malditoftof培训培训讲学.ppt

2020 3 7 BrukerConfidential 1 MALDITOF质谱培训周春喜博士布鲁克公司应用工程师 欢迎参加 2020 3 7 BrukerConfidential 2 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 2020 3 7 BrukerConfidential 3 质谱法 MASSSPECTROMETRY Analyticalmethoddeterminingthemolecularmassofions 质谱仪 MASSSPECTROMETER Analyticalinstrumentforthedeterminationofthemolecularmassofions 2020 3 7 BrukerConfidential 4 常见的离子源 FastAtomBombardment FABIonBombardment SIMSFieldDesorption FDLaserIonizationElectroSprayIonization ESIMatrixAssistedLaserDesorption Ionization MALDI基质辅助激光解析电离 2020 3 7 BrukerConfidential 5 常见的质量分析器 QuadrupolMagneticSectorIonTrapICRFTMSTime of Flight TOF飞行时间 2020 3 7 BrukerConfidential 6 Laser SampleTarget Detector Clock Time of Flight MolecularMass MALDI TOF的基本原理 2020 3 7 BrukerConfidential 7 EnablingLifeScienceToolsBasedonMassSpectrometryTM Ek zqUEk 1 2mV2 V2 2qUz m 2qU m z 2020 3 7 BrukerConfidential 8 MALDITOFMS的构成 2020 3 7 BrukerConfidential 9 反射模式下TOF的分辨率高于线性模式 LinearTOF MS ReflectionTOF MS 2020 3 7 BrukerConfidential 10 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 2020 3 7 BrukerConfidential 11 灵敏度分辨率准确度 EnablingLifeScienceToolsBasedonMassSpectrometryTM 质谱仪的主要性能指标 2020 3 7 BrukerConfidential 12 质量分辨率Itisameasureofamassspectrometer sabilitytodistinguishtwocompoundsofnearlyequalmass 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 2020 3 7 BrukerConfidential 13 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 2020 3 7 BrukerConfidential 14 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 2020 3 7 BrukerConfidential 15 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 2020 3 7 BrukerConfidential 16 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 2020 3 7 BrukerConfidential 17 ReasonsforPeakbroadeningorpoorResolution PreparationInitialEnergySpreadCoulombrepulsionandFragmentationofionsduetohighlaserpowerChargingupofcontaminationintheionoptics 2020 3 7 BrukerConfidential 18 初始能量不均 2020 3 7 BrukerConfidential 19 初始能量不均导致谱峰变宽 分辨率降低 2020 3 7 BrukerConfidential 20 如何补偿初始能量的不均 Uacc L Ionsource Field freedriftregion M1 Detector m z Int MH 2020 3 7 BrukerConfidential 21 延时提取产生聚焦效果 IS1 L Ionsource Field freedriftregion M1 Detector Epot t tdelay 2020 3 7 BrukerConfidential 22 延时提取产生聚焦效果 IS1 L Ionsource Field freedriftregion M1 Detector Epot t tdelay 2020 3 7 BrukerConfidential 23 延时提取产生聚焦效果 IS1 L Ionsource Field freedriftregion Detector Epot t tdelay IS2 dEpot t tdelay Tuningparameter IS2tdelay 2020 3 7 BrukerConfidential 24 Reflectron IS1 Ionsource M LinearDetector R2 Urefl ReflectorDetector 1 1 1 2 2 2 IS2 1 2 IncreasingofResolutionandSignal toNoisebyfocusingtheionsinTimeandSpace gridlessdesign 2020 3 7 BrukerConfidential 25 数据采集速率DigitizationRate EnablingLifeScienceToolsBasedonMassSpectrometryTM 2020 3 7 BrukerConfidential 26 低质量端的大量背景信号 K Matrix Matrixdimer Analyte 2020 3 7 BrukerConfidential 27 抑制基质峰的方法 偏转法Deflection Withthedeflectoron ionsaredeflectedofftheionopticalaxisandcannotreachthedetector 降压法Detectorgating Withdetectorgatingon thevoltageonthedetectorisreducedduringtheearlypartofeverytimeperiod Onlythelineardetectorisequippedwiththegatingoption EnablingLifeScienceToolsBasedonMassSpectrometryTM 2020 3 7 BrukerConfidential 28 偏转法 EnablingLifeScienceToolsBasedonMassSpectrometryTM 2020 3 7 BrukerConfidential 29 降压法 Preventsaturationofthedetectorbydeflectionoflowmassionsorrampingthedetectorvoltage detectorgating Onlyionsofinterestreachthedetector 2020 3 7 BrukerConfidential 30 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 2020 3 7 BrukerConfidential 31 常用的基质 EnablingLifeScienceToolsBasedonMassSpectrometryTM 2020 3 7 BrukerConfidential 32 MatrixAnalyteSinapinicacidproteins glycos polarpolymersDHBproteins glycos sugars polarpolymerssuper DHBproteins glycos sugars polarpolymersalpha cyanopeptidesHABApeptides proteinsTHAP1oligonucleotidesHPA1oligonucleotidesDithranol2nonpolarpolymersIAA2nonpolarpolymersAdditives 1 ammoniumcitrate2 Ag TFA 基质的选择 2020 3 7 BrukerConfidential 33 点样方法 DriedDroplet matrix analyte samplesolution sampledeposition Analytedispersedthroughoutmatrixcrystals EnablingLifeScienceToolsBasedonMassSpectrometryTM 2020 3 7 BrukerConfidential 34 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 2020 3 7 BrukerConfidential 35 AnchorChip 普通靶 疏水涂层靶 2020 3 7 BrukerConfidential 36 疏水涂层靶 普通靶AnchorChip 2mm 600um 2020 3 7 BrukerConfidential 37 杂质对样品信号的抑制 Caseindigestwith200mMKCland0 1 Tween20 Caseindigestaftercleanup EnablingLifeScienceToolsBasedonMassSpectrometryTM 2020 3 7 BrukerConfidential 38 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 2020 3 7 BrukerConfidential 39 MALDI TOF TOF质谱仪 2020 3 7 BrukerConfidential 40 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 2020 3 7 BrukerConfidential 41 LinearTOF Source1 Detector 源后裂解 post sourcedecay PSD 2020 3 7 BrukerConfidential 42 PSD碎片被反射镜分离 但不能一次全部被检测 2020 3 7 BrukerConfidential 43 triggerdiode N2laser PostSourceDecay PSD highreflectorvoltage 2020 3 7 BrukerConfidential 44 triggerdiode N2laser PostSourceDecay PSD mediumreflectorvoltage 2020 3 7 BrukerConfidential 45 triggerdiode N2laser PostSourceDecay PSD lowreflectorvoltage 2020 3 7 BrukerConfidential 46 MALDITOF TOF的原理 LIFT LIFTanalysis ClassicalPSD Ekin 1 2mv2 Source 2020 3 7 BrukerConfidential 47 Target LIFT Selector MALDITOF TOFScheme PotentialProfileduringLIFT LIFT EnablingLifeScienceToolsBasedonMassSpectrometryTM 2020 3 7 BrukerConfidential 48 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 2020 3 7 BrukerConfidential 49 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 2020 3 7 BrukerConfidential 50 Terminology LIFT LaserInducedFragmentationTechniquePatentedverysensitivemethodtoacquireTOF TOFspectrawithhigh energydetectionofproductionsviaBrukerinnovativepotential lift inultraflexFragmentionsare lifted post accelerated ontohigherenergy EnablingLifeScienceToolsBas