《疾病蛋白质组学》PPT课件.ppt

第四讲疾病蛋白质组学 一 diseaseproteomics 一 基本概念和总体研究概况 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 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 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 二 心血管疾病蛋白质组学CardiovascularProteomics thecardiovascular CV systemiscomposedofanumberofspecializedcelltypesincludingcardiacmyocytes fibroblast neurons endothelialandsmoothmusclecellsandnewlydiscoveredstemandprogenitorcells Todate theproteomeofthesecellsarenotwellcharacterizednorhastheinterplaybetweenthecelltypesbeenestablishedinhealthordisease ThisremainsasignificantchallengeasCVdiseaseisthenumberonekillerworldwide ResearchFocus Themyofilamentproteome Redoxmodificationsinthecardiacproteome Cardiacbiomarkers SecretorymicrovesiclesProteomicsofthesecretome Themyofilamentproteome Themyofilament 肌丝 proteinsareresponsibleforthecontractilenatureofthecardiacmyocytes themyofilamentsubproteomeallowsthehearttoactasapump Themyofilamentproteinsarehighlyregulatedbyanumberofspecificpost translationalmodifications PTMs someofwhichhavebeendiscoveredthroughproteomicstudies PTMsofmyofilamentproteinscandirectlyimpactonthecontractilityoftheheart 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 Structureofaregionoftheoverlapregionofacardiacsarcomereindiastoleontheleftandduringsystoleontherightwithindicationsofmajorandfunctionallysignificantproteinphosphorylationsites Post translationalmodificationsofmyofilamentproteins Samplepreparation Therearetwocommonlyusedmyofilamentprotein 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 DetectionMethodsforProteinmodification phosphorylationchanges 1 D IEF phosphorylationsignificantlydecreasesproteinpIvalues Westernblotswithphosphorylation site specificantibodiesMSanalysis MALDI TOFcoupledwithphosphatasetreatmentorPostsourcedecay PSD immobilizedmetalaffinitycolumn IMAC enrichmentandLCseparationfollowedbyMS MSanalysis Immobilizedmetalaffinitycolumn IMAC Schematicofaffinitybindingofphosphopeptidestoimmobilizedmetalionaffinitycolumns DetectionMethodsforProteinmodification Proteindegradation 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 文献阅读 ProteomicsClin Appl 2008 ChaoYuan R JohnSolaro Myofilamentproteins Fromcardiacdisorderstoproteomicchanges p788 799 WenhaiJin AnnaT Brown AnneM Murphy Cardiacmyofilaments fromproteometopathophysiology p800 810 2 Redoxmodificationsinthecardiacproteome Myocardialischemiaresultsinoxidativestress whichinvolvesthemitochondriaandmany allaspectsofmyocytefunction Duetothesusceptibilityofcardiacproteintooxidativedamage proteomicscanhelptodiscover quantify andcharacterizetheredoxsignalingandoxidativePTMs NitricoxideisakeymediatorofCVcellularresponseinacuteandchronicdiseasesettings Newapproachesintheproteomicscanhelpidentifyanddefineimportantpathwayofnitricoxide inducedPTMs Outlineofpotentialconsequencesofoxidativestressincellsystem Oxidantscanreactwithproteinstocauseoneoftwobroadconsequences Theycanoxidisecellularcomponentssuchasproteins renderingthemdysfunctional whichnegativelyaffectscellfunctionandpromotesdisease Inthisscenario antioxidantscanpreventthecellularproteinsfrombeingoxidisedandsoprovideprotection Incontrast oxidantscaninduceregulatorypost translationaloxidativeproteinmodifications whichareimportantforstressadaptation Thus antioxidantscaninterferewithhomeostaticcontrolandmightexplainwhyantioxidanttherapiescanbedetrimentalinsomecases MechanismsofROSgeneration Sequentialreductionofmolecularoxygentogeneratesuperoxide hydrogenperoxideandthenhydroxylradical Listofaminoacidsparticularlysusceptibletomodification 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 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 Commonlyobservedoxidativemodificationsofproteinaminoacids A cysteine B methionine C tyrosine D tryptophan Alltheaminoacidsareschematicallyrepresentedaspartofapolypeptidechain However thenamesshownarethoseoffreeaminoacidsforconvenience Listofthemostutilizedmethodsinredoxproteomics BiotinswitchmethodAhypotheticalproteinisindicatedwithcysteinesineitherthefreethiol disulphide ornitrosothiolconformations Inthefirststep freethiolsareblockedusingMMTS Next nitrosylatedcysteineresiduesareselectivelyreducedwithascorbateandthenewlygeneratedfreethiolsarefinallyS biotinylatedwithbiotin HPDP ThebiotinylatedproteinscanbedetecteddirectlybyWesternblottingwithantibodiesspecificforbiotinorusingavidinorstreptavidin Antibodiescanberadiolabelled fluorescentlyorenzymaticallylabelled asisknownintheart Additionally taggedproteinscanalsobeisolatedfromaffinitycolumnsorbeads PSH proteinsulphhydrylgroups PSNO Snitrosatedproteins 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 labelledpairsdefinestherelativeabundanceofitsparentproteinsbetweenthetwocellstates quantificationofproteincysteineoxidation Listofcardiacproteinsdemonstratedtoundergooxidativemodification Ref ProteomicsClin Appl 2008 2 823 836 3 Cardiacbiomarkers DiagnosisofMIreliesonthedetectioninserumofacardiacspecificisoformofthemyofilamentprotein troponinIwhichisreleasedintothebloodwhenthecardiacmyocytediesduesevereischemiaEarlierdetectionofMIordiagnosisofmyocardialischemiapriortocelldeathwillhelptoallowevenearlierinterventiontosave potentiallyviable heartmuscle proteomicdiscoverypipelineforanalysisofhumanplasmasamplesforpatientswithinducedandcontrolMIhelpedtosetthestageforearlierdetectionofpatentsathighrisk CurrentgoldstandardmarkersofCVdistress i electrophysiologicalandfunctionalchangesasmonitoredbyelectrocardiographyandechocardiographyrespectively ii elevatedserumlevelsofcardiacspecificproteins myofilamentproteinsandcardiactroponin Iand T myocardialinfarction brainnatriureticpeptideandinflammation relatedproteins includingC reactiveprotein CRP heartfailure cardiacenzymeslactatedehydrogenaseandcreatinekinase CK Severalapproachescurrentlyusedtoquantitativelyprofileglobalproteomicexpressionpatterns fluorescence2 DDIGEcoupledtoMSanalysisProteinarraysinvitroandinvivostableisotopelabelLC MStechniquesSignificantcostofusinglabeledreagentsinlarge scalestudies theapparentbiasofthesetechniquestowardslabelingtherelativelymostabundantspeciesinacomplexmixture Morerecently label free differential d MS 无标记的质谱定量方法 Workflowforlabel freedMSanalysisofplasmasamples A Workflowchart Thesixstagesoftheprocessarerepresentedwithinthisfigureincludingsamplepreparation additionofinternalstandardsandMSanalysis Eachstageplaysanimportantroleinleadingtoasuccessfulofdeterminationofmeaningfuldifferentials 4 Secretorymicrovesicles VascularsecretoryproteinandmembranevesiclescanaffecthomeostasisandcommunicationwithinentireCVsysteminresponsetoinjury Schematicfigureoftheuseofproteomicsforthecharacterizationofthenon cellularproteinfractionsrelevantinatherosclerosis Thefigurerepresentsanatheroscleroticplaqueanditscellularcomponents Thecellsinvolvedinatheromaformationreleasesolubleproteinsandmembranebodiesthatmodifythevascularmicroenvironment Proteomicscanbeappliedtothecharacterizationofthesenon cellularcomponentsoftheatheroscleroticmicroenvironment Thelimitationsofplasmaproteomics plasmaandserumareroutinelyusedforbiomarkerdiscoveryinproteomics thehigh abundanceproteins notablyalbuminandimmunoglobulins whichtogetherwithhaptoglobulin antitrypsinandtransferrin typicallyconstitutemorethan90 ofthetotalproteinmassinhumanplasma prospectivebiomarkers pg ng ml albumin 35 50mg mlthelimitedabilityofproteomicstodetectlow abundanceplasmaproteins Proteomicsofextracellularsecretoryvesicles 3 Matrixvesiclesareextracellularmembraneparticlesobservedintheinitialstagesofarterialcalcificationandcontainhighlevelsofcalcium bindingacidicphospholipids 4 Apoptoticbodiesarelargeparticlesreleasedfromcellsatthelaterstagesofprogrammedcelldeathandcharacterizedbylargediameter nuclearcontent andsurfaceligandsforphagocyticcellreceptors 5 Heterogeneouspopulationorsecretorymicrovesicles 1 Microparticlesarereleasedfromtheplasmamembraneofstimulatedorapoptoticcells Theirproteincompositionmayvaryinresponsetodifferentstimuli highshearstress apoptosis etc 2 Exosomesarethesmallestofthesecretorymembraneparticlesandaresecretedasaconsequenceofthefusionoftheplasmamembranewiththemultivesicularbodies MVB MVBarelatecomponentsoftheendocyticpathway Thecriticalpatho physiologicalroleofmicroparticles Inthevascularcontext microparticlesarereleasedbyendothelialcells smoothmusclecells lymphocytes monocytes erythrocytesandplatelets Plasmalevelsofmicroparticlesaremarkedlyelevatedinpatientswithveinthrombosis acutecoronarydisease ischemicstroke diabetes myocardialinfarction andhypertension Microparticlesshowpro coagulantactivity pro inflammatory andpro atheroscleroticactivities modulatingtheendothelialsecretionofprostacyclinandnitricoxide promotemonocyte endotheliuminteractionbydirecttransferofarachidonicacidtotheplasmamembrane physicallymediateleukocyte leukocyteandleukocyte endotheliuminteractionsviadirectbindingofcellsurfacereceptors Proteomicsofmicroparticles Proteomicanalysisofproteinexpressioninhumanplasmamicroparticles MicroparticlesderivedfromtheperipheralbloodbycentrifugationwerelysedandlabelledwithCydyes greenandredcolourinAandB respectively UsingDIGE microparticleandmicroparticle depletedplasmaproteinswereco separatedinlargeformat2 Dgels ImageswereacquiredonafluorescencescannerandproteinsidentifiedbyLC MS MS Actinandhaemoglobinareenrichedinmicroparticles comparedtomicroparticle depletedplasma characterisationofmicroparticlesreleasedbyaparticularcelltypeinvitrobyproteomics Besidestheinvestigationofthemixtureofmicroparticlescontainedinhumanplasma 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 TheroleofExosomes modulateimmuneresponseregulatehaemostaticbalancesupportthrombingenerationandinduceexpressionandsecretionofplasminogenactivatorinhibitor 1byendothelialcellsattenuatingfibrinolysisandpromotingpro thromboticconditionsabilitytobeabsorbedtothecellsurfaceandmediatecell cellinteractionsinthecardiovascularsystem Proteomicsofexosomes dendriticcell 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 regulatethesecretionofplasminogenactivat