Summary

A combined review, manual and reference for the successful analysis of proteins using the classical approach of 2-D gel electrophoresis, mass spectrometry and related sequence database inquiries. The first section, written in a textbook style, introduces the entire technology, while the second section represents a comprehensive laboratory manual spanning the full range of methods from sample preparation to protein identification. Alternative methods and procedures are only suggested for those cases where the "default" procedure would fail to deliver adequate results. The third section is a unique troubleshooting guide, designed to answer many of the frequently asked questions regarding proteome analysis. The final section contains a thorough reference list to guide interested readers towards further detail. Intended for all those wishing to go beyond the theoretical aspects of proteome analysis, this book is targeted at research groups within academia and industry, course instructors, research assistants and graduate students. Special feature: Updates of recipes as well as animations illustrating crucial methodological steps will be available on the Web at http://www.wiley-vch.de/books/info/3-527-30354-5

Author Biography

Reiner Westermeier gained his doctorate in 1981 at the Technical University of Munich, Germany. There he remained engaged in the development of new electrophoresis systems and applications until 1983, a period that also involved several international collaborations and lecturing tours.
From 1984 to 1987 Reiner Westermeier was employed as an electrophoresis products and applications specialist at LKB Instrument GmbH thereafter until 1990 at Pharmacia Biotech. He founded ETC Elektrophorese-Technik in 1991 for the development of new electrophoresis methods, media, and equipment. From January 1996 to July 1997 he was manager of scientific support Europe at Pharmacia Biotech in Freiburg, Germany. He has been at Amersham Pharmacia Biotech, later GE Healthcare since July 1997, and its Senior Scientist for Proteomics since January 2002.
Reiner Westermeier is the author of several publications and two books, both published by WILEY-VCH.
Tom Naven and Hans-Rudolf H+¦pker are also working for GE Healthcare as proteomics scientists.

Preface	p. XI
Foreword	p. XIII
Abbreviations, Symbols, Units	p. XV
Introduction	p. 1
History	p. 1
Critical Points	p. 8
Challenges of the Protein Samples	p. 8
Challenges of the Analysis Systems	p. 11
Proteomics Strategies	p. 12
Proteome Mapping	p. 12
Differential Analysis	p. 12
Time Point Experiments	p. 13
Verification of Targets or Biomarkers	p. 13
Integration of Results into Biological Context	p. 13
Systems Biology	p. 13
Concept of Experimental Planning	p. 14
Biological Replicates	p. 14
Pooling of Samples: Yes or No?	p. 14
Pre-fractionation of Samples: Yes or No?	p. 14
Which is the Best Workflow to Start With?	p. 15
Proteomics Technology
Electrophoretic Techniques	p. 19
The Principle of Electrophoresis and Some Methodological Background	p. 19
Free Flow Electrophoretic Methods	p. 20
Gels for Electrophoretic Techniques	p. 21
Electroendosmosis Effects	p. 21
Polyacrylamide Gel Electrophoresis	p. 22
The Polyacrylamide Gel	p. 22
SDS Polyacrylamide Gel Electrophoresis	p. 27
Blue Native Electrophoresis	p. 32
Cationic Detergent Electrophoresis	p. 34
Blotting	p. 35
Electrophoretic Transfer	p. 36
Protein Detection on the Membrane	p. 36
Isoelectric Focusing	p. 38
Theoretical Background	p. 39
Preparation of IEF Gels	p. 44
Isoelectric Focusing in Proteomics	p. 45
Two-dimensional Electrophoresis	p. 53
Sample Preparation	p. 53
Pre-labeling of Proteins for Difference Gel Electrophoresis	p. 68
First Dimension: Isoelectric Focusing in IPG Strips	p. 77
Second Dimension: SDS Electrophoresis	p. 100
Detection of Protein Spots	p. 119
Image Analysis	p. 125
Image Acquisition	p. 125
Image Analysis and Evaluation	p. 129
Use of 2-D Electrophoresis Data	p. 137
Spot Handling	p. 137
Spot Picking	p. 139
Protein Cleavage	p. 141
Liquid Chromatography Techniques	p. 151
Basic Principles of Important Liquid Chromatography Techniques	p. 151
Ion Exchange Chromatography	p. 153
Reversed Phase Chromatography	p. 162
Affinity Chromatography	p. 167
Gel Filtration	p. 172
Strategic Approach and General Applicability	p. 174
Liquid Chromatography Techniques and Applications in Proteome Analysis	p. 176
Peptide Separation	p. 176
2DLC Peptide Separation	p. 179
Affinity Chromatography and LC-MS/MS	p. 187
Protein Pre-fractionation	p. 189
Practical Considerations and Application of LC-based Protein Pre-fractionation	p. 194
Sample Extraction and Preparation	p. 196
Experimental Setup	p. 197
Ion Exchange Chromatography and Protein Pre-fractionation	p. 198
Reversed Phase Chromatography and Protein Pre-fractionation	p. 205
Fraction Size and Number of Fractions	p. 210
Critical Review and Outlook	p. 211
Mass Spectrometry	p. 215
Ionization	p. 218
Matrix Assisted Laser Desorption Ionization	p. 218
Electrospray Ionization	p. 222
Ion Separation	p. 225
Time-of-Flight Analyzer	p. 225
Triple Quadrupole Analyzer	p. 227
Quadrupole Ion Trap	p. 228
Quadrupole Time-of-Flight	p. 230
Hybrid Triple Quadrupole Linear Ion Trap	p. 231
TOF/TOF Analyzer	p. 231
Fourier Transform Ion Cyclotron	p. 232
Orbitrap	p. 233
Generating MS Data for Protein Identification	p. 233
Peptide Mass Fingerprint	p. 234
Peptide Mass Fingerprint Combined With Composition Information	p. 237
Peptide Mass Fingerprint Combined With Partial Sequence Information	p. 238
Tandem Mass Spectrometry	p. 242
Protein Characterization	p. 258
Phosphorylation Analysis	p. 259
Affinity Chromatography	p. 260
Chemical Derivatization	p. 261
Glycosylation	p. 263
Protein Quantification Using Mass Spectrometry	p. 264
Stable Isotope Labeling Approaches	p. 264
Isotope-coded Affinity Tags	p. 265
Stable Isotope Labeling with Amino Acids in Cell Culture	p. 266
AQUA	p. 267
iTRAQ	p. 267
Non-labeling Software Approaches	p. 268
MS Strategies	p. 271
Bottom up Approach	p. 271
Top down Approach	p. 272
Functional Proteomics: Studies of Protein-Protein Interactions	p. 273
Non-immunological Methods	p. 273
Separation of Intact Multi-protein Complexes	p. 273
Probing with Interaction Partners	p. 273
Surface Plasmon Resonance	p. 274
Antibody-based Techniques	p. 275
Western Blotting and Dot Blots	p. 275
Protein Microarrays	p. 276
Practical Manual of Proteome Analysis	p. 279
Equipment, Consumables, Reagents	p. 281
Sample Preparation	p. 287
Fluorescence Difference Gel Electrophoresis	p. 299
Isoelectric Focusing	p. 309
SDS Polyacrylamide Gel Electrophoresis	p. 323
Scanning of Gels Containing Pre-labeled Proteins	p. 357
Staining of Gels	p. 361
Image Analysis and Evaluation of DIGE Gels	p. 373
Spot Excision	p. 383
Sample Destaining	p. 387
Protein Digestion	p. 389
Microscale Desalting and Concentrating of Sample	p. 393
Chemical Derivatization of the Peptide Digest	p. 397
MS Analysis	p. 399
Calibration of MALDI-ToF MS	p. 403
Preparing for a Database Search	p. 407
Trouble Shooting	p. 411
Two-dimensional Electrophoresis	p. 413
Sample Preparation	p. 413
Isoelectric focusing in IGPG strips	p. 414
SDS PAGE	p. 416
Staining	p. 417
DIGE Fluorescence Labeling	p. 418
Results in 2-D Electrophoresis	p. 421
Mass Spectrometry	p. 429
References	p. 433
Glossary of Terms	p. 461
Index	p. 473
Legal Statements	p. 481
Table of Contents provided by Ingram. All Rights Reserved.

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