Proteomics in Practice: A Guide to Successful Experimental Design, Second Edition

492 Pages·2008·6.086 MB·English

by  Dr. Reiner Westermeier, Dr. Tom Naven, Hans?Rudolf Hopker(auth.)

ReinerWestermeier,TomNaven,
andHans-RudolfHçpker
ProteomicsinPractice
ProteomicsinPractice.AGuidetoSuccessfulExperimentalDesign2ndEd.
ReinerWestermeier,TomNaven,andHans-RudolfHçpker
Copyright(cid:1)2008WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:978-3-527-31941-1
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Reiner Westermeier, Tom Naven, and
Hans-Rudolf Hçpker
Proteomics in Practice
A Guide to Successful Experimental Design
Second,CompletelyRevisedEdition
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ISBN 978-3-527-31941-1
V
Contents
Preface XI
Foreword XIII
Abbreviations,Symbols,Units XV
Introduction 1
1 History 1
2 CriticalPoints 8
2.1 ChallengesoftheProteinSamples 8
2.1 ChallengesoftheAnalysisSystems 11
3 ProteomicsStrategies 12
3.1 ProteomeMapping 12
3.2 DifferentialAnalysis 12
3.3 TimePointExperiments 13
3.4 VerificationofTargetsorBiomarkers 13
3.5 IntegrationofResultsintoBiologicalContext 13
3.6 SystemsBiology 13
4 ConceptofExperimentalPlanning 14
4.1 BiologicalReplicates 14
4.2 PoolingofSamples:YesorNo? 14
4.3 Pre-fractionationofSamples:YesorNo? 14
4.4 WhichistheBestWorkflowtoStartWith? 15
PartI: ProteomicsTechnology
1 ElectrophoreticTechniques 19
1.1 ThePrincipleofElectrophoresisandSomeMethodological
Background 19
1.1.1 FreeFlowElectrophoreticMethods 20
1.1.2 GelsforElectrophoreticTechniques 21
1.1.3 ElectroendosmosisEffects 21
1.2 PolyacrylamideGelElectrophoresis 22
ProteomicsinPractice.AGuidetoSuccessfulExperimentalDesign2ndEd.
ReinerWestermeier,TomNaven,andHans-RudolfHçpker
Copyright(cid:1)2008WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:978-3-527-31941-1
VI Contents
1.2.1 ThePolyacrylamideGel 22
1.2.2 SDSPolyacrylamideGelElectrophoresis 27
1.2.3 BlueNativeElectrophoresis 32
1.2.4 CationicDetergentElectrophoresis 34
1.3 Blotting 35
1.3.1 ElectrophoreticTransfer 36
1.3.2 ProteinDetectionontheMembrane 36
1.4 IsoelectricFocusing 38
1.4.1 TheoreticalBackground 39
1.4.2 PreparationofIEFGels 44
1.4.3 IsoelectricFocusinginProteomics 45
1.5 Two-dimensionalElectrophoresis 53
1.5.1 SamplePreparation 53
1.5.2 Pre-labelingofProteinsforDifferenceGelElectrophoresis 68
1.5.3 FirstDimension:IsoelectricFocusinginIPGStrips 77
1.5.4 SecondDimension:SDSElectrophoresis 100
1.5.5 DetectionofProteinSpots 119
1.6 ImageAnalysis 125
1.6.1 ImageAcquisition 125
1.6.2 ImageAnalysisandEvaluation 129
1.6.3 Useof2-DElectrophoresisData 137
1.7 SpotHandling 137
1.7.1 SpotPicking 139
1.7.2 ProteinCleavage 141
2 LiquidChromatographyTechniques 151
2.1 BasicPrinciplesofImportantLiquidChromatography
Techniques 151
2.1.1 IonExchangeChromatography 153
2.1.2 ReversedPhaseChromatography 162
2.1.3 AffinityChromatography 167
2.1.4 GelFiltration 172
2.2 StrategicApproachandGeneralApplicability 174
2.3 LiquidChromatographyTechniquesandApplicationsinProteome
Analysis 176
2.3.1 PeptideSeparation 176
2.3.2 2DLCPeptideSeparation 179
2.3.3 AffinityChromatographyandLC-MS/MS 187
2.3.4 ProteinPre-fractionation 189
2.4 PracticalConsiderationsandApplicationofLC-basedProtein
Pre-fractionation 194
2.4.1 SampleExtractionandPreparation 196
2.4.2 ExperimentalSetup 197
2.4.3 IonExchangeChromatographyand
ProteinPre-fractionation 198
Contents VII
2.4.4 ReversedPhaseChromatographyand
ProteinPre-fractionation 205
2.4.5 FractionSizeandNumberofFractions 210
2.5 CriticalReviewandOutlook 211
3 MassSpectrometry 215
3.1 Ionization 218
3.1.1 MatrixAssistedLaserDesorptionIonization 218
3.1.2 ElectrosprayIonization 222
3.2 IonSeparation 225
3.2.1 Time-of-FlightAnalyzer 225
3.2.2 TripleQuadrupoleAnalyzer 227
3.2.3 QuadrupoleIonTrap 228
3.2.4 QuadrupoleTime-of-Flight 230
3.2.5 HybridTripleQuadrupoleLinearIonTrap 231
3.2.6 TOF/TOFAnalyzer 231
3.2.7 FourierTransformIonCyclotron 232
3.2.8 Orbitrap 233
3.3 GeneratingMSDataforProteinIdentification 233
3.3.1 PeptideMassFingerprint 234
3.3.2 PeptideMassFingerprintCombinedWithComposition
Information 237
3.3.3 PeptideMassFingerprintCombinedWithPartialSequence
Information 238
3.3.4 TandemMassSpectrometry 242
3.4 ProteinCharacterization 258
3.4.1 PhosphorylationAnalysis 259
3.4.2 AffinityChromatography 260
3.4.3 ChemicalDerivatization 261
3.4.4 Glycosylation 263
3.5 ProteinQuantificationUsingMassSpectrometry 264
3.5.1 StableIsotopeLabelingApproaches 264
3.5.2 Isotope-codedAffinityTags 265
3.5.3 StableIsotopeLabelingwithAminoAcidsinCellCulture 266
3.5.4 AQUA 267
3.5.5 iTRAQ 267
3.5.6 Non-labelingSoftwareApproaches 268
3.6 MSStrategies 271
3.6.1 BottomupApproach 271
3.6.2 TopdownApproach 272
4 FunctionalProteomics:StudiesofProtein–ProteinInteractions 273
4.1 Non-immunologicalMethods 273
4.1.1 SeparationofIntactMulti-proteinComplexes 273
4.1.2 ProbingwithInteractionPartners 273
VIII Contents
4.1.3 SurfacePlasmonResonance 274
4.2 Antibody-basedTechniques 275
4.2.1 WesternBlottingandDotBlots 275
4.2.2 ProteinMicroarrays 276
PartII: PracticalManualofProteomeAnalysis 279
Equipment,Consumables,Reagents 281
Step1:SamplePreparation 287
Step2:FluorescenceDifferenceGelElectrophoresis 299
Step3:IsoelectricFocusing 309
Step4:SDSPolyacrylamideGelElectrophoresis 323
Step5:ScanningofGelsContainingPre-labeledProteins 357
Step6:StainingofGels 361
Step7:ImageAnalysisandEvaluationofDIGEGels 373
Step8:SpotExcision 383
Step9:SampleDestaining 387
Step10:ProteinDigestion 389
Step11:MicroscaleDesaltingandConcentratingofSample 393
Step12:ChemicalDerivatizationofthePeptideDigest 397
Step13:MSAnalysis 399
Step14:CalibrationofMALDI-ToFMS 403
Step15:PreparingforaDatabaseSearch 407
PartIII: TroubleShooting 411
1 Two-dimensionalElectrophoresis 413
1.1 SamplePreparation 413
1.2 IsoelectricfocusinginIGPGstrips 414
1.3 SDSPAGE 416
1.4 Staining 417
Contents IX
1.5 DIGEFluorescenceLabeling 418
1.6 Resultsin2-DElectrophoresis 421
2 MassSpectrometry 429
References 433
GlossaryofTerms 461
Index 473
LegalStatements 481