Proteomics in Practice

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1 Reiner Westermeier, Torn Naven Hans-Rudolf Höpker Proteomics in Practice A Guide to Successful Experimental Design

2 2008 Wiley-VCH Verlag- Weinheim

3 Preface Foreword XI XIII Abbreviations, Symbols, Units XV Introduction 1 1 History 1 2 Critical Points Challenges of the Protein Samples Challenges of the Analysis Systems 11 3 Proteomics Strategies Proteome Mapping Differential Analysis Time Point Experiments Verification of Targets or Biomarkers Integration of Results into Biological Context Systems Biology 13 4 Concept of Experimental Planning Biological Replicates Pooling of Samples: Yes or No? Pre-fractionation of Samples: Yes or No? Which is the Best Workflow to Start With? 15 Part I: Proteomics Technology 1 Electrophoretic Techniques The Principle of Electrophoresis and Some Methodological Background Free Flow Electrophoretic Methods Gels for Electrophoretic Techniques Electroendosmosis Effects Polyacrylamide Gel Electrophoresis 22

4 1.2.1 The Polyacrylamide Gel SDS Polyacrylamide Gel Electrophoresis Blue Native Electrophoresis Cationic Detergent Electrophoresis Blotting Electrophoretic Transfer Protein Detection on the Membrane Isoelectric Focusing Theoretical Background Preparation of IEF Gels Isoelectric Focusing in Proteomics Two-dimensional Electrophoresis Sample Preparation Pre-labeling of Proteins for Difference Gel Electrophoresis First Dimension: Isoelectric Focusing in IPG Strips Second Dimension: SDS Electrophoresis Detection of Protein Spots I mage Analysis I mage Acquisition I mage Analysis and Evaluation Use of 2-D Electrophoresis Data Spot Handling Spot Picking Protein Cleavage Liquid Chromatography Techniques Basic Principles of Important Liquid Chromatography Techniques Ion Exchange Chromatography Reversed Phase Chromatography Affinity Chromatography Gel Filtration Strategic Approach and General Applicability Liquid Chromatography Techniques and Applications in Proteome Analysis Peptide Separation DLC Peptide Separation Affinity Chromatography and LC-MS/MS Protein Pre-fractionation Practical Considerations and Application of LC-based Protein Pre-fractionation Sample Extraction and Preparation Experimental Setup Ion Exchange Chromatography and Protein Pre-fractionation 198

5 2.4.4 Reversed Phase Chromatography and Protein Pre-fractionation Fraction Size and Number of Fractions Critical Review and Outlook Mass Spectrometry Ionization Matrix Assisted Laser Desorption Ionization Electrospray Ionization Ion Separation Time-of-Flight Analyzer Triple Quadrupole Analyzer Quadrupole Ion Trap Quadrupole Time-of-Flight Hybrid Triple Quadrupole Linear Ion Trap TOF/TOF Analyzer Fourier Transform Ion Cyclotron Orbitrap Generating MS Data for Protein Identification Peptide Mass Fingerprint Peptide Mass Fingerprint Combined With Composition Information Peptide Mass Fingerprint Combined With Partial Sequence Information Tandem Mass Spectrometry Protein Characterization Phosphorylation Analysis Affinity Chromatography Chemical Derivatization Glycosylation Protein Quantification Using Mass Spectrometry Stable Isotope Labeling Approaches Isotope-coded Affinity Tags Stable Isotope Labeling with Amino Acids in Cell Culture AQUA itraq Non-labeling Software Approaches MS Strategies Bottom up Approach Top down Approach Functional Proteomics: Studies of Protein-Protein Interactions Non-immunological Methods Separation of Intact Multi-protein Complexes Probing with Interaction Partners 273

6 4.1.3 Surface Plasmon Resonance Antibody-based Techniques Western Blotting and Dot Blots 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 DICE 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 Ill: Trouble Shooting Two-dimensional Electrophoresis Sample Preparation Isoelectric focusing in IGPG strips SDS PAGE Staining 417

7 1.5 DIGE Fluorescence Labeling Results in 2-D Electrophoresis Mass Spectrometry 429 References 433 Glossary of Terms 461 Index 473 Legal Statements 481

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