蛋白质芯片技术介绍ppt课件

Thefutureisinyourhands Theproteinchip 1 Nowadays thepoteomics 蛋白质的 researchisoneofageneraldirectionofthedevelopmentoflifescienceaftergenomics 基因组学的 research Proteinstructureandfunctionhavethedirectimpactonthechangeoflifeactivity Andthelevelofgenetranscription 转录 canonlyreflectsthechangeofgeneexpressionproductsinacertainextent However trulyfunctionalproteinprocessesafterthetranscription translationregulationprocessing controlingtoformandsoonsteps Thusthedirectstudyofproteintotheinterpretation 翻译 ofthereallifephenomenonisofgreatimprotance Butresearchonmeansandmethods 手段方法 oftheproteindoesn thaveabigdevelopment sothesearchforeffectiveandfastproteinanalysistechnologyhasbecomeoneofthemostimportantstep 2 Thencomestheproteinchip 3 Theproteinchip 蛋白芯片 technologyisquicklydevelopedinrecentyearsinchemicalbiologyasahighandnewtechnology Itisthebasicprincipleismakevariousproteinsorderlyfixedonallkindsofmediumcarrier 介质载体 asatestchip Andthen usingthetagantibodies 抗体 thatarespecifiedbythefluorescent 荧光 substancematchwiththeprotein Andthecorresponding 对应的 proteinwillmatchwiththechip WhatisProteinmicrochip 4 Then thefluorescentontheantibodywillindicatethecorresponding 对应的 numberofproteinanditsexpression Alltheotherantibodiesthatdoesn tmakecomplementarycombinationwillbewashedafterusingthefluorescentscanner 荧光扫描仪 orlaserscanningtechnology 激光扫描共聚技术 Throughanalysisthefluorescenceintensity 强度 ofeachpointonthechipandinteractionrelationshipbetweenproteinandprotein thepurposeofachievingdeterminingallsortsoffunctionofgeneexpressionwillbedone 5 Therearethreetypesofproteinmicroarraysthatarecurrentlyusedtostudythebiochemicalactivitiesofproteins Alltypesofchips 6 Analyticalmicroarraysarealsoknownascapturearrays Inthistechnique alibraryofantibodies aptamersoraffibodiesisarrayedonthesupportsurface Theseareusedascapturemoleculessinceeachbindsspecificallytoaparticularprotein Thearrayisprobedwithacomplexproteinsolutionsuchasacelllysate Analysisoftheresultingbindingreactionsusingvariousdetectionsystemscanprovideinformationaboutexpressionlevelsofparticularproteinsinthesampleaswellasmeasurementsofbindingaffinitiesandspecificities Thistypeofmicroarrayisespeciallyusefulincomparingproteinexpressionindifferentsolutions Forinstancetheresponseofthecellstoaparticularfactorcanbeidentifiedbycomparingthelysatesofcellstreatedwithspecificsubstancesorgrownundercertainconditionswiththelysatesofcontrolcells Anotherapplicationisintheidentificationandprofilingofdiseasedtissues 7 Functionalproteinmicroarrays alsoknownastargetproteinarrays areconstructedbyimmobilisinglargenumbersofpurifiedproteinsandareusedtoideantifyprotein protein protein DNA protein RNA protein phospholipid andprotein smallmoleculeinteractions toassayenzymaticactivityandtodetectantibodiesanddemonstratetheirspecificity Theydifferfromanalyticalarraysinthatfunctionalproteinarraysarecomposedofarrayscontainingfull lengthfunctionalproteinsorproteindomains Theseproteinchipsareusedtostudythebiochemicalactivitiesoftheentireproteomeinasingleexperiment 8 Reversephaseproteinmicroarray RPPA involvecomplexsamples suchastissuelysates Cellsareisolatedfromvarioustissuesofinterestandarelysed Thelysateisarrayedontothemicroarrayandprobedwithantibodiesagainstthetargetproteinofinterest Theseantibodiesaretypicallydetectedwithchemiluminescent fluorescentorcolorimetricassays Referencepeptidesareprintedontheslidestoallowforproteinquantificationofthesamplelysates RPAsallowforthedeterminationofthepresenceofalteredproteinsorotheragentsthatmaybetheresultofdisease Specifically post translationalmodifications whicharetypicallyalteredasaresultofdiseasecanbedetectedusingRPAs 9 Theadvantagesofproteinchip 1 Allofthecoarsebiologicalsamples 粗生物样品 canbedirectlyanalyzed serum urine bodyfluids 直接用粗生物样品 血清 尿 体液 进行分析2 Itcanrapidlydetectmultiplebiomarkersatthesametime同时快速发现多个生物标记物3 Asmallamountofsample只需要小量样品 约2 10 L 4 Highfluxabilityofvalidation高通量的验证能力 一次性验证多个蛋白质分子 foundlowabundanceproteins发现低丰度蛋白质 即在总蛋白提取物中含量很少的蛋白 10 6 Determinatethehydrophobicprotein测定疏水蛋白质 疏水蛋白质具有不同寻常的生化性质 很难用常规的蛋白分析方法检测到其存在 7 Inthesamesystem makecimcdiscoveryanddetectionalltogether Usingmonoclonalantibodychiptoidentifytheunknownantigen protein toreducethedeterminationofproteinsequenceofwork 在同一系统中集发现和检测为一体 利用单克隆抗体芯片 鉴定未知抗原 蛋白质 以减少测定蛋白质序列的工作量 8 Itcanquantitativelyusemonoclonalantibodychip Asaresultofthatthecombinationtochipantibodyisquantitative itcanbeusedtomeasurequantityofantigen 可以定量利用单克隆抗体芯片 由于结合至芯片上的抗体是定量的 故可以测定抗原量 9 Usingmonoclonalantibodymicroarrayscancomplethefunctionofinsufficientcells suchasdissolvecells determinatingtheintracellularantigens What smore itssensitivityismuchhigherthanthat 利用单克隆抗体芯片 可互补流式细胞仪不足的功能 如将细胞溶解 可测定细胞内的抗原 而且灵敏度远高于流式细胞仪 11 Therearefivemajorareaswhereproteinchipsarebeingapplied diagnostics 诊断 proteomics proteinfunctionalanalysis antibodycharacterization andtreatmentdevelopment Diagnosticsinvolvesthedetectionofantigensandantibodiesinbloodsamples theprofilingofseratodiscovernewdiseasebiomarkers themonitoringofdiseasestatesandresponsestotherapyinpersonalizedmedicine themonitoringofenvironmentandfood Proteomicspertainstoproteinexpressionprofilingi e whichproteinsareexpressedinthelysateofaparticularcell Applications 12 Proteinfunctionalanalysisistheidentificationofprotein proteininteractions e g identificationofmembersofaproteincomplex protein phospholipidinteractions smallmoleculetargets enzymaticsubstrates particularlythesubstratesofkinases andreceptorligands Antibodycharacterizationischaracterizingcross reactivity specificityandmappingepitopes Treatmentdevelopmentinvolvesthedevelopmentofantigen specifictherapiesforautoimmunity cancerandallergies theidentificationofsmallmoleculetargetsthatcouldpotentiallybeusedasnewdrugs 13 1 Establishingafast cheap highproteinexpressionandpurificationmethodofflux highfluxanddefiningeachantibodypreparationofantibodyaffinityspecificity Thefirstgenerationofproteindetectionchipwillrelymainlyonantibodiesandothermacromolecules clearly withthesematerialpreparationcomplexchips scaleproductionwillexistalotofpracticalproblems inparticular theidealsolutionistoadoptthemethodofchemicalsynthesisoflarge scalepreparationofantibodies 建立快速 廉价 高通量的蛋白质表达和纯化方法 高通量制备抗体并定义每种抗体的亲和特异性 第一代蛋白检测芯片将主要依赖于抗体和其他大分子 显然 用这些材料制备复杂的芯片 尤其是规模生产会存在很多实际问题 理想的解决办法是采用化学合成的方法大规模制备抗体 Theproblemthatproteinchiphave 14 2 Improvingthematrixmaterialsurfacetreatmenttechnologytoreducethenonspecificcombinationofproteins 改进基质材料的表面处理技术以减少蛋白质的非特异性结合 3 Speedingupthelatticeofchipproduction Provideappropriatetemperatureandhumiditytomaintainthestabilityandthebiologicalactivityofproteinchipsurface 提高芯片制作的点阵速度 提供合适的温度和湿度以保持芯片表面蛋白质的稳定性及生物活性 4 Detectingthegeneralmethodofhighsensitivityandresolution toachievetheintegrationofimaginganddataanalysis 研究通用的高灵敏度 高分辨率检测方法 实现成像与数据分析一体化 15 16