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Methods in Molecular Biology 2596 Kay Ohlendieck Editor Diff erence Gel Electrophoresis Methods and Protocols Second Edition M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Difference Gel Electrophoresis Methods and Protocols Second Edition Edited by Kay Ohlendieck Maynooth University, Maynooth, Ireland Editor KayOhlendieck MaynoothUniversity Maynooth,Ireland ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2830-0 ISBN978-1-0716-2831-7 (eBook) https://doi.org/10.1007/978-1-0716-2831-7 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. 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Preface TheaimofthissecondeditionofDifferenceGelElectrophoresisistoprovideacomprehensive updateoftechnicaldevelopmentsandnovelbioanalyticalapplicationsofthiskeymethodof mass spectrometry-based proteomics. Two-dimensional gel electrophoresis is one of the mostfrequentlyusedproteinseparationtechniquesinthebiologicalsciencesandproteomic investigations. DifferenceGelElectrophoresisemploysthedirectlabelingofproteomeswithfluorescent dyesprior tolarge-scalegelelectrophoreticseparationenablinghigh-throughputcompara- tive analyses. Labeling of two or more samples with different fluorescent dyes followed by separationonthesametwo-dimensionalgelsystemeliminatesgel-to-gelvariations,making this advanced separation technique a robust and highly suitable method for proteomic profilingsurveyswithdiversebiologicalapplications. Difference Gel Electrophoresis presents an introduction to the development of this method and outlines the principles of differential protein labeling and two-dimensional gel electrophoresis. The bioanalytical integration of the difference gel electrophoresis technique into optimized proteomic workflows using advanced mass spectrometry for protein identificationisprovided,aswellasdetailedstep-by-stepprotocolsfortheapplicationofthis methodinbasicbiologicalresearchandappliedbiomarkerdiscovery.Essentialbioinformat- ics tools used routinely in systems biology and standard biochemical and cell biological approaches for the independent verification of proteomic data sets are described. Detailed protocols of difference gel electrophoresis applications are provided for a wide range of systematicproteomicapplicationsinbasicbiology,pathobiology,andappliedbiochemistry. Following an introduction into the versatility of two-dimensional gel electrophoresis (Chap.1),thisneweditionoutlinestheconceptoftop-downproteomicstostudyindividual proteoforms (Chap. 2) and discusses analysis software used in protein identification approaches (Chap. 3). A section on the application of difference gel electrophoresis gives detaileddescriptionsofnativegelelectrophoresisforthequantitativeandfunctionalanalysis of protein interactions (Chap. 4), comparative fluorescence gel electrophoresis (Chap. 5), the identification of ubiquitination associated proteins (Chap. 6), the characterization of phosphorylationasakeypost-translationalmodification(Chap.7),biomarkerdiscoveryin blood cancers (Chap. 8), studying formalin-fixed and paraffin-embedded tissue sections (Chap.9),thefractionationandcharacterizationofserum/plasmasamples(Chap.10),the simultaneous analysis of several biological specimens on the same two-dimensional gel (Chap. 11), and the proteomic identification of saliva proteins as non-invasive diagnostic biomarkers(Chaps.12and13). An extensive section of Difference Gel Electrophoresis on comparative tissue proteomics outlinesdetailedprotocolsonthegel-basedanalysisofclinicalspecimens(Chap.14),animal tissue (Chap. 15), liver tissue from animal models (Chaps. 16 and 17), testis tissue (Chap. 18), human biopsy material (Chap. 19), subcellular muscle fractions (Chap. 20), andfishtissues(Chap.21).Thedescriptionofauxiliarymethodsofdifferencegelelectropho- resis includes sample preparation, protein determination, protein digestion, subcellular fractionation, immunodepletion of abundant protein species prior to gel electrophoretic analysis, and an overview of useful bioinformatics analysis tools (Chaps. 22, 23, 24, 25, and26). v vi Preface For the independent confirmation of key findings from gel-based proteomic studies, various routine biochemical and cell biological techniques are employed. The most fre- quently used verification approaches are described in a separate section that outlines the usefulness and applications of enzyme assays (Chaps. 27 and 28), immunoblot analysis (Chap. 29), immunofluorescence microscopy (Chap. 30), and histological and histochem- icalmicroscopy(Chap.31)inmodernproteomics. Maynooth,Ireland KayOhlendieck Contents Preface ..................................................................... v Contributors................................................................. xi PART I INTRODUCTION 1 Two-DimensionalGelElectrophoresisand2D-DIGE........ ....... ........ 3 PaulaMeleady PART II DIFFERENCE GEL ELECTROPHORESIS 2 Top-DownProteomicsandComparative2D-DIGEAnalysis......... ........ 19 KayOhlendieck 3 DIGEAnalysisSoftwareandProteinIdentificationApproaches....... ........ 39 PaulDowling PART III DIGE APPLICATIONS 4 NativeDIGEforQuantitativeandFunctionalAnalysisofProtein Interactomes ....... ....... ........ ....... ....... ........ ....... ..... ... 53 DikshaDaniandNorbertA.Dencher 5 ComparativeTwo-DimensionalFluorescenceGelElectrophoresis .... ...... .. 71 DoreenAckermannandSimoneKönig 6 IdentificationofUbiquitination-AssociatedProteinsUsing2D-DIGE......... 83 PaulDowlingandDespinaBazou 7 DIGE-BasedPhosphoproteomicAnalysis.... ....... ........ ....... ........ 97 TarasStasykandLukasAlfonsHuber 8 DIGE-BasedBiomarkerDiscoveryinBloodCancers......... ....... ........ 105 KatieDunphyandPaulDowling 9 DIGESaturationLabelingforScarceAmountsofProtein fromFormalin-FixedParaffin-Embedded(FFPE)Tissue...... ....... ........ 113 PaulDowling 10 DIGEAnalysisofProteoMiner™FractionatedSerum/PlasmaSamples ...... . 119 SandraMurphyandPaulDowling 11 Comparative3-Sample2D-DIGEAnalysisofSkeletalMuscles ....... ........ 127 KayOhlendieck 12 ProteomicIdentificationofSalivaProteinsasNoninvasive DiagnosticBiomarkers ..... ........ ....... ....... ........ ....... ........ 147 EleanorM.O’Sullivan,PaulDowling,DieterSwandulla, andKayOhlendieck 13 AnalysisoftheSalivaProteomeUsing2D-DIGE.... ........ ....... ........ 169 PaulDowlingandEleanorM.O’Sullivan vii viii Contents PART IV COMPARATIVE DIGE TISSUE PROTEOMICS 14 DIGEAnalysisofClinicalSpecimens........ ....... ........ ....... ........ 177 CeciliaGelfiandDanieleCapitanio 15 DIGEAnalysisofAnimalTissues .... ....... ....... ........ ....... ........ 201 AlessioDiLuca,RuthHamill,AnneMariaMullen, andGiulianoElia 16 2D-DIGEProteomicAnalysisofMouseLiverWithin1Week........ ........ 217 ShotaroKamataandIsaoIshii 17 2D-DIGEAnalysisofLiverDiseaseinMice......... ........ ... .... ...... .. 231 A¨ıshaCallebaut,RitaDerua,LutOverbergh, andVeerleJanssens 18 Two-DyeVersusThree-DyeDIGEforComparativeTestis TissueProteomicAnalysis........... ....... ....... ........ ....... ........ 245 AshlingHolland 19 Two-CyDye-Based2D-DIGEAnalysisofAgedHumanMuscle BiopsySpecimens.......... ........ ....... ....... ........ ....... ........ 265 KayOhlendieck 20 IdentificationofSubproteomicMarkersforSkeletal MuscleProfiling.... ...... ....... .. ... .... ....... ........ ....... ........ 291 PaulDowling,StephenGargan,DieterSwandulla, andKayOhlendieck 21 DIGEAnalysisofFishTissues....... ....... ....... ........ ....... ........ 303 JoannaNynca,MariolaA.Dietrich,andAndrzejCiereszko PART V AUXILIARY DIGE METHODS 22 SamplePreparationandProteinDetermination for2D-DIGEProteomics........... ....... ..... .. ...... .. ..... .. ........ 325 StephenGarganandKayOhlendieck 23 ProteinDigestionfor2D-DIGEAnalysis .... ....... ........ ....... ........ 339 SandraMurphyandKayOhlendieck 24 SubcellularFractionationforDIGE-BasedProteomics ....... ....... ........ 351 SandraMurphy 25 DIGEAnalysisofImmunodepletedPlasma ......... ........ ....... ........ 363 PaulDowlingandKayOhlendieck 26 BioinformaticAnalysisoftheSubproteomicProfile ofCardiomyopathicTissue........ .. ....... ....... ........ ....... ........ 377 SandraMurphy,MargitZweyer,DieterSwandulla, andKayOhlendieck PART VI DIGE VERIFICATION ANALYSIS 27 ElucidatingCellularMetabolismandProteinDifferenceData fromDIGEProteomicsExperimentsUsingEnzymeAssays.......... ........ 399 AndrewDowd Contents ix 28 EnzymeAssayMethodstoValidateDIGEProteomicsData.......... ........ 421 AndrewDowd 29 ImmunoblotAnalysisofDIGE-BasedProteomics ........... ....... ........ 429 MartinLandsbergerandHeinrichBrinkmeier 30 VerificationofProteinChangesDeterminedby2D-DIGEBased ProteomicsUsingImmunofluorescenceMicroscopy ......... ....... ........ 445 MargitZweyer,KayOhlendieck,andDieterSwandulla 31 HistologicalandHistochemicalMicroscopyUsedtoVerify 2D-DIGEPathoproteomics......... ....... ....... ........ ....... ........ 465 MargitZweyer,KayOhlendieck,andDieterSwandulla Index ...................................................................... 481