丙烯酰胺胶中肽提取和蛋白消化技术Digestion of Proteins and Extraction of Peptides from an Acrylamide Gel相关交流-蚂蚁淘在线

丙烯酰胺胶中肽提取和蛋白消化技术Digestion of Proteins and Extraction of Peptides from an Acrylamide Gel

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DigestionofProteinsandExtractionofPeptidesfromanAcrylamideGel

SherryNiessen,IanMcLeodandJohnR.YatesIIIDepartmentofCellBIOLOGy,TheScrippsResearchInstitute,LaJolla,California.ExcerptedFromProtein:ProteinInteractions,SecondEditionEditedbyEricaA.GolemisandPeterD.Adams.

ABSTRACT
AnalysisofcellularproteinsbyMassSpectrometryrequirestheisolationofproteincomplexes,andenzymaticdigestionintopeptides.Afterproteinsareseparatedintobandsthroughelectrophoresisonanacrylamidegel,thisprotocolprovidesamethodforreduction,alkylationandin-geldigestionintopeptides,aswellasextractionoftheresultantpeptides.

MATERIALS
Buffers,Solutions,andReagents Acetonitrile Ammoniumbicarbonate(NH₄HCO₃),100mM CaCl₂,1M Digestionbuffer 50mMNH₄HCO₃,5mMCaCl₂ Formicacid90%,5% Milli-Qwater Iodoacetamide(IAA),55mM(preparefresh) K₃Fe(CN)₆ Na₂S₂O₃·5H₂O ProteinsingelslicesasobtainedinProtocol1,containedinamicrofugetube Tris(2-carboxyethyl)-phosphinehydrochloride(TCEP) Trypsin,sequencinggrade,modified SpecialEquipment Speedvacuum Incubator,presetto37°C
METHOD
RemovalofSilverStainBeforetheproteinsaredigested,theCoomassieorsilverstainmustberemovedfromthegel. Ifproteinswerevisualizedbysilverstain,firstremovesilverstain(steps1-3).However,ifproteinswerevisualizedbyCoomassie,beginatstep4.Ifthesilver-stainedgelslicewasstoredinaceticacid,removethestoragesolutionandrinsetheslicewithwater. Foreachgelslice,preparea30-mMsolutionofK₃Fe(CN)₆anda100-mMsolutionofNa₂S₂O₃·5H2O.Mix200µlofeachsolutiontogether(total400µl)andtransferthemixturetothemicrofugetubecontainingthegelpiece.Incubatefor5minwithgentlevortexing. Removetheliquidandwasheachgelslice3timeswith500µlofwater.Incubatetheslicesin100µlof100mMNH₄HCO₃for30min.Discardtheliquidandproceedwithstep4. RemovalofCoomassieStainingandTreatmentofGelSlices Atthispoint,thegelslicesareeitherstainedwithCoomassieorhavejusthadthesilverstainremoved.Ineithercase,washthegelsliceswithwaterfor15min.Allstepsshouldbeperformedwithlightvortexing.Thevolumeofsolventsaddedinthewashandelutionstepsshouldbetwicethatofthecutgelpieces(~100µl). Removetheliquidremainingfromstep4andadd100µlofwater,andthen100µlofacetonitriletothegelpieces.Incubatefor15minatroomtemperature. Removetheliquidandaddasufficientvolumeofacetonitriletocoverthegelpieces(~100µl).Thepieceswilldehydrate,shrinkingandturningwhiteandsticky.Afterthis,removetheacetonitrile. Rehydratetheslicesbyadding100µlof100mMNH₄HCO₃.Wait5min,andthenadd100µlofacetonitrile.Incubatefor15minatroomtemperature.IfCoomassiestainingwasusedtovisualizetheproteinsandastrongbluecolorpersists,incubateforafurther15minwithcontinuousgentlevortexing.Removetheliquidanddrydownthegelpiecesinaspeedvacuumfor15min. Reduction,Alkylation,andIn-gelDigestion Rehydratethegelpiecesusingasufficientvolumeof10mMTCEPin100mMNH₄HCO₃tocoverthem(~100µl).Incubatefor20-30minatroomtemperature. Removetheliquidandquicklyaddthesamevolume(~100µl)of55mMIAAin100mMNH₄HCO₃.Incubatefor30minatroomtemperatureinthedark.ItisimportantthattheIAAisfreshlyprepared. Removetheliquidanddehydratethegelbyadding100µlofacetonitrile.Afterthegelpieceshaveshrunkandturnedwhiteandsticky,removetheacetonitrile.Add100µlof100mMNH₄HCO₃andincubatefor5minatroomtemperaturetoallowrehydration. Add100µlofacetonitriletocreatea1:1mixof100mMNH₄HCO₃andacetonitrile,andincubatefor15min. Removetheliquidanddrythegelslicescompletelyinaspeedvacuum,foratleast30min.Rehydratein100µlofdigestionbuffercontaining12.5mg/mltrypsin.Incubteat37°Covernight. ExtractionofPeptides Centrifugethetrypsindigestsbrieflyinamicrocentrifugeandtransferthepeptides(inthesupernatant)toa0.5-mlmicrofugetubeandreserve.Inthemeantime,add100µlof25mMNH₄HCO₃tothegelpieces(makesuretheyarecoveredbythesolution)andincubatefor15min.Thenadd100µlofacetonitrileandincubateforafurther15min. Recovertheliquidandtransferittothe0.5-mltubecontainingthepeptides.Begindryingthepeptidesinaspeedvacuum,whilecontinuingwithsteps14and15.Add100µlof5%formicacidtothegelslicesandincubatefor15min.Next,add100µlofacetonitriletotheslicesandincubatefor15min. Recoverthesupernatantfromtheslicesandtransferittothe0.5-mltubecontainingthepeptides.Repeatstep14.Continuedryingthesampleinthe0.5-mlmicrofugetubeinthespeedvacuumfor~2hr,untilthereisonly10µlremaining.Atthispoint,thepeptidesarereadyforanalysis.AnalysiscanbedonebyESIMS,ESIMS/MS(tandemMS),orMALDI/TOF.

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丙烯酰胺胶中肽提取和蛋白消化技术Digestion&nbsp;of&nbsp;Proteins&nbsp;and&nbsp;Extraction&nbsp;of&nbsp;Peptides&nbsp;from&nbsp;an&nbsp;Acrylamide&nbsp;Gel

丙烯酰胺胶中肽提取和蛋白消化技术Digestion of Proteins and Extraction of Peptides from an Acrylamide Gel