疾病蛋白质组学课件

疾病蛋白质组学PPT课件疾病蛋白质组学PPT课件疾病蛋白质组学PPT课件疾病蛋白质组学PPT课件疾病蛋白质组学PPT课件疾病蛋白质组1一、基本概念和总体研究概况一、基本概念和总体研究概况2疾病蛋白质组学

diseaseproteomics运用蛋白质组学研究手段，通过比较正常和病理情况下细胞、组织或体液中蛋白质在组成成分、表达水平、表达位置和修饰状态上的差异，寻找疾病诊断和预后的特异性蛋白质(群)，包括特异性抗原及相关抗原、受体、酶等，以及药物治疗的靶标等。通过深入了解这些疾病特异性蛋白质的结构和功能，揭示疾病过程中细胞内全部蛋白质的活动规律，为多种疾病发生、发展机制的阐明和早期诊断及治疗提供理论根据和解决途径。疾病蛋白质组学

diseaseproteomics运用蛋白3研究进展肿瘤蛋白质组：研究细胞的增殖、分化、异常转化、肿瘤形成白血病、乳腺癌、结肠癌、膀胱癌、前列腺癌、肺癌、肾癌、肝细胞癌和神经母细胞瘤等联合激光捕获微切割技术(Lasercapturemierodisseetion，LCM)，直接从肿瘤组织中提取纯肿瘤细胞，以克服组织内异质性的问题，为肿瘤蛋白质组研究提供了技术上的保障。鉴定了一批肿瘤相关蛋白，为肿瘤的早期诊断、药靶的发现、疗效和预后的判断提供了重要依据。在心脏、肺部、内分泌系统、神经系统疾病、药物成瘾性、环境毒理学、传染病、内耳相关疾病等方面，蛋白质组研究成果也为其提供了新的诊疗方向。国内：重点在肝病、恶性肿瘤、心血管、神经系统疾病和新发传染病等方面研究进展肿瘤蛋白质组：4存在问题和发展趋势利用蛋白质组研究的人类疾病的范围虽然日趋扩大，但仍停留在初级比较阶段。进一步鉴定、验证，发展成应用于临床的生物标志物开展全方位的蛋白质组相互作用网络的分析进一步提高蛋白分离和鉴定的通量、灵敏度和规模；提高生物信息学应用范围与准确率，进行信息综合，准确地分析蛋白质的相互作用，界定相互作用连锁群；存在问题和发展趋势利用蛋白质组研究的人类疾病的范围虽然日趋扩5二、心血管疾病蛋白质组学

CardiovascularProteomicsthecardiovascular(CV)systemiscomposedofanumberofspecializedcelltypesincludingcardiacmyocytes,fibroblast,neurons,endothelialandsmoothmusclecellsandnewlydiscoveredstemandprogenitorcells.Todate,theproteomeofthesecellsarenotwellcharacterizednorhastheinterplaybetweenthecelltypesbeenestablishedinhealthordisease.ThisremainsasignificantchallengeasCVdiseaseisthenumberonekillerworldwide.二、心血管疾病蛋白质组学

Cardiovascular6ResearchFocusThemyofilamentproteome.Redoxmodificationsinthecardiacproteome.Cardiacbiomarkers.SecretorymicrovesiclesProteomicsofthesecretomeResearchFocusThemyofilament7ThemyofilamentproteomeThemyofilament（肌丝）proteinsareresponsibleforthecontractilenatureofthecardiacmyocytes.themyofilamentsubproteomeallowsthehearttoactasapump.Themyofilamentproteinsarehighlyregulatedbyanumberofspecificpost-translationalmodifications(PTMs)someofwhichhavebeendiscoveredthroughproteomicstudies.PTMsofmyofilamentproteinscandirectlyimpactonthecontractilityoftheheart.ThemyofilamentproteomeThemy8Asimplifiedillustrationofthecardiacmyofilamentproteins.Thethickfilamentproteinsconsistofmyosinheavychain(MHC),myosin-bindingproteinC(MyBP-C),andtwomyosinlightchains(MLC1andMLC2).Thethinfilamentproteinsconsistofactin,tropomyosin(Tm),andthethreecomponentsoftroponin;troponinI(TnI),troponinC(TnC)andtroponinT(TnT).Phosphorylationsitesonthemyofilamentproteinsareindicatedwithasmalldiamond.Thelargescaffoldingprotein,titin,whichspansthesarcomere,isnotincludedinthisillustration.肌球蛋白重链(MHC):myosinheavychain肌球蛋白轻链-1,2(MLC1,2):myosinlightchain-1,2肌动蛋白：Actin肌球蛋白结合蛋白C(MyBP-c):myosinbindingproteinC）肌钙蛋白(TnT,TnI,TnC):troponinT,I，C-原肌球蛋白(Tm)：-tropomyosin肌联蛋白:titinAsimplifiedillustrationoft9Structureofaregionoftheoverlapregionofacardiacsarcomereindiastoleontheleftandduringsystoleontherightwithindicationsofmajorandfunctionallysignificantproteinphosphorylationsites.Structureofaregionoftheo10Post-translationalmodificationsofmyofilamentproteinsPost-translationalmodificatio11疾病蛋白质组学课件12SamplepreparationTherearetwocommonlyusedmyofilamentprotein-enrichmentstrategies.Bothmethodsarecompatiblewith1-DEand2-DEanalysis：TFA(trifluoroaceticacid,三氟醋酸)extraction：cellsarelysedwithlowionicbuffer,andmyofilamentproteinsareextractedfromtheresultingpelletwith1%TFAv/v.appliedtoextractmyofilamentproteinsfromminuteamounts(20,50mg)ofbiopsysamples.（ref:Proteomics2002,2,978–987.)Myofibrilisolation：intactmyofibrilscanbeisolatedformdetergent-skinned(detergentextraction)heartmuscleandstoredin50%glycerolat-20C.(ref:FASEBJ.2005,19,1137–1139.)SamplepreparationTherearetw13DetectionMethodsforProteinmodificationphosphorylationchanges:1-D-IEF(phosphorylationsignificantlydecreasesproteinpIvalues)Westernblotswithphosphorylation-site-specificantibodiesMSanalysis:MALDI-TOFcoupledwithphosphatasetreatmentorPostsourcedecay(PSD)immobilizedmetalaffinitycolumn(IMAC)enrichmentandLCseparationfollowedbyMS/MSanalysisDetectionMethodsforProtein14Immobilizedmetalaffinitycolumn(IMAC)Schematicofaffinitybindingofphosphopeptidestoimmobilizedmetalionaffinitycolumns.Immobilizedmetalaffinitycol15DetectionMethodsforProteinmodificationProteindegradation:1-D-gelseparationfollowedbyWesternblot2-DE,2-DDIGEdirectsequencingfromtheNterminusorMS(exactsiteofdegradation)oxidationandnitrosylation:gelelectrophoresis(changeapparentMWandpIvalues)nano-ESILC/MS/MS(identifynitrotyrosineresidues)“top-down”MS(傅里叶转换离子回旋共振质谱）DetectionMethodsforProtein16文献阅读ProteomicsClin.Appl.(2008)ChaoYuan,R.JohnSolaro.Myofilamentproteins:Fromcardiacdisorderstoproteomicchanges(p788-799)WenhaiJin,AnnaT.Brown,AnneM.Murphy.Cardiacmyofilaments:fromproteometopathophysiology

(p800-810)

文献阅读ProteomicsClin.Appl.(20172.RedoxmodificationsinthecardiacproteomeMyocardialischemiaresultsinoxidativestress,whichinvolvesthemitochondriaandmany/allaspectsofmyocytefunction.Duetothesusceptibilityofcardiacproteintooxidativedamage,proteomicscanhelptodiscover,quantify,andcharacterizetheredoxsignalingandoxidativePTMs.NitricoxideisakeymediatorofCVcellularresponseinacuteandchronicdiseasesettings.Newapproachesintheproteomicscanhelpidentifyanddefineimportantpathwayofnitricoxide-inducedPTMs.2.Redoxmodificationsinthe18OutlineofpotentialconsequencesofoxidativestressincellsystemOxidantscanreactwithproteinstocauseoneoftwobroadconsequences.Theycanoxidisecellularcomponentssuchasproteins,renderingthemdysfunctional,whichnegativelyaffectscellfunctionandpromotesdisease.Inthisscenario,antioxidantscanpreventthecellularproteinsfrombeingoxidisedandsoprovideprotection.Incontrast,oxidantscaninduceregulatorypost-translationaloxidativeproteinmodifications,whichareimportantforstressadaptation.Thus,antioxidantscaninterferewithhomeostaticcontrolandmightexplainwhyantioxidanttherapiescanbedetrimentalinsomecases.Outlineofpotentialconsequen19MechanismsofROSgeneration.Sequentialreductionofmolecularoxygentogeneratesuperoxide,hydrogenperoxideandthenhydroxylradical.Listofaminoacidsparticularlysusceptibletomodification.MechanismsofROSgeneration.20DiagramshowingtheproductionofNOandRNS,withtheireffectsonbiologicaltargets.Athighconcentrations,NOreactsmainlywithoxygensuperoxideformingperoxynitrite(ONOO)andperoxynitrousacid(ONOOH).Inthisway,NOisintimatelylinkedwithROS.Moreover,thereactionofNOwithO2leadstotheformationofthehighlypoisonousnitrogendioxide(NO2),dinitrogentetroxide(N2O4),orboth.Atlowconcentrations,thedirecteffectsofNOpredominate(dashedarrow)andhaemsandredoxmetalsatiron–sulphurcentresinproteinsarethemaintargets.Ni-NOR,nitrite:nitricoxidereductase;Ni,nitritereductase;NOS,nitricoxidesynthase;NR,nitratereductase;RSNOs,S-nitrosothiols.Diagramshowingtheproduction21Structureofcommonredoxmodificationsofaminoacidsidechains.ROSandRNScanchemicallymodifyaminoacids,particularlythesidechainsofthoseoutlinedhere.Clearly,cysteinethiolsaresubjecttoadiverserangeofalterations.亚磺酸磺酸次磺酸亚砜

亚硝基硫醇

羰基化

硝基化酪氨酸Structureofcommonredoxmodi22Commonlyobservedoxidativemodificationsofproteinaminoacids(A)cysteine;(B)methionine;(C)tyrosine;(D)tryptophan.Alltheaminoacidsareschematicallyrepresentedaspartofapolypeptidechain.However,thenamesshownarethoseoffreeaminoacidsforconvenience.Commonlyobservedoxidativemo23ListofthemostutilizedmethodsinredoxproteomicsFrom:JournalofExperimentalBotany,Vol.59,No.14,pp.3781–3801,2008Listofthemostutilizedmeth24Biotinswitchmethod

Ahypotheticalproteinisindicatedwithcysteinesineitherthefreethiol,disulphide,ornitrosothiolconformations.Inthefirststep,freethiolsareblockedusingMMTS.Next,nitrosylatedcysteineresiduesareselectivelyreducedwithascorbateandthenewlygeneratedfreethiolsarefinallyS-biotinylatedwithbiotin-HPDP.ThebiotinylatedproteinscanbedetecteddirectlybyWesternblottingwithantibodiesspecificforbiotinorusingavidinorstreptavidin.Antibodiescanberadiolabelled,fluorescentlyorenzymaticallylabelled,asisknownintheart.Additionally,taggedproteinscanalsobeisolatedfromaffinitycolumnsorbeads.PSH,proteinsulphhydrylgroups;PSNO,Snitrosatedproteins.BiotinswitchmethodThebiot25IsotopeCodedAffinityTagging(ICAT)

(a).Thereagentconsistsofthreemoieties:anaffinitytagbiotin,alinkerthatcanincorporatesstableisotopes,andamaleimide(顺丁烯二酰亚胺)groupwhichreactsspecificallywiththethiolgroupofcysteine.

Twolabelledformsofthereagentareused,theheavycontainingeightdeuteriums(氘)andthelightwithnone.

(b)ProteinsfromtwodifferentcellstatesarelabelledwiththelightorheavyICATreagents.

Thesamplesarethencombinedanddigested.

TheICAT-labelledpeptidesareisolatedbyaffinitychromatographyusinganavidincolumnandthenanalysedHPLC-MS(/MS)directlyorbyMALDIofthecollectedHPLCfractions.

TheratioofthepeaksareasforspecificICAT-labelledpairsdefinestherelativeabundanceofitsparentproteinsbetweenthetwocellstatesquantificationofproteincysteineoxidationIsotopeCodedAffinityTagging26ListofcardiacproteinsdemonstratedtoundergooxidativemodificationRef:ProteomicsClin.Appl.2008,2,823–836Listofcardiacproteinsdemon273.CardiacbiomarkersDiagnosisofMIreliesonthedetectioninserumofacardiacspecificisoformofthemyofilamentprotein,troponinIwhichisreleasedintothebloodwhenthecardiacmyocytediesduesevereischemiaEarlierdetectionofMIordiagnosisofmyocardialischemiapriortocelldeathwillhelptoallowevenearlierinterventiontosave“potentiallyviable”teomicdiscoverypipelineforanalysisofhumanplasmasamplesforpatientswithinducedandcontrolMIhelpedtosetthestageforearlierdetectionofpatentsathighrisk.3.CardiacbiomarkersDiagnosis28CurrentgoldstandardmarkersofCVdistress(i)electrophysiologicalandfunctionalchangesasmonitoredbyelectrocardiographyandechocardiographyrespectively(ii)elevatedserumlevelsofcardiacspecificproteins：myofilamentproteinsandcardiactroponin-Iand-T（myocardialinfarction）brainnatriureticpeptideandinflammation-relatedproteins,includingC-reactiveprotein(CRP),（heartfailure）.cardiacenzymeslactatedehydrogenaseandcreatinekinase(CK)Currentgoldstandardmarkers29Severalapproachescurrentlyusedtoquantitatively

proproteomicexpressionpatternsfluorescence2-DDIGEcoupledtoMSanalysisProteinarraysinvitroandinvivostableisotopelabelLC-MStechniquesSignificantcostofusinglabeledreagentsinlarge-scalestudies.theapparentbiasofthesetechniquestowardslabelingtherelativelymostabundantspeciesinacomplexmixture,Morerecently,“label-free”differential(d)MS(无标记的质谱定量方法)Severalapproachescurrentlyu30疾病蛋白质组学课件31Workflowforlabel-freedMSanalysisofplasmasamples.(A)Workflowchart.Thesixstagesoftheprocessarerepresentedwithinthisfigureincludingsamplepreparation,additionofinternalstandardsandMSanalysis.Eachstageplaysanimportantroleinleadingtoasuccessfulofdeterminationofmeaningfuldifferentials.Workflowforlabel-freedMSan324.SecretorymicrovesiclesVascularsecretoryproteinandmembranevesiclescanaffecthomeostasisandcommunicationwithinentireCVsysteminresponsetoinjury.Schematicfigureoftheuseofproteomicsforthecharacterizationofthenon-cellularproteinfractionsrelevantinatherosclerosis.Thefigurerepresentsanatheroscleroticplaqueanditscellularcomponents.Thecellsinvolvedinatheromaformationreleasesolubleproteinsandmembranebodiesthatmodifythevascularmicroenvironment.Proteomicscanbeappliedtothecharacterizationofthesenon-cellularcomponentsoftheatheroscleroticmicroenvironment.4.SecretorymicrovesiclesVasc33Thelimitationsofplasmaproteomicsplasmaandserumareroutinelyusedforbiomarkerdiscoveryinproteomics.thehigh-abundanceproteins,notablyalbuminandimmunoglobulins,whichtogetherwithhaptoglobulin,antitrypsinandtransferrin,typicallyconstitutemorethan90%spectivebiomarkers:pg~ng/ml;albumin:35–50mg/mlthelimitedabilityofproteomicstodetectlow-abundanceplasmaproteinsThelimitationsofplasmaprot34Proteomicsofextracellularsecretory

vesicles(3)Matrixvesiclesareextracellularmembraneparticlesobservedintheinitialstagesofarterialcalcificationandcontainhighlevelsofcalcium-bindingacidicphospholipids.(4)Apoptoticbodiesarelargeparticlesreleasedfromcellsatthelaterstagesofprogrammedcelldeathandcharacterizedbylargediameter,nuclearcontent,andsurfaceligandsforphagocyticcellreceptors.(5)Heterogeneouspopulationorsecretorymicrovesicles.(1)Microparticlesarereleasedfromtheplasmamembraneofstimulatedorapoptoticcells.Theirproteincompositionmayvaryinresponsetodifferentstimuli(highshearstress,apoptosis,etc.).(2)Exosomesarethesmallestofthesecretorymembraneparticlesandaresecretedasaconsequenceofthefusionoftheplasmamembranewiththemultivesicularbodies(MVB).MVBarelatecomponentsoftheendocyticpathway.Proteomicsofextracellularse35Thecriticalpatho-physiologicalroleofmicroparticlesInthevascularcontext,microparticlesarereleasedbyendothelialcells,smoothmusclecells,lymphocytes,monocytes,erythrocytesandplatelets.Plasmalevelsofmicroparticlesaremarkedlyelevatedinpatientswithveinthrombosis,acutecoronarydisease,ischemicstroke,diabetes,myocardialinfarction,andhypertension.Microparticlesshowpro-coagulantactivity,pro-inflammatory,andpro-atheroscleroticactivities.modulatingtheendothelialsecretionofprostacyclinandnitricoxide;promotemonocyte-endotheliuminteractionbydirecttransferofarachidonicacidtotheplasmamembrane;physicallymediateleukocyte-leukocyteandleukocyte-endotheliuminteractionsviadirectbindingofcellsurfacereceptorsThecriticalpatho-physiologic36ProteomicsofmicroparticlesProteomicanalysisofproteinexpressioninhumanplasmamicroparticles.MicroparticlesderivedfromtheperipheralbloodbycentrifugationwerelysedandlabelledwithCydyes(greenandredcolourinAandB,respectively).UsingDIGE,microparticleandmicroparticle-depletedplasmaproteinswereco-separatedinlargeformat2-Dgels.ImageswereacquiredonafluorescencescannerandproteinsidentifiedbyLC-MS/MS.Actinandhaemoglobinareenrichedinmicroparticles,comparedtomicroparticle-depletedplasma.ProteomicsofmicroparticlesPr37characterisationofmicroparticlesreleasedbyaparticularcelltypeinvitrobyproteomicsBesidestheinvestigationofthemixtureofmicroparticlescontainedinhumanplasma,proteomicscanbeappliedtothecharacterisationofmicroparticlesreleasedbyaparticularcelltypeinvitro.plateletmicroparticles(J.ProteomeRes.2005;4:1516–1521)surfaceproteinstypicalofplatelets,suchasintegrinaIIb,integrinb3andP-selectin,andchemokines,suchasCXCL4,CXCL7andCCL5,380proteinsnotpreviouslyidentifiedinplateletsEndothelialcellsinresponsetostimulationwith(TNFa).(Proteomics2005;5:4443–4455)cytoskeletonandcytoskeleton-bindingproteins(tubulin,actin,cofilin,vimentin,etc.)membrane-associatedproteinsthatcontroltransportandsignalling(caveolin,annexins,dynein,etc.)foldingchaperones(calnexin,calreticulin,etc.)Adhesionmolecules,suchasICAM-1andintegrinsb1,a5anda2characterisationofmicroparti38TheroleofExosomesmodulateimmuneresponseregulatehaemostaticbalancesupportthrombingenerationandinduceexpressionandsecretionofplasminogenactivatorinhibitor-1byendothelialcellsattenuatingfibrinolysisandpromotingpro-thromboticconditionsabilitytobeabsorbedtothecellsurfaceandmediatecell-cellinteractionsinthecardiovascularsystemTheroleofExosomesmodulatei39Proteomicsofexosomesdendriticcell-derivedexosomes(J.Immunol.2001,166,7309–7318.)endocyticproteinswereabundantcomponentsoftheproteomeofexosomes.21newexosomalproteinswereidentified,includingcytoskeleton-relatedproteins,suchascofilin,profilinIorelongationfactor1a,andintracellularmembranetransportproteins,suchasannexins,rab7,11,rap1B,andsyntenin.aseriesofapoptosis-relatedproteins,includingthioredoxinperoxidaseII,Alix,14-3-3,andgalectin-3.mast-cellderivedexosomes(Arterioscler.Thromb.Vasc.Biol.2005,25,1744–1749)regulatethesecretionofplasminogenactivatorinhibitor-1byendothelialcellspossiblybyprothrombinasecomplexTNFaangiotensinogenprecursors.Proteomicsofexosomesdendriti405.Proteomicsofthesecretome“secretome”referredtothecomplexcollectionofproteinssecretedbyaparticulartypeofcell-oftenmaintainedinvitro.theanalysisofthesecretomeofdifferentbloodandvascularcelltypescouldbeofcriticalimportanceintheclarificationofheterogeneouscell-cellinteractionsandtheirregulationbyautocrineandparacrinefactors.Thelimitedcomplexityofthesecretomemakesitsuitablefortheapplicationofaproteomicapproach5.Proteomicsofthesecretome41ChallengestoProteomicsofthesecretomeItisdifficulttocompletelyavoidcross-contaminationwithproteinsoftheserumsupplementcommonlyusedincellcultures,althoughtheconditionedmediumisusuallysampledafterextensivewashingandduringincubationinserum-freemedium.Anyvariationinthecarry-overofserumproteinshasaprofoundimpactonquantitativecomparisonsCelldeathandcytoplasmicproteinreleaseintheculturemediumisasourceoffalsepositives,whichfurtherimpairsthereliabilityofproteomicanalysisofthesecretome.ChallengestoProteomicsofth42Technicalinnovationstoimprovethecapabilityofsecretomeanalysisprotein-enrichmentbyprecipitation(e.g.carrier-assistedTCAprecipitation)[Proteomics2007,7:1757–1770]high-abundanceserumproteindepletion(e.g.sodiumchloride/ethanolprecipitation)[Proteomics2005,5:2656–2664]),LCfraction(e.g.RPtC2Sorbent)[J.ProteomeRes.2006,5:899–906])dialysis/ultrafiltrationmethods[J.Microbiol.Methods2007,68:396–402].Technicalinnovationstoimpro43SUMMARY一、疾病蛋白质组学（diseaseproteomics）概念和总体研究概况二、心血管疾病蛋白质组学Themyofilamentproteome.Redoxmodificationsinthecardiacproteome.Cardiacbiomarkers.SecretorymicrovesiclesProteomicsofthesecretomeSUMMARY一、疾病蛋白质组学（diseaseprote44谢谢！谢谢！45疾病蛋白质组学PPT课件疾病蛋白质组学PPT课件疾病蛋白质组学PPT课件疾病蛋白质组学PPT课件疾病蛋白质组学PPT课件疾病蛋白质组46一、基本概念和总体研究概况一、基本概念和总体研究概况47疾病蛋白质组学

diseaseproteomics运用蛋白质组学研究手段，通过比较正常和病理情况下细胞、组织或体液中蛋白质在组成成分、表达水平、表达位置和修饰状态上的差异，寻找疾病诊断和预后的特异性蛋白质(群)，包括特异性抗原及相关抗原、受体、酶等，以及药物治疗的靶标等。通过深入了解这些疾病特异性蛋白质的结构和功能，揭示疾病过程中细胞内全部蛋白质的活动规律，为多种疾病发生、发展机制的阐明和早期诊断及治疗提供理论根据和解决途径。疾病蛋白质组学

diseaseproteomics运用蛋白48研究进展肿瘤蛋白质组：研究细胞的增殖、分化、异常转化、肿瘤形成白血病、乳腺癌、结肠癌、膀胱癌、前列腺癌、肺癌、肾癌、肝细胞癌和神经母细胞瘤等联合激光捕获微切割技术(Lasercapturemierodisseetion，LCM)，直接从肿瘤组织中提取纯肿瘤细胞，以克服组织内异质性的问题，为肿瘤蛋白质组研究提供了技术上的保障。鉴定了一批肿瘤相关蛋白，为肿瘤的早期诊断、药靶的发现、疗效和预后的判断提供了重要依据。在心脏、肺部、内分泌系统、神经系统疾病、药物成瘾性、环境毒理学、传染病、内耳相关疾病等方面，蛋白质组研究成果也为其提供了新的诊疗方向。国内：重点在肝病、恶性肿瘤、心血管、神经系统疾病和新发传染病等方面研究进展肿瘤蛋白质组：49存在问题和发展趋势利用蛋白质组研究的人类疾病的范围虽然日趋扩大，但仍停留在初级比较阶段。进一步鉴定、验证，发展成应用于临床的生物标志物开展全方位的蛋白质组相互作用网络的分析进一步提高蛋白分离和鉴定的通量、灵敏度和规模；提高生物信息学应用范围与准确率，进行信息综合，准确地分析蛋白质的相互作用，界定相互作用连锁群；存在问题和发展趋势利用蛋白质组研究的人类疾病的范围虽然日趋扩50二、心血管疾病蛋白质组学

CardiovascularProteomicsthecardiovascular(CV)systemiscomposedofanumberofspecializedcelltypesincludingcardiacmyocytes,fibroblast,neurons,endothelialandsmoothmusclecellsandnewlydiscoveredstemandprogenitorcells.Todate,theproteomeofthesecellsarenotwellcharacterizednorhastheinterplaybetweenthecelltypesbeenestablishedinhealthordisease.ThisremainsasignificantchallengeasCVdiseaseisthenumberonekillerworldwide.二、心血管疾病蛋白质组学

Cardiovascular51ResearchFocusThemyofilamentproteome.Redoxmodificationsinthecardiacproteome.Cardiacbiomarkers.SecretorymicrovesiclesProteomicsofthesecretomeResearchFocusThemyofilament52ThemyofilamentproteomeThemyofilament（肌丝）proteinsareresponsibleforthecontractilenatureofthecardiacmyocytes.themyofilamentsubproteomeallowsthehearttoactasapump.Themyofilamentproteinsarehighlyregulatedbyanumberofspecificpost-translationalmodifications(PTMs)someofwhichhavebeendiscoveredthroughproteomicstudies.PTMsofmyofilamentproteinscandirectlyimpactonthecontractilityoftheheart.ThemyofilamentproteomeThemy53Asimplifiedillustrationofthecardiacmyofilamentproteins.Thethickfilamentproteinsconsistofmyosinheavychain(MHC),myosin-bindingproteinC(MyBP-C),andtwomyosinlightchains(MLC1andMLC2).Thethinfilamentproteinsconsistofactin,tropomyosin(Tm),andthethreecomponentsoftroponin;troponinI(TnI),troponinC(TnC)andtroponinT(TnT).Phosphorylationsitesonthemyofilamentproteinsareindicatedwithasmalldiamond.Thelargescaffoldingprotein,titin,whichspansthesarcomere,isnotincludedinthisillustration.肌球蛋白重链(MHC):myosinheavychain肌球蛋白轻链-1,2(MLC1,2):myosinlightchain-1,2肌动蛋白：Actin肌球蛋白结合蛋白C(MyBP-c):myosinbindingproteinC）肌钙蛋白(TnT,TnI,TnC):troponinT,I，C-原肌球蛋白(Tm)：-tropomyosin肌联蛋白:titinAsimplifiedillustrationoft54Structureofaregionoftheoverlapregionofacardiacsarcomereindiastoleontheleftandduringsystoleontherightwithindicationsofmajorandfunctionallysignificantproteinphosphorylationsites.Structureofaregionoftheo55Post-translationalmodificationsofmyofilamentproteinsPost-translationalmodificatio56疾病蛋白质组学课件57SamplepreparationTherearetwocommonlyusedmyofilamentprotein-enrichmentstrategies.Bothmethodsarecompatiblewith1-DEand2-DEanalysis：TFA(trifluoroaceticacid,三氟醋酸)extraction：cellsarelysedwithlowionicbuffer,andmyofilamentproteinsareextractedfromtheresultingpelletwith1%TFAv/v.appliedtoextractmyofilamentproteinsfromminuteamounts(20,50mg)ofbiopsysamples.（ref:Proteomics2002,2,978–987.)Myofibrilisolation：intactmyofibrilscanbeisolatedformdetergent-skinned(detergentextraction)heartmuscleandstoredin50%glycerolat-20C.(ref:FASEBJ.2005,19,1137–1139.)SamplepreparationTherearetw58DetectionMethodsforProteinmodificationphosphorylationchanges:1-D-IEF(phosphorylationsignificantlydecreasesproteinpIvalues)Westernblotswithphosphorylation-site-specificantibodiesMSanalysis:MALDI-TOFcoupledwithphosphatasetreatmentorPostsourcedecay(PSD)immobilizedmetalaffinitycolumn(IMAC)enrichmentandLCseparationfollowedbyMS/MSanalysisDetectionMethodsforProtein59Immobilizedmetalaffinitycolumn(IMAC)Schematicofaffinitybindingofphosphopeptidestoimmobilizedmetalionaffinitycolumns.Immobilizedmetalaffinitycol60DetectionMethodsforProteinmodificationProteindegradation:1-D-gelseparationfollowedbyWesternblot2-DE,2-DDIGEdirectsequencingfromtheNterminusorMS(exactsiteofdegradation)oxidationandnitrosylation:gelelectrophoresis(changeapparentMWandpIvalues)nano-ESILC/MS/MS(identifynitrotyrosineresidues)“top-down”MS(傅里叶转换离子回旋共振质谱）DetectionMethodsforProtein61文献阅读ProteomicsClin.Appl.(2008)ChaoYuan,R.JohnSolaro.Myofilamentproteins:Fromcardiacdisorderstoproteomicchanges(p788-799)WenhaiJin,AnnaT.Brown,AnneM.Murphy.Cardiacmyofilaments:fromproteometopathophysiology

(p800-810)

文献阅读ProteomicsClin.Appl.(20622.RedoxmodificationsinthecardiacproteomeMyocardialischemiaresultsinoxidativestress,whichinvolvesthemitochondriaandmany/allaspectsofmyocytefunction.Duetothesusceptibilityofcardiacproteintooxidativedamage,proteomicscanhelptodiscover,quantify,andcharacterizetheredoxsignalingandoxidativePTMs.NitricoxideisakeymediatorofCVcellularresponseinacuteandchronicdiseasesettings.Newapproachesintheproteomicscanhelpidentifyanddefineimportantpathwayofnitricoxide-inducedPTMs.2.Redoxmodificationsinthe63OutlineofpotentialconsequencesofoxidativestressincellsystemOxidantscanreactwithproteinstocauseoneoftwobroadconsequences.Theycanoxidisecellularcomponentssuchasproteins,renderingthemdysfunctional,whichnegativelyaffectscellfunctionandpromotesdisease.Inthisscenario,antioxidantscanpreventthecellularproteinsfrombeingoxidisedandsoprovideprotection.Incontrast,oxidantscaninduceregulatorypost-translationaloxidativeproteinmodifications,whichareimportantforstressadaptation.Thus,antioxidantscaninterferewithhomeostaticcontrolandmightexplainwhyantioxidanttherapiescanbedetrimentalinsomecases.Outlineofpotentialconsequen64MechanismsofROSgeneration.Sequentialreductionofmolecularoxygentogeneratesuperoxide,hydrogenperoxideandthenhydroxylradical.Listofaminoacidsparticularlysusceptibletomodification.MechanismsofROSgeneration.65DiagramshowingtheproductionofNOandRNS,withtheireffectsonbiologicaltargets.Athighconcentrations,NOreactsmainlywithoxygensuperoxideformingperoxynitrite(ONOO)andperoxynitrousacid(ONOOH).Inthisway,NOisintimatelylinkedwithROS.Moreover,thereactionofNOwithO2leadstotheformationofthehighlypoisonousnitrogendioxide(NO2),dinitrogentetroxide(N2O4),orboth.Atlowconcentrations,thedirecteffectsofNOpredominate(dashedarrow)andhaemsandredoxmetalsatiron–sulphurcentresinproteinsarethemaintargets.Ni-NOR,nitrite:nitricoxidereductase;Ni,nitritereductase;NOS,nitricoxidesynthase;NR,nitratereducta