分子生物学分子生物学 (24).pdf
Chapter 10Proteomics research methods 10.2Research method of proteomeexpression pattern-Two-dimensional electrophoresisResearch methods of proteomeexpression patternsStudy the composition of the proteomeThe main technologies are two-dimensionalgel electrophoresis,protein identificationtechnology and bioinformatics technologyrepresented by mass spectrometry.10.2.1 Sample preparation in proteome researchThe whole protein components of cells or tissueswere used for proteome analysis；Pre-classification of samples may increase thesample loading and detection sensitivity of low-abundance proteins.The main basis of sample pre-classificationProtein solubility:soluble protein,insolubleprotein,etc;Protein localization:membrane proteins,nuclear proteins,etc;Protein organelle positioning:mitochondria,Golgi bodies,chloroplast,etc.Tissue level proteome sample preparationReason:Clinical samples are usually mixtureof various cells or tissues,and even in differentstatus.For example,cancerous epithelial cellsin tumors are always mixed with blood vesselsand stromal cells.Laser capture microdissection(LCM)Specific cells orcell groups can beaccurately isolatedfrom tissue sectionsdirectly under themicroscope.LCM purified lung adenocarcinoma cellsA：Lung adenocarcinoma tissue before LCM cutting;B：Lung adenocarcinoma tissue after LCM cutting;C：Collected lung adenocarcinoma cellsABC10.2.2 Sample separation in proteome researchtwo-dimensional electrophoresis（2-DE)a method to separate various proteins byutilizing the difference in isoelectric point andmolecular weight of proteins,combining withthe application of chemical properties of gel.working principle:Separation of proteins by first-dimensionalisoelectric focusing electrophoresisaccording to different isoelectric points；Transfer the first-dimentional separationsto the second direction SDS-polyacrylamidegel,and then separate according to theprotein molecular mass.（）（）（）（）（）（）（）（）low pHhigh pHThe buffer in polyacrylamide gel produced a pH gradient across the gel under electric field.Each protein will migrate to the locus where pH is consistent with its PI.The first-dimension isoelectric focusing(IEF）Advantages of solid phase pH gradient isoelectric focusingovercomes the shortcomings of cathode driftof carrier amphoteric electrolyte;Precisely set the pH gradient;In particular,the second round of analysiscan be performed in a narrow pH range,greatlyimproving resolution and repeatability.Two-dimensional gel electrophoresisThe horizontal direction reflected thedifference in protein pI,while the verticaldirection reflected the difference inmolecular weight.The second-dimensional SDS-PAGE 3-4 mm70-240 mmgelPlastic backingIEFSDS-PAGESDS-PAGEsample preparation;the first dimension:solid phase pH gradient isoelectric focusing;the second dimension:SDS-PAGE;dyeing:Coomassie brilliant blue or silver staining;image analysis:direct observation or automatic scanning.The main steps of 2-DE:Gel staining1stdimension IEF2nddimension SDS-PAGEImaging and data evaluationSpot excision and mass spectrometrySamplepreparation2-DE flow chartThe map of 2-DEDysfunctions,CancerDrugsSimultaneous protein profiling to understand the function and regulation of thousands of proteinsCell specific expressionTemperatureMetabolic stateStressCulture conditionsDifferential Expression ABE.Coli grown at 30 CE.Coli grown at 42 CAdvantages of 2-DEisolation of proteins in the range of 10 to 100 KD;high sensitivity and high resolution;convenient processing for image analysis by computermatch with mass spectrometry analysis.Disadvantages of 2-DEextremely acidic/basic proteins,hydrophobicproteins,extremely large/small proteins,andlow-abundance proteins are difficult to separateeffectively;in-gel enzymolysis process is time-consumingand laborious,and is difficult to automaticallyconnect mass spectrometry.New non-gel technologyLiquid Chromatography(LC)Partition chromatographyAdsorption chromatographyIon exchange chromatographyExclusion chromatographyCapillary Electrophoresis(CE)LC-MS/MSThe protein mixture was separated by liquidchromatography instead of 2-DE,then entered intothe MS system to obtain the peptide molecularweight,and then obtained partial sequenceinformation through the tandem MS.Finally,protein query and identification was performed bycomputer network.Question:What happened to the protein in SDS-PAGE electrophoresis?Whats the pointof that?
