单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,液质分析条件的优化策略（简化板）,液相色,谱,谱与液,质,质联用,仪,仪使用,的,的要点,质量校,正,正的正,确,确,对于相,关,关分析,要,要有合,适,适的支,持,持软件,（,（Maxnet，TargeLyness）,合适的,液,液相色,谱,谱平台,合适的,质,质谱接,口,口方式,液相色,谱,谱分析,中,中合适,的,的色谱,柱,柱的选,用,用,质谱检,测,测模式,的,的选择,必要的,后,后液流,补,补充,质量校,正,正的正,确,确（Myoglobin校,正,正）,质量校,正,正的正,确,确（Myoglobin校,正,正）,质量校,正,正的正,确,确（CsI校,正,正）的,肽,肽测定,质量校正的,正,正确（CsI校正）的,蛋,蛋白测定,质量校正的,关,关键点,针对不同的,分,分析采用不,同,同的校正，,一,一般蛋白质,采,采用Myoglobin，对于小,分,分子分析建,议,议采用CsI校正。,对于采用Myoglobin校正,的,的建议采用,高,高次方的校,正,正曲线（3-4）；对CsI校正,的,的建议采用,低,低次方的校,正,正曲线（1-2）,合适的液相,色,色谱平台,能提供一个,连,连续、稳定,的,的液流环境,真空脱气设,备,备,系统的死体,积,积尽可能小,，,，减少管路,的,的长度,输液泵的设,计,计能适用于,微,微径柱的要,求,求,二极管阵列,检,检测器的池,体,体积与质谱,仪,仪匹配,必要的液相,色,色谱辅助配,件,件,必要的液相,色,色谱与质谱,仪,仪的软件操,作,作平台,合适的液相,色,色谱柱,柱内径：2.1mm,柱长度：,根据分析的,目,目的选择50mm或150mm,柱填料选用,新,新型的填料,：,：,Symmetry，Discovery，Vydac，Zorbax，Xterra，Intersil，Diamonsil等,LC/MSFlowInjectionAnalysisof Peptides andProteinsby Reversed-Phase HPLC,1.0%HOAc,min,Abundance,10.00,12.00,14.00,16.00,2.00,4.00,6.00,8.00,50000,100000,150000,200000,250000,0.2%TFA,2.00,4.00,6.00,8.00,10.00,12.00,14.00,16.00,50000,100000,150000,200000,250000,min,Abundance,Reverse-Phase LC/MSSolvents,ACN,MeOH,H,2,O,Isopropanol,Normal-Phase LC/MS Solvents(for APCI-MS),Hexane,Methylene Chloride,Acetone,Ethanol,CompatibleLC/MS Buffersand Modifiers:,Formic acid,aceticacid,ammoniumacetate,ammonium formate,ammoniumhydroxide,trifluoroacetic acid,TFA concentration shouldbe 0.1%v/v,Keepvolatile bufferconcentrations20mM tominimizeESI ion suppression,AvoidNon-volatile Buffers,Alkali-metal phosphates,borates,etc.,Suitable Solvents for LC/MS,Volatile buffer,minimize instrument downtime,Buffer concentration:,High,ionsuppression decreases ESI sensitivity,Low system adequately buffered?,pH range permitted by stationary phase,Methanol or acetonitrile,Startwithacetonitrile,01111,Change Retention toImprove Resolution Select Solvents/Modifiers that are MSCompatible,Useful pHRanges forVolatileBuffers,Buffers normally used inLC/MS:,01094,Buffer,pK,a,pH range,Formate,3.8,2.8 4.8,Acetate,4.8,3.8 5.8,9.2,8.2 10.2,Triethylamine,11.0,10 12,Diethylamine,10.5,9.5 11.5,?,Ammonia,7,6 8,Buffer Concentrations/Additiveamounts:,10 to50 mM,formic,aceticacids0.01-1%v/v,trifluoroaceticacid0.1%v/v,alkylaminetypebases 0.1%v/v,Effect ofBuffer onAnalyte Response,Phosphatebuffers suppress theMS response ofcaffeineat all pHsandalsothe MS responseof Oxazepam atpH 8.Volatilebuffers(formate,acetate,ammonia)generally providegoodresponses.,01121,Mobile phase:,A-10mM bufferpH 6.0;,B methanol,Gradient:5%to75%in 4min,Mobile Phase pHeffect onESI,Column:HyPURITY,C18 5,m,50 x2.1mm,Aqueous mobilephases:,0.1%Formic acid,pH 3,Ammoniumformate 20mM,pH 5,Ammoniumacetate 20mM,pH 8.2,Ammonium acetate20 mM,pH 9,Ammoniumacetate 20mM,Aqueous/methanol(50:50),Flowrate:0.2ml/min,Temperature:25,C,Detection:+ESI,450,C,4.5kV,20V,-ESI,450C,3.5kV,20V,Scan:120,480u,Analytes:,Nortriptyline,Propranolol,Tetracycline,Caffeine,Paracetamol,Tryptophan,Salicylicacid,Nicotinicacid,01113,Effect ofMobile Phase pHon+ESI Response,01115,Effect ofMobile Phase pHon-ESI Response,01116,Solvent System,50/50 MeOH/H2O,50/50 ACN/H2O,100%H2O,100%MeOH,100%ACN,50/50 MeOH/H2O 1%Acetic,50/50 MeOH/H2O 0.1%Formic,50/50 ACN/H2O 1%Acetic,50/50 ACN/H2O 0.1%Formic,50/50 MeOH/H2O 5mM NH4OAc,50/50 MeOH/H2O 10mM NH4OAc,50/50 MeOH/H2O 0.1%TFA,50/50 MeOH/H2O 0.05%TFA,50/50 MeOH/H2O 0.02%TFA,50/50 ACN/H2O 0.1%TFA,50/50 ACN/H2O 0.05%TFA,50/50 ACN/H2O 0.02%TFA,50/50 MeOH/H2O 0.1%NH4OH,50/50 ACN/H2O 0.1%NH4OH,0,100000,200000,300000,400000,500000,600000,Ion Signal,Counts M+H,+,Solution Chemistry Effects onPositive Ion ESI-MSof Leu-Enkephalin,LC/MSSensitivity vs.Mobile PhaseModifierGluCDigest ofBS,5%Acetic,0.001%TFA,0.005%TFA,0.01%TFA,Zorbax 300SB-C3,(,2.1 x150,mm,),HP1100 MSD,Reversed-phaseHPLC/MS analysis ofa GluC digest of BSAwasusedas amodelto test the recovery andpeakshapeof peptides using varying concentrations ofTFA or 5%acetic acid asa mobile-phaseadditive in combination with the Zorbax300SB-C3.,Digestionof BSA wascarried out 37,C overnight,usingGluCin a1:20ratiowithBSA(by weight).The final mixturecontained1M urea and 25mM sodiumphosphate.,A significant increase insensitivity ofpeptideswas observed for most peptidesanalyzedusing5%aceticacidrather than TFA.,ReducingTFAconcentrationto0.001%causedonlyaminorimprovementinsensitivity.Somepeptidesweremuchlessaffectedbyadditivechangethanothers.,0for5min,0-40%B/55minthen40-100%B/20min,F=0.2mL/min,A=5%AceticAcid,B=ACN,MSD1TIC,MS,Stable,StericallyProtectedC3BondedPhaseinLCandLC/MSApplications,R.D.Ricker(1),B.E.Boyes(1),J.P.Nawrocki(2),andL.K.Pannell(2)(1)AgilentTechnologies,Inc.LCApplicatonsLab538FirstStateBlvd,Newport,DE19804-3552USA.(2)StructuralMassSpectrometryGroupNIDDK,NIH,Bethesda,MD,20892USA.EasternAnalyticalSymposium,Nov.,1999,ProposedMechanismforTFASignalSuppressionandtheTFA-Fix,-,(M+H),+,+CH,3,COO,-,(M+H),+,CH,3,COO,-,0”,Weak Ion Pairing with Acid-Anion,g g,CF,3,COO,-,+RCOOH,CF,3,COOH,+RCOO,-,Acid Competition(TFA more volatile),(M+H),+,+CF,3,COO,-,(M+H),+,CF,3,