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1、micrOTOF-Q 应用培训,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,micrOTOF-Q 应用培训培训目的：潘晨松 博士,培训安排,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 复习及操作考查 HyStar LC/MS 练习小分子混合
2、样品和蛋白/多肽样品测定 DataAnalysis软件(2) 练习处理LC/MS数据,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,培训安排,液质联用型高分辨串联质谱仪 电喷雾四级杆飞行时间 (ESI-Q-TOF),商品名：micrOTOF-Q,液质联用型高分辨串联质谱仪 商品名：micrOTOF-Q,Dry Gas Heater,Orthogonal Accelerator,Glass Capil
3、lary,Collision Gas Supply,Hexapole,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,离子传输与聚焦,分辨离子,Ion Generation,Ion Transmission and focus,Ion Resolution,micrOTOF-Q,Dry Gas HeaterOrthogonal Accel,Ion Generation: Atmospheric Pressure Ionization (API),Ion Generation: Atmospheric Pr,Atmospheric Pressure Ionizati
4、on- Electrospray Ionization (API-ESI),Ion generation for MS analysis Nebulization Desolvation Coulomb explosions Desorption,Under proper source conditions, individual ions only will enter the capillary. An unstable signal or capillary current may indicate a need for adjustment of gases, flow rate, o
5、r spray needle. See the Users Manual for Troubleshooting tips.,Atmospheric Pressure Ionizatio,Nebulizer Gas,Sample,Dry Gas,Capillary ( 4 kV),Spray Needlegrounded,Dry Gas,Generation of Ions - Nebulization,Droplet formation in presence of electrical field at the needle tip within the spray chamber,HV,
6、Nebulizer GasSampleDry GasCapi,Generation of Ions-Desolvation,Generation of Ions-Desolvation,Generation of Ions-Ion Evaporation,Generation of Ions-Ion Evapora,To obtain spectral information, ions need to be created and introduced to the instrument for interrogationMust consider flow rates of Sample:
7、NebulizerDry gas,Generation of Ions - ESI source,The 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 o
8、rder to prevent droplets from entering the glass capillary.,To obtain spectral information,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 s
9、eparates atmospheric pressure from first vacuum stage parts: drying gas heater glass capillary,Generation of Ions Desolvation unit,The desolvation unit contains,Electrospray Factors Affecting Ionization,Needle set-up Inner Needle Position Nebulizer Pressure Needle Condition,High voltage electrodes C
10、apillary Voltage settings Condition of Capillary and Chamber High Voltage Elements Condition of Insulators,Solution Chemistry Flow Rate Solution pH Sample pKa Solution Conductivity,Electrospray Solution ChemistryMobile phase pH has a major effect for analytes that are ions in solution. Basic pH (7.0
11、; 9 preferred) for negative ionsAcid pH (7.0; 5 preferred) for positive ions*Manipulation 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 Sample,Electrospray Factors A
12、ffectin,Electrospray Buffers,Choose buffers carefully for TOF instrumentsElectrospray Buffer 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% A
13、mmonium salts favor production of single ammonium adducts. General buffers Ammonium acetate Ammonium formate Triethylamine Other volatile solvents (0.5%) Basic solutions favor negative ion mode. Ammonium hydroxide (pH 10-11)*TFA can act as an ion suppressant avoid use.,Electrospray BuffersChoose buf
14、,APCI,APCI LC/MSAdvantagesComplementary to API-Electrospray for less polar analytes.Good sensitivity for compounds of intermediate MW and polarity. Less sensitive to solution chemistry effects than API-ES. Tolerates higher flow rates without decrease in sensitivity. DisadvantagesLess useful for ther
15、mally labile compounds. Requires some compound volatility.,Classical APCI process:The mobile phase and analyte are nebulized. The droplets are vaporized. The mobile phase molecules are ionized by electrons from the corona discharge. The analyte molecules are ionized by the mobile phase ions.,APCIAPC
16、I LC/MSClassical APCI p,Atmospheric Pressure Ionization,Atmospheric Pressure Ionization (API) is a family of techniques which include:Electrospray (ESI)Solvent and sample are nebulized by use of a gas stream.Small droplets are dried and undergo electrospray ion formation.Ions in solution are desolva
17、ted and desorbed under the influence of high potential electrostatic fields.Atmospheric Pressure Chemical Ionization (APCI)Solvent and sample are nebulized and completely vaporized by a heater.Ionization occurs both during the vaporization process and as the solvent and sample enter the corona regio
18、n of the APCI source.,Relative Applicability of LC/MS Techniques,Electrospray LC/MSAdvantagesSoftest ionization availableLC/MS interface with best sensitivityExtends mass range for multiply charged analytesWorks with a wide range of moderate to high polarity compoundsLow maintenanceDisadvantagesSolu
19、tion chemistry influences ionization processWorks only for moderate to high polarity analytesAdduct ions (other than M+H) possible with some analytesSome sensitivity loss at higher flow rates (1ml/min),Atmospheric Pressure Ionizatio,Dry Gas Heater,Orthogonal Accelerator,Glass Capillary,Collision Gas
20、 Supply,Hexapole,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,离子传输与聚焦,分辨离子,Ion Generation,Ion Transmission and focus,Ion Resolution,Ion Transmission and Focus,Dry Gas HeaterOrthogonal Accel,电喷雾四级杆飞行时间Qtof理论课件,电喷雾四级杆飞行时间Qtof理论课件,布鲁克公司独家专利设计：离子漏斗式传输系统,离子流方向,离子传输率大大提高，从而提高灵敏度。增宽传输离子质量范围,离子流方向,传统的锥式离子传输,布鲁克公司
21、独家专利设计：离子流方向离子传输率大大提高，,Quadrupole Mass Analyzer,DCACResonant ionNonresonant io,Collision Cell,Parent ion,Fragments,Argon Gas,Energy,MS : Ions were cooled by argon gasMS/MS: Fragments were produced by argon gas and energy,Collision Cell+Parent ion Fr,Dry Gas Heater,Orthogonal Accelerator,Glass Capill
22、ary,Collision Gas Supply,Hexapole,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,离子传输与聚焦,分辨离子,Ion Generation,Ion Transmission and focus,Ion Resolution,Ion Resolution,Dry Gas HeaterOrthogonal Accel,Contains last lens package (lens4 & 5) Accelerates the ions for measuring the time of flight,Fill mode - pusher
23、 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 vertical direction (towar
24、ds the reflector),Ion Optics Orthogonal Accelerator,Ion beam,Ion beam,Contains last lens package (l,In 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
25、 regione = electronic charge (1.06022 x 10-19 C),TOF Theory,In the ideal case:mi = mass of,The 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 fli
26、ght tube.The reflector corrects the energy dispersion of ions with 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
27、 kinetic energy from electric field. E = 1/2mv2,Resolution of Ions TOF Assembly,m1 = m2, but E1 E2,Orthogonal Detector accelerator,The aim of an electrostatic re,Think of the TOF operation as a drag race between vehicles of different sizes, but all having identical engines: “Start line” = orthogonal
28、 accelerator; “Finish line” = TOF detector Just as all vehicles have the same engine (i.e., horsepower), all ions are pulsed 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.,START,FINISH,Principle
29、of the TOF Mass Analyzer,Think of the TOF operation as,A detector converts an ion signal into an electrical signal. Here, the detector is a micro-channel plate detector. It has millions of small pores which are coated inside with a semi-conductive layer. Each of these channels work as an independent
30、 electron multiplier.Micro-channel plates are effective for the detection of signals over a large dynamic range without saturation. In addition, the time response of this detection system is very rapid, therefore avoiding deterioration of peak resolution.,Multiplication process in a channel,Microcha
31、nnel Plate Detector,A detector converts an ion sig,Q- Separation,CID,micrOTOFQ MS2 Possibilities,TOF-MS,Q- SeparationCIDDry Gas Heater,质谱基本术语： 分辨率 Mass Resolution同位素分布模式 Isotope Patterns单同位素质量 Monoisotopic Mass.,质谱基本术语：,It is a measure of a mass spectrometers ability to distinguish two compounds of
32、nearly equal mass.,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”,“not so easy”,Resolution,What is meant by Resolution?,It is a measure of a mass spec,The narrower the FWHM, the higher the resolution. With the same FWH
33、M, higher masses will have higher resolution than lower masses.,Resolution = (m/z) / FWHM,FWHM Full Width at Half Maximum,How do we measure mass resolution?,The narrower the FWHM, the hig,Isotopic Distribution Patterns,Isotopes are atoms with the same number of protons in the nuclei, but with differ
34、ent 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 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
35、.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.,Isotopic Distribution Patterns,pQLYENKPRRPYIL MW 1672.9,Res. 1,000,1,682,1,680,1,678,1,676,1,674,1,672,100,90,80,70,60,
36、50,40,30,20,10,1673.9,Average Mass,40,1,682,1,680,1,678,1,676,1,674,1,672,100,90,80,70,60,50,30,20,10,0,Res. 10,000,M+H+,M+H+1+,M+H+2+,M+H+3+,M+H+4+,1672.9,Monoisotopic Mass,Neurotensin,Resolution = (m/z) / FWHM,Average and monoisotopic masses,Exact mass or accurate mass is the mass of the monoisoto
37、pic 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,pQLYENKPRRPYIL Res. 1,0001,682,multiple charged ion,2+,1+,multiple charged ion m/z =M,Determining Multiple Ch
38、arge States,Example: 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 (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 = 0.25,2+,3+,1+,Deter
39、mining Multiple Charge St,质量准确度计算,Mass accuracy can be expressed as a percentage or ppm:,e.g. % mass accuracy =,Very small percentage errors (0.01%) are expressed as parts per million or ppm. 0.01% = 100ppm,Measured mass: 1296.970 True mass: 1296.685 0.02% error,ppm = 0.001 per thousand,质量准确度计算Mass
40、accuracy can be ex,micrOTOF Q Tuning,The goal of tuning is to maximize the intensity and/or resolution of ions within an m/z range of interest.,To achieve this, the parameters of the source and TOF portions of the instrument must be properly set.,Source Parameters largely affect the intensity; Optic
41、s Parameters - determine the m/z range over which ions are transmitted.TOF Parameters largely affect the resolution.,micrOTOF Q TuningThe goal of t,micrOTOF Q Tuning,micrOTOF Q Tuning,Tune Parameters,Ion Optics,Thus we give the rule of thumb: the larger the mass, the higher the value for the tuning
42、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,Tune ParametersIon OpticsThus,Scans,Summations,RollingAvg. of 1,A single spectrum is obtained each time th
43、e 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 averaged spectrum is weighte
44、d proportionally to its recency.,RollingAvg. of 2,Rolling Average and Summation,Summing and averaging increases the statistical reliability of your data set.,ScansSummationsRollingAvg. of,7,8,9,10,11,12,13,Time min,0.0,0.5,1.0,1.5,2.0,2.5,8,x10,Intens.,mAU,Rolling 5: TIC All,Rolling averages togethe
45、r is used for infusion of low level samples 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
46、intensity make up part of the data point after the peak has eluted.,7,8,9,10,11,12,13,Time min,0.0,0.5,1.0,1.5,2.0,2.5,8,x10,Intens.,mAU,Rolling off: TIC All,Rolling off,Peak tailing Loss of peak resolution Decreased peak intensity,Rolling 5,To smooth chromatographic data, rolling of one to three is
47、 acceptable. Rolling values higher than this can cause considerable tailing and loss of peak 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 reducti
48、on, increase the number of summations. Rolling averaged scans together increases the statistical reliability of your data set.,Rolling Averages and Chromatography,Too much Rolling,Notice:,78910111213Time min0.00.51.0,Helpful Tips,Typical ESI Source Conditions,Typical Currents (V): Capillary 3000 450
49、0 End Plate -500 (up to 2000 with buffers)ESI Common Solvents:ESI Common BuffersMethanolEthanolAcetic AcidPropanolIsopropanolFormic AcidButanolWaterAmmonium Acetate and FormateAcetoneAcetonitrileSodium AcetateChloroformFormamidePotassium AcetateTetrahydrofuranAcetic AcidESI Solvents Requiring Modifi
50、er: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/mL :pmol/mL =(mg/mL * 106)/protein MW,milli = 10-3micro = 10-6nano = 10-9pico = 10-12femto =10-15
