1、micrOTOF-Q 应用培训应用培训培训目的：培训目的：了解ESI-Q-TOF的基本原理 掌握仪器的基本操作和维护 数据处理软件的使用潘晨松 博士布鲁克道尔顿公司应用技术支持工程师培训安排培训安排第一天第一天 基本理论简介及使用中应注意的要点 microTOF Control软件介绍(1)测样练习（tunemix、小分子、多肽、蛋白质）microTOF Control软件(2):Calibration;DataAnalysis 软件介绍(1)练习DataAnalysis软件的使用 学员练习校正及测样第二天第二天 复习及操作考查 HyStar LC/MS 练习小分子混合样品和蛋白/多肽样品测定
2、 DataAnalysis软件(2)练习处理LC/MS数据第三天第三天 复习及操作考查 软件介绍与练习（LibraryEditor,Isotope Pattern,Generate Molecular Formular,ReportDesigner.)练习处理LC/MS数据 样品测定样品测定 答疑、讨论液质联用型液质联用型高分辨高分辨串联质谱仪串联质谱仪 电喷雾四级杆飞行时间电喷雾四级杆飞行时间(ESI-Q-TOF)商品名：商品名：micrOTOF-QDry Gas HeaterOrthogonal AcceleratorGlass CapillaryCollision Gas SupplyH
3、exapole电喷雾离子源去溶剂系统飞行管检测器反射镜四级杆Q碰撞池产生离子产生离子离子传输与聚焦离子传输与聚焦分辨离子分辨离子Ion GenerationIon Transmission and focusIon ResolutionmicrOTOF-QIon Generation:Atmospheric Pressure Ionization(API)Ion generation for MS analysis Nebulization Desolvation Coulomb explosions DesorptionUnder proper source conditions,indiv
4、idual ions only will enter the capillary.An unstable signal or capillary current may indicate a need for adjustment of gases,flow rate,or spray needle.See the Users Manual for Troubleshooting tips.Nebulizer GasSampleDry GasCapillary(4 kV)Spray NeedlegroundedDry GasGeneration of Ions-NebulizationDrop
5、let formation in presence of electrical field at the needle tip within the spray chamberGeneration of Ions-DesolvationGeneration of Ions-Ion EvaporationTo obtain spectral information,ions need to be created and introduced to the instrument for interrogationMust consider flow rates of Sample:Nebulize
6、rDry gasGeneration of Ions-ESI sourceThe sample is introduced via the nebulizer.The ESI process is supported by nitrogen gas.The drying gas heats up the source and dries the spray.It acts as a counter current flow to the nebulized sample ensuring complete evaporation of the solvent in order to preve
7、nt droplets from entering the glass capillary.The desolvation unit contains the dry gas heating block and capillary housing.The glass capillary interfaces the atmospheric pressure region of the spray chamber with the fist vacuum stage of the ion optics separates atmospheric pressure from first vacuu
8、m stage parts:drying gas heater glass capillaryGeneration of Ions Desolvation unitElectrospray Factors Affecting IonizationNeedle set-up Inner Needle Position Nebulizer Pressure Needle ConditionHigh voltage electrodes Capillary Voltage settings Condition of Capillary and Chamber High Voltage Element
9、s Condition of InsulatorsSolution Chemistry Flow Rate Solution pH Sample pKa Solution ConductivityElectrospray Solution ChemistryMobile phase pH has a major effect for analytes that are ions in solution.Basic pH(7.0;9 preferred)for negative ionsAcid pH(7.0;5 preferred)for positive ions*Manipulation
10、of pH can enhance performance for analytes that are not normally ionized in solution.Electrospray Sample ChemistryPositive Ion Mode Negative Ion Mode Base +acid Sample Acid +base SampleRNRRRNRHRH AOCORHOCOR:B+H:B+:-+A-Electrospray BuffersChoose buffers carefully for TOF instrumentsElectrospray Buffe
11、r Selection:Volatile buffers are used to modify mobile phase pH.May be added in mobile phase as a post-column addition.Acidic solutions favor positive ion mode.Formic acid,0.1-1.0%Acetic acid,0.1-1.0%Ammonium salts favor production of single ammonium adducts.General buffers Ammonium acetate Ammonium
12、 formate Triethylamine Other volatile solvents(离子传输率大大提高，从而离子传输率大大提高，从而提高灵敏度。提高灵敏度。增宽传输离子质量范围增宽传输离子质量范围 离子流方向传统的锥式离子传输传统的锥式离子传输DCACResonant ionNonresonant ionQuadrupole Mass Analyzer Collision Cell+Parent ion FragmentsArgon Gas EnergyMS:Ions were cooled by argon gasMS/MS:Fragments were produced by a
13、rgon gas and energy Dry Gas HeaterOrthogonal AcceleratorGlass CapillaryCollision Gas SupplyHexapole电喷雾离子源去溶剂系统飞行管检测器反射镜四级杆Q碰撞池产生离子产生离子离子传输与聚焦离子传输与聚焦分辨离子分辨离子Ion GenerationIon Transmission and focusIon ResolutionIon Resolution Contains last lens package(lens4&5)Accelerates the ions for measuring the t
14、ime of flight Fill mode -pusher and puller are grounded,corrector is on“fill potential -the region between pusher and puller is filled with ions Extract mode -pusher and puller are set to“high”voltages opposite to each other (about 400V)-corrector is on extract voltage -ions are accelerated into ver
15、tical direction(towards the reflector)Ion Optics Orthogonal AcceleratorIn the ideal case:mi=mass of analyte ionzi=charge of analyte ionE=extraction fieldti=flight time of ionls=length of source(orthogonal acceleration stage)ld=length of the field-free drift regione=electronic charge(1.06022 x 10-19
16、C)TOF Theory22 =disiilteElzmThe aim of an electrostatic reflector,also called reflectron is to improve mass resolution.It creates a retarding field that acts as an ion mirror by deflecting the ions and sending them back through the flight tube.The reflector corrects the energy dispersion of ions wit
17、h the same m/z ratio.Indeed,ions with more kinetic energy will penetrate the reflectron more deeply and will spend more time in the reflectron.Thus they reach the detector at the same time as slower ions of the same m/z.Ions receive kinetic energy from electric field.E=1/2mv2Resolution of Ions TOF A
18、ssemblym1=m2,but E1 E2Orthogonal Detector acceleratorThink of the TOF operation as a drag race between vehicles of different sizes,but all having identical engines:“Start line”=orthogonal accelerator;“Finish line”=TOF detector Just as all vehicles have the same engine(i.e.,horsepower),all ions are p
19、ulsed up the flight tube with the same kinetic energy.Since m=2E/v2,the smaller vehicles/ions will reach the finish line/detector before the larger ones.STARTFINISHPrinciple of the TOF Mass AnalyzerA detector converts an ion signal into an electrical signal.Here,the detector is a micro-channel plate
20、 detector.It has millions of small pores which are coated inside with a semi-conductive layer.Each of these channels work as an independent electron multiplier.Micro-channel plates are effective for the detection of signals over a large dynamic range without saturation.In addition,the time response
21、of this detection system is very rapid,therefore avoiding deterioration of peak resolution.Multiplication process in a channelMicrochannel Plate DetectorQ-SeparationCIDDry Gas HeaterDual Ion FunnelAnalyticalQuadrupoleCollision CellOrthogonal AcceleratorDetectorReflectronFlight TubeGlass CapillaryCol
22、lision Gas SupplyAPI Spray ChamberSprayerHexapolemicrOTOFQ MS2 PossibilitiesTOF-MSIn Source CIDMS3质谱基本术语：质谱基本术语：分辨率分辨率 Mass Resolution同位素分布模式同位素分布模式 Isotope Patterns单同位素质量单同位素质量 Monoisotopic Mass.It is a measure of a mass spectrometers ability to distinguish two compounds of nearly equal mass.600800
23、10001200140016001800200022002400m/z 100 200 300 400 500 600 700 800 900 1000 1100a.i.161816231628 m/z“easy”“not so easy”ResolutionWhat is meant by Resolution?The narrower the FWHM,the higher the resolution.With the same FWHM,higher masses will have higher resolution than lower masses.Resolution=(m/z
24、)/FWHMFWHM Full Width at Half Maximum1521.97431522.9772+MS,0.6-0.9min#(40-57)0100200300400Intens.152015211522152315241525m/zm/zHow do we measure mass resolution?Isotopic Distribution Patterns13121009080706050403020100C11221211201009080706050403020100C101,2061,2041,2021,2001009080706050403020100C1001
25、2,03012,02012,01012,0001009080706050403020100C1000Isotopes are atoms with the same number of protons in the nuclei,but with different numbers of neutrons.Only 21 elements have only one stable isotope.All other elements are mixtures of at least 2 stable isotopes,and the proportions of these isotopes
26、can vary greatly depending on the element.Carbon has 2 stable isotopes,C-12 and C-13,with natural abundance of 98.892%and 1.108%respectively.As the number of carbons increase in a molecule,the isotopic distribution pattern will reflect the mass contribution of the isotopes with their extra neutrons.
27、pQLYENKPRRPYIL MW 1672.9Res.1,0001,6821,6801,6781,6761,6741,672100908070605040302010 1673.9Average Mass401,6821,6801,6781,6761,6741,67210090807060503020100Res.10,000M+H+M+H+1+M+H+2+M+H+3+M+H+4+1672.9Monoisotopic MassNeurotensinResolution=(m/z)/FWHMAverage and monoisotopic massesExact mass or accurat
28、e mass is the mass of the monoisotopic peak measured accurately to within a few millimass units.Lower resolution results in an“average”-inaccurate determination of peak center -calculated average mass is inaccurate at best multiple charged ion m/z=M+nHnH质荷质荷比的计算：比的计算：591.7975592.2961592.7964593.2987
29、+MS,0.1-0.3min#(3-9)0123455x10Intens.591.5592.0592.5 593.0593.5594.0m/z556.2761557.2774558.2810+MS,0.1-0.3min#(3-9)012345x10Intens.556557558559m/z2+1+Determining Multiple Charge StatesExample:m1=1000,isotope m2=1001Charge(z)=1 1000/1,1001/1 m/z=1000,1001Charge(z)=2 1000/2,1001/2 m/z=500,500.5Charge(
30、z)=3 1000/3,1001/3 m/z=333.33,333.66The m/z difference is incremental to the charge.Charge 2=0.5Charge 3=0.333Charge 4=0.252+3+质量准确度计算质量准确度计算Mass accuracy can be expressed as a percentage or ppm:e.g.%mass accuracy =Measurement errorTrue massx 100%Very small percentage errors(0.01%)are expressed as p
31、arts per million or ppm.0.01%=100ppme.g.ppm=Measurement errorTrue massx 106Measured mass:1296.970 True mass:1296.685 0.02%errorppm=0.001 per thousandmicrOTOF Q TuningThe goal of tuning is to maximize the intensity and/or resolution of ions within an m/z range of interest.To achieve this,the paramete
32、rs of the source and TOF portions of the instrument must be properly set.Source Parameters largely affect the intensity;Optics Parameters-determine the m/z range over which ions are transmitted.TOF Parameters largely affect the resolution.micrOTOF Q TuningTune ParametersIon OpticsThus we give the ru
33、le of thumb:the larger the mass,the higher the value for the tuning parameters.How the voltages affect the ions is largely mass dependent.and smaller mass ions require less energy to control them.Larger mass ions need more energy to direct them into the trap,ScansSummationsRollingAvg.of 1A single sp
34、ectrum is obtained each time the TOF is pulsed.A data point is the spectrum that is saved and/or displayed in the GUI.To make one data point,several spectra are summed together.When rolling average is turned on,each data point is an average of itself with the“X”number of data points prior.Each avera
35、ged spectrum is weighted proportionally to its recency.RollingAvg.of 2less weightmore weightSumming and averaging increases the statistical reliability of your data set.78910111213Time min0.00.51.01.52.02.58x10Intens.mAURolling 5:TIC AllRolling averages together is used for infusion of low level sam
36、ples to increase the signal to noise(S/N)ratio for the low intensity ions of interest.Rolling provides a smoothing effect to the data and tailing can be observed in a chromatographic peak if there is too much rolling.This is because older scans with higher intensity make up part of the data point af
37、ter the peak has eluted.78910111213Time min0.00.51.01.52.02.58x10Intens.mAURolling off:TIC AllPeak tailing Loss of peak resolution Decreased peak intensityTo smooth chromatographic data,rolling of one to three is acceptable.Rolling values higher than this can cause considerable tailing and loss of p
38、eak shape and separation,which could result in more work to extract data during processing.Rolling does not affect the number of data points and thus does not affect the size of the data file.For data reduction,increase the number of summations.Rolling averaged scans together increases the statistic
39、al reliability of your data set.Too much RollingNotice:Helpful Tips m/z Species Contaminant 102(M+H)+triethylamine(TEA)123(M+H)+dimethylaminopyridine(DMAP)130(M+H)+diisopropylethylamine(DIPEA)144(M+H)+Tripropylamine(TPA)153(M+H)+1,8-diazabicyclo5.4.0undec-7-ene(DBU)182(M+H)+diTMS diaminopyridine 225
40、 449(M+H)+(2M+H)+Dicyclohexyl urea(DCU)239/241(M.HCl)2-Cl+triethylamine 242 M+tetrabutylammonium(C4H9)4N+391 413 454 803(M+H)+(M+Na)+(M+Na+CH3CN)+(2M+Na)+Pthalic acid ester/DOP or DEHP diisooctyl phthalate(plasticiser)Typical ESI Source ConditionsTypical Currents(V):Capillary 3000 4500 End Plate-500
41、(up to 2000 with buffers)ESI Common Solvents:ESI Common BuffersMethanolEthanolAcetic AcidPropanolIsopropanolFormic AcidButanolWaterAmmonium Acetate and FormateAcetoneAcetonitrileSodium AcetateChloroformFormamidePotassium AcetateTetrahydrofuranAcetic AcidESI Solvents Requiring Modifier:BenzeneCarbon DisulfideCarbon tetrachlorideCyclohexaneHexaneLigroinDichloromethaneToluene*DO NOT USE TFA.Useful Conversions1 mM=1000 mM1 mM=1 pmol/mL1 mg/mL=1 mg/mLTo convert mg/mL or mg/mL to pmol/m mL:pmol/mL=(mg/mL*106)/protein MWmilli=10-3m micro=10-6nano=10-9pico=10-12femto=10-15atto=10-18
