疾病蛋白质组学

疾病蛋白质组学第1页/共44页一、基本概念和总体研究概况第2页/共44页疾病蛋白质组学

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。第3页/共44页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第4页/共44页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；第5页/共44页二、心血管疾病蛋白质组学

CardiovascularProteomicsthecardiovascular(CV)systemiscomposedofanumberofspecializedcelltypesincludingcardiacmyocytes,fibroblast,neurons,endothelialandsmoothmusclecellsandnewlydiscoveredstemandprogenitorcells.Todate,theproteomeofthesecellsarenotwellcharacterizednorhastheinterplaybetweenthecelltypesbeenestablishedinhealthordisease.ThisremainsasignificantchallengeasCVdiseaseisthenumberonekillerworldwide.第6页/共44页ResearchFocusThemyofilamentproteome.Redoxmodificationsinthecardiacproteome.Cardiacbiomarkers.SecretorymicrovesiclesProteomicsofthesecretome第7页/共44页ThemyofilamentproteomeThemyofilament（肌丝）proteinsareresponsibleforthecontractilenatureofthecardiacmyocytes.themyofilamentsubproteomeallowsthehearttoactasapump.Themyofilamentproteinsarehighlyregulatedbyanumberofspecificpost-translationalmodifications(PTMs)someofwhichhavebeendiscoveredthroughproteomicstudies.PTMsofmyofilamentproteinscandirectlyimpactonthecontractilityoftheheart.第8页/共44页Asimplifiedillustrationofthecardiacmyofilamentproteins.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:titin第9页/共44页Structureofaregionoftheoverlapregionofacardiacsarcomereindiastoleontheleftandduringsystoleontherightwithindicationsofmajorandfunctionallysignificantproteinphosphorylationsites.第10页/共44页Post-translationalmodificationsofmyofilamentproteins第11页/共44页第12页/共44页SamplepreparationTherearetwocommonlyusedmyofilamentprotein-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.)第13页/共44页DetectionMethodsforProteinmodificationphosphorylationchanges:1-D-IEF(phosphorylationsignificantlydecreasesproteinpIvalues)Westernblotswithphosphorylation-site-specificantibodiesMSanalysis:MALDI-TOFcoupledwithphosphatasetreatmentorPostsourcedecay(PSD)immobilizedmetalaffinitycolumn(IMAC)enrichmentandLCseparationfollowedbyMS/MSanalysis第14页/共44页Immobilizedmetalaffinitycolumn(IMAC)Schematicofaffinitybindingofphosphopeptidestoimmobilizedmetalionaffinitycolumns.第15页/共44页DetectionMethodsforProteinmodificationProteindegradation: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）第16页/共44页文献阅读ProteomicsClin.Appl.(2008)ChaoYuan,R.JohnSolaro.Myofilamentproteins:Fromcardiacdisorderstoproteomicchanges(p788-799)WenhaiJin,AnnaT.Brown,AnneM.Murphy.Cardiacmyofilaments:fromproteometopathophysiology

(p800-810)

第17页/共44页2.RedoxmodificationsinthecardiacproteomeMyocardialischemiaresultsinoxidativestress,whichinvolvesthemitochondriaandmany/allaspectsofmyocytefunction.Duetothesusceptibilityofcardiacproteintooxidativedamage,proteomicscanhelptodiscover,quantify,andcharacterizetheredoxsignalingandoxidativePTMs.NitricoxideisakeymediatorofCVcellularresponseinacuteandchronicdiseasesettings.Newapproachesintheproteomicscanhelpidentifyanddefineimportantpathwayofnitricoxide-inducedPTMs.第18页/共44页OutlineofpotentialconsequencesofoxidativestressincellsystemOxidantscanreactwithproteinstocauseoneoftwobroadconsequences.Theycanoxidisecellularcomponentssuchasproteins,renderingthemdysfunctional,whichnegativelyaffectscellfunctionandpromotesdisease.Inthisscenario,antioxidantscanpreventthecellularproteinsfrombeingoxidisedandsoprovideprotection.Incontrast,oxidantscaninduceregulatorypost-translationaloxidativeproteinmodifications,whichareimportantforstressadaptation.Thus,antioxidantscaninterferewithhomeostaticcontrolandmightexplainwhyantioxidanttherapiescanbedetrimentalinsomecases.第19页/共44页MechanismsofROSgeneration.Sequentialreductionofmolecularoxygentogeneratesuperoxide,hydrogenperoxideandthenhydroxylradical.Listofaminoacidsparticularlysusceptibletomodification.第20页/共44页DiagramshowingtheproductionofNOandRNS,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.第21页/共44页Structureofcommonredoxmodificationsofaminoacidsidechains.ROSandRNScanchemicallymodifyaminoacids,particularlythesidechainsofthoseoutlinedhere.Clearly,cysteinethiolsaresubjecttoadiverserangeofalterations.亚磺酸磺酸次磺酸亚砜

亚硝基硫醇

羰基化

硝基化酪氨酸第22页/共44页Commonlyobservedoxidativemodificationsofproteinaminoacids(A)cysteine;(B)methionine;(C)tyrosine;(D)tryptophan.Alltheaminoacidsareschematicallyrepresentedaspartofapolypeptidechain.However,thenamesshownarethoseoffreeaminoacidsforconvenience.第23页/共44页ListofthemostutilizedmethodsinredoxproteomicsFrom:JournalofExperimentalBotany,Vol.59,No.14,pp.3781–3801,2008第24页/共44页Biotinswitchmethod

Ahypotheticalproteinisindicatedwithcysteinesineitherthefreethiol,disulphide,ornitrosothiolconformations.Inthefirststep,freethiolsareblockedusingMMTS.Next,nitrosylatedcysteineresiduesareselectivelyreducedwithascorbateandthenewlygeneratedfreethiolsarefinallyS-biotinylatedwithbiotin-HPDP.ThebiotinylatedproteinscanbedetecteddirectlybyWesternblottingwithantibodiesspecificforbiotinorusingavidinorstreptavidin.Antibodiescanberadiolabelled,fluorescentlyorenzymaticallylabelled,asisknownintheart.Additionally,taggedproteinscanalsobeisolatedfromaffinitycolumnsorbeads.PSH,proteinsulphhydrylgroups;PSNO,Snitrosatedproteins.第25页/共44页IsotopeCodedAffinityTagging(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-labelledpairsdefinestherelativeabundanceofitsparentproteinsbetweenthetwocellstatesquantificationofproteincysteineoxidation第26页/共44页ListofcardiacproteinsdemonstratedtoundergooxidativemodificationRef:ProteomicsClin.Appl.2008,2,823–836第27页/共44页3.CardiacbiomarkersDiagnosisofMIreliesonthedetectioninserumofacardiacspecificisoformofthemyofilamentprotein,troponinIwhichisreleasedintothebloodwhenthecardiacmyocytediesduesevereischemiaEarlierdetectionofMIordiagnosisofmyocardialischemiapriortocelldeathwillhelptoallowevenearlierinterventiontosave“potentiallyviable”teomicdiscoverypipelineforanalysisofhumanplasmasamplesforpatientswithinducedandcontrolMIhelpedtosetthestageforearlierdetectionofpatentsathighrisk.第28页/共44页CurrentgoldstandardmarkersofCVdistress(i)electrophysiologicalandfunctionalchangesasmonitoredbyelectrocardiographyandechocardiographyrespectively(ii)elevatedserumlevelsofcardiacspecificproteins：myofilamentproteinsandcardiactroponin-Iand-T

（myocardialinfarction）brainnatriureticpeptideandinflammation-relatedproteins,includingC-reactiveprotein(CRP),（heartfailure）.cardiacenzymeslactatedehydrogenaseandcreatinekinase(CK)第29页/共44页Severalapproachescurrentlyusedtoquantitatively

profileglobalproteomicexpressionpatternsfluorescence2-DDIGEcoupledtoMSanalysisProteinarraysinvitroandinvivostableisotopelabelLC-MStechniquesSignificantcostofusinglabeledreagentsinlarge-scalestudies.theapparentbiasofthesetechniquestowardslabelingtherelativelymostabundantspeciesinacomplexmixture,Morerecently,“label-free”differential(d)MS(无标记的质谱定量方法)第30页/共44页第31页/共44页Workflowforlabel-freedMSanalysisofplasmasamples.(A)Workflowchart.Thesixstagesoftheprocessarerepresentedwithinthisfigureincludingsamplepreparation,additionofinternalstandardsandMSanalysis.Eachstageplaysanimportantroleinleadingtoasuccessfulofdeterminationofmeaningfuldifferentials.第32页/共44页4.SecretorymicrovesiclesVascularsecretoryproteinandmembranevesiclescanaffecthomeostasisandcommunicationwithinentireCVsysteminresponsetoinjury.Schematicfigureoftheuseofproteomicsforthecharacterizationofthenon-cellularproteinfractionsrelevantinatherosclerosis.Thefigurerepresentsanatheroscleroticplaqueanditscellularcomponents.Thecellsinvolvedinatheromaformationreleasesolubleproteinsandmembranebodiesthatmodifythevascularmicroenvironment.Proteomicscanbeappliedtothecharacterizationofthesenon-cellularcomponentsoftheatheroscleroticmicroenvironment.第33页/共44页Thelimitationsofplasmaproteomicsplasmaandserumareroutinelyusedforbiomarkerdiscoveryinproteomics.thehigh-abundanceproteins,notablyalbuminandimmunoglobulins,whichtogetherwithhaptoglobulin,antitrypsinandtransferrin,typicallyconstitutemorethan90%spectivebiomarkers:pg~ng/ml;albumin:35–50mg/mlthelimitedabilityofproteomicstodetectlow-abundanceplasmaproteins第34页/共44页Proteomicsofextracellularsecretory

vesicles(3)Matrixvesiclesareextracellularmembraneparticlesobservedintheinitialstagesofarterialcalcificationandcontainhighlevelsofcalcium-bindingacidicphospholipids.(4)Apoptoticbodiesarelargeparticlesreleasedfromcellsatthelaterstagesofprogrammedcelldeathandcharacterizedbylargediameter,nuclearcontent,andsurfaceligandsforphagocyticcellreceptors.(5)Heterogeneouspopulationorsecretorymicrovesicles.(1)Microparticlesarereleasedfromtheplasmamembraneofstimulatedorapoptoticcells.Theirproteincompositionmayvaryinresponsetodifferentstimuli(highshearstress,apoptosis,etc.).(2)Exosomesarethesmallestofthesecretorymembraneparticlesandaresecretedasaconsequenceofthefusionoftheplasmamembranewiththemultivesicularbodies(MVB).MVBarelatecomponentsoftheendocyticpathway.第35页/共44页Thecriticalpatho-physiologicalroleofmicroparticlesInthevascularcontext,microparticlesarereleasedbyendothelialcells,smoothmusclecells,lymphocytes,monocytes,erythrocytesandplatelets.Plasmalevelsofmicroparticlesaremarkedlyelevatedinpatientswithveinthrombosis,acutecoronarydisease,ischemicstroke,diabetes,myocardialinfarction,andhypertension.Microparticlesshowpro-coagulantactivity,pro-inflammatory,andpro-atheroscleroticactivities.modulatingtheendothelialsecretionofprostacyclinandnitricoxide;promotemonocyte-endotheliuminteractionbydirecttransferofarachidonicacidtotheplasmamembrane;physicallymediateleukocyte-leukocyteandleukocyte-endotheliuminteractionsviadirectbindingofcellsurfacereceptors第36页/共44页ProteomicsofmicroparticlesProteomicanalysisofproteinexpressioninhumanplasmamicroparticles.MicroparticlesderivedfromtheperipheralbloodbycentrifugationwerelysedandlabelledwithCydyes(greenandredcolourinAandB,respectively).UsingDIGE,microparticleandmicroparticle-depletedplasmaproteinswereco-separatedinlargeformat2-Dgels.ImageswereacquiredonafluorescencescannerandproteinsidentifiedbyLC-MS/MS.Actinandhaemoglobinareenrichedinmicroparticles,comparedtomicroparticle-depletedplasma.第37页/共44页characterisationofmicroparticlesreleasedbyaparticularcelltypeinvitrobyproteomicsBesidestheinvestigationofthemixtureofmicroparticlescontainedinhumanplasma,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,a5anda2第38页/共44页TheroleofExosomesmodulateimmuneresponseregulatehaemostaticbalancesupportthrombingenerationandinduceexpressionandsecretionofplasminogenactivatorinhibitor-1byendothelialcellsattenuatingfibrinolysisandpromotingpro-thromboticconditionsabilitytobeabsorbedtothecellsurfaceandmediatecell-cellinteractionsinthecardiovascularsystem第39页/共44页Proteomicsofexosomesdendriticcell-derivedexosomes

(J.Immunol.2001,166,7309–7318.)endocyticproteinswereabundantcomponentsoftheproteomeofexosomes.21newexosomalproteinswereidentified,includingcytoskeleton-relatedproteins,suchascofilin,profilinIorelongationfactor1a,andintracellularmembranetransportproteins,suchasannexins,rab7,11,rap1B,andsyntenin.aseriesofapoptosis-relatedproteins,includingthioredoxinperoxidaseII,