MALDI-TOFTOF培训ppt课件.ppt

MALDITOF质谱培训周春喜博士布鲁克公司应用工程师,欢迎参加,1,MALDITOF质谱仪的基本原理多肽和蛋白质样品的制备仪器操作和数据采集(FlexControl)数据分析(FlexAnalysis,BioTools,etc),培训内容,2,质谱法(MASSSPECTROMETRY),Analyticalmethoddeterminingthemolecularmassofions!,质谱仪(MASSSPECTROMETER),Analyticalinstrumentforthedeterminationofthemolecularmassofions.,3,常见的离子源,FastAtomBombardment,FABIonBombardment,SIMSFieldDesorption,FDLaserIonizationElectroSprayIonization,ESIMatrixAssistedLaserDesorption/Ionization,MALDI基质辅助激光解析电离,4,常见的质量分析器,QuadrupolMagneticSectorIonTrapICRFTMSTime-of-Flight,TOF飞行时间,5,Laser,SampleTarget,Detector,Clock,Time-of-Flight,MolecularMass,MALDI-TOF的基本原理,6,EnablingLifeScienceToolsBasedonMassSpectrometryTM,Ek=zqUEk=1/2mV2,V2=2qUz/m=2qU/(m/z),7,MALDITOFMS的构成,8,反射模式下TOF的分辨率高于线性模式,LinearTOF-MS,ReflectionTOF-MS,9,线性模式适合大分子（蛋白质，核酸，多聚物等）分辨率和质量准确度低于反射模式，通常测得的为平均分子量反射模式适合分子量5000Da的物质，如多肽，寡糖等。分辨率高，可准确测得单同位素分子量。,10,灵敏度分辨率准确度,EnablingLifeScienceToolsBasedonMassSpectrometryTM,质谱仪的主要性能指标,11,质量分辨率Itisameasureofamassspectrometersabilitytodistinguishtwocompoundsofnearlyequalmass.,600,800,1000,1200,1400,1600,1800,2000,2200,2400,m/z,100,200,300,400,500,600,700,800,900,1000,1100,a.i.,1618,1623,1628,m/z,“easy”,“notsoeasy”,m=1620Dm=0.2R=8100,EnablingLifeScienceToolsBasedonMassSpectrometryTM,Resolution=(m/z)/Dm,Dm:FullWidthatHalfMaximum(FWHM),12,质量准确度,Massaccuracycanbeexpressedasapercentageorppm:,e.g.%massaccuracy=,Verysmallpercentageerrors(0.01%)areexpressedaspartspermillionorppm.0.01%=100ppm,Measuredmass:1296.970Truemass:1296.6850.02%error,ppm=0.001perthousand,EnablingLifeScienceToolsBasedonMassSpectrometryTM,13,ACTH(18-39)C112H166N27O36,R=40002465.2m/z,R=20002466.21m/z,R=10002466.36m/z,R=5002466.58m/z,分辨率影响准确度,EnablingLifeScienceToolsBasedonMassSpectrometryTM,14,如何提高准确度,分辨率和灵敏度,仪器调节-仪器校准-延时提取-激光功率-基质抑制-数据采集频率样品制备-基质选择-点靶方法-样品纯化,15,仪器校准,校正方程:m/z=At2+Bt+C内标法:样品与标准品点在同一靶点外标法:样品与标准品点在不同靶点,16,ReasonsforPeakbroadeningorpoorResolution!,PreparationInitialEnergySpreadCoulombrepulsionandFragmentationofionsduetohighlaserpowerChargingupofcontaminationintheionoptics,17,初始能量不均,18,初始能量不均导致谱峰变宽，分辨率降低,19,如何补偿初始能量的不均？,Uacc,L,Ionsource,Field-freedriftregion,M1+,Detector,m/z,Int.,MH+,20,延时提取产生聚焦效果,IS1,L,Ionsource,Field-freedriftregion,M1,Detector,Epot,ttdelay,21,延时提取产生聚焦效果,IS1,L,Ionsource,Field-freedriftregion,M1,Detector,Epot,ttdelay,+,+,+,+,22,延时提取产生聚焦效果,IS1,L,Ionsource,Field-freedriftregion,Detector,Epot,ttdelay,Tuningparameter:IS2tdelay,23,Reflectron,IS1,Ionsource,M,LinearDetector,R2,Urefl,ReflectorDetector,1,1,1,2,2,2,IS2,+,1,+,+,+,2,+,+,IncreasingofResolutionandSignal-toNoisebyfocusingtheionsinTimeandSpace!,gridlessdesign,24,数据采集速率DigitizationRate,EnablingLifeScienceToolsBasedonMassSpectrometryTM,25,低质量端的大量背景信号,K+,Matrix+,Matrixdimer+,Analyte+,26,抑制基质峰的方法,偏转法Deflection:Withthedeflectoron,ionsaredeflectedofftheionopticalaxisandcannotreachthedetector.降压法Detectorgating:Withdetectorgatingon,thevoltageonthedetectorisreducedduringtheearlypartofeverytimeperiod.Onlythelineardetectorisequippedwiththegatingoption.,EnablingLifeScienceToolsBasedonMassSpectrometryTM,27,偏转法,EnablingLifeScienceToolsBasedonMassSpectrometryTM,28,降压法,Preventsaturationofthedetectorbydeflectionoflowmassionsorrampingthedetectorvoltage(detectorgating!),Onlyionsofinterestreachthedetector!,29,Matrix-HCCA,H,基质吸收激光的能量，并将部分能量传递给样品。帮助样品离子化。,Matrix,样品分子,TimeofFlight,基质的作用,30,常用的基质,EnablingLifeScienceToolsBasedonMassSpectrometryTM,31,MatrixAnalyteSinapinicacidproteins/glycos/polarpolymersDHBproteins/glycos/sugars/polarpolymerssuper-DHBproteins/glycos/sugars/polarpolymersalpha-cyanopeptidesHABApeptides/proteinsTHAP1oligonucleotidesHPA1oligonucleotidesDithranol2nonpolarpolymersIAA2nonpolarpolymersAdditives:1)ammoniumcitrate2)Ag-TFA,基质的选择,32,点样方法：DriedDroplet,matrix,analyte,samplesolution,sampledeposition,Analytedispersedthroughoutmatrixcrystals,EnablingLifeScienceToolsBasedonMassSpectrometryTM,33,样品厚度不均匀的后果,Uacc,A,B,UB,UA,A,B,UB,UA,E=1-2kV/mm,Example:Dx=50mm,Dtof=3-4nsorDm=0.27Daat2465Da,A,B,B,A,34,AnchorChip,普通靶,疏水涂层靶,35,疏水涂层靶,普通靶AnchorChip,2mm,600um,36,杂质对样品信号的抑制,Caseindigestwith200mMKCland0.1%Tween20,Caseindigestaftercleanup,EnablingLifeScienceToolsBasedonMassSpectrometryTM,37,样品之间的信号抑制,1300,1800,2300,m/z,500,1000,1500,2000,2500,3000,3500,4000,4500,5000,5500,6000,6500,a.i.,Equimolarmixturesmayproduceunequalpeakintensities.Competitionforchargeaffectsionabundances.-morebasicanalytesfavored,EnablingLifeScienceToolsBasedonMassSpectrometryTM,38,MALDI-TOF/TOF质谱仪,39,EnablingLifeScienceToolsBasedonMassSpectrometryTM,LID(激光诱导解离)Laser“puts”somuchenergyintoamoleculethatthemolecule“fragments”CID(碰撞诱导解离)MoleculesflythroughacollisioncellandfragmentasaresultofcollisionswithcollisiongasesLIDandCIDareusedintheAutoFlexastwoalternativeandcomplementarydissociationmethods.,诱导解离的方式,40,LinearTOF,Source1,Detector,源后裂解(post-sourcedecay,PSD),41,PSD碎片被反射镜分离,但不能一次全部被检测,42,triggerdiode,N2laser,PostSourceDecay(PSD),highreflectorvoltage,43,triggerdiode,N2laser,PostSourceDecay(PSD),mediumreflectorvoltage,44,triggerdiode,N2laser,PostSourceDecay(PSD),lowreflectorvoltage,45,MALDITOF-TOF的原理：LIFT,LIFTanalysis,ClassicalPSD,Ekin=1/2mv2,Source,46,Target,LIFT,Selector,MALDITOF/TOFScheme,PotentialProfileduringLIFT,LIFT,EnablingLifeScienceToolsBasedonMassSpectrometryTM,47,MALDITOF-TOF的原理：LIFT,质谱图,离子检测器,离子反射器,母离子选择器,MALDI离子源,LIFT子离子加速器,48,TOF/TOF二级质谱图,AngiotensinIIat1046,931.513,1046.555,354.201,400.216,110.064,517.260,647.358,756.390,263.147,0,1000,2000,3000,4000,5000,6000,Intens.a.u.,100,200,300,400,500,600,700,800,900,1000,m/z,49,Terminology,LIFT:LaserInducedFragmentationTechniquePatentedverysensitivemethodtoacquireTOF/TOFspectrawithhigh-energydetectionofproductionsviaBrukerinnovativepotentialliftinultraflexFragmentionsare“lifted”(post-accelerated)ontohigherenergy.,EnablingLifeScienceToolsBasedon