1.2 Probing with Interaction Partners......Page 296
4.1.3 Surface Plasmon Resonance......Page 297
4.2.1 Western Blotting and Dot Blots......Page 298
4.2.2 Protein Microarrays......Page 299
Part II: Practical Manual of Proteome Analysis......Page 302
Equipment, Consumables, Reagents......Page 304
Step 1: Sample Preparation......Page 310
Step 2: Fluorescence Difference Gel Electrophoresis......Page 322
Step 3: Isoelectric Focusing......Page 332
Step 4: SDS Polyacrylamide Gel Electrophoresis......Page 346
Step 5: Scanning of Gels Containing Pre-labeled Proteins......Page 380
Step 6: Staining of Gels......Page 384
Step 7: Image Analysis and Evaluation of DIGE Gels......Page 396
Step 8: Spot Excision......Page 406
Step 9: Sample Destaining......Page 410
Step 10: Protein Digestion......Page 412
Step 11: Microscale Desalting and Concentrating of Sample......Page 416
Step 12: Chemical Derivatization of the Peptide Digest......Page 420
Step 13: MS Analysis......Page 422
Step 14: Calibration of MALDI-ToF MS......Page 426
Step 15: Preparing for a Database Search......Page 430
Part III Trouble Shooting......Page 434
1.1 Sample Preparation......Page 436
1.2 Isoelectric focusing in IGPG strips......Page 437
1.3 SDS PAGE......Page 439
1.4 Staining......Page 440
1.5 DIGE Fluorescence Labeling......Page 441
1.6 Results in 2-D Electrophoresis......Page 444
2 Mass Spectrometry......Page 452
References......Page 456
Glossary of Terms......Page 484
Index......Page 496
Legal Statements......Page 504