Mass Spectrometry for Biotechnology 1 质谱仪的概念？ Mass Spectrometry:Mass Spectrometry: A method to “weigh” moleculesA method to “weigh” molecules A simple measurement of A simple measurement of mass is used to confirm the mass is used to confirm the identity of a molecule, but it identity of a molecule, but it can be used for much can be used for much moremore Other information can be inferred from Other information can be inferred from a weight measurement.a weight measurement. Post-translational Post-translational modificationsmodifications Molecular Molecular interactionsinteractions ShapeShape SequenceSequence Physical dimensionsPhysical dimensions etc.etc. Definition An analytical device that determines the molecular weight of chemical components(and obtains structural information) by separating molecular(and product) ions according to their m/z; 能使物质粒子离子化并根据其质荷比（m/z）不同 ，通过适当的电场、 磁场将它们按空间、时间先 后或者轨道稳定与否进行分离并检测强度后进行 物质分析的仪器。质谱的重要特点之一是检测物 质的离子的质/荷比。 2 质谱仪技术及其发展 The Five Mass Spectrometry Nobel Prize Pioneers Joseph John Thomson 1906 Nobel Prize for Physics “in recognition of the great merits of his theoretical and experimental investigations on the conduction of electricity by gases“ Francis William Aston 1922 Nobel Prize for Chemistry “for his discovery, by means of his mass spectrograph, of isotopes, in a large number of non-radioactive elements, and for his enunciation of the whole-number rule“ Wolfgang Paul 1989 Nobel Prize for Physics “for the development of the ion trap technique“ John Bennet Fenn 2002 Nobel Prize for Chemistry “for the development of soft desorption ionisation methods (ESI) for mass spectrometric analyses of biological macromolecules“ Koichi Tanaka 2002 Nobel Prize for Chemistry “for the development of soft desorption ionisation methods (MALDI) for mass spectrometric analyses of biological macromolecules“ Preface and Introduction(2) 3.Definition and basic components: 1)An analytical device that determines the molecular weight of chemical components(and obtains structural information) by separating molecular(and product) ions according to their m/z; 2)Consists of three components: ionization source(ion production); mass analyzer(ion separation); ion detector(ion detection); Preface and Introduction(3) Pre-Separation Mass SpectrometerMass Spectrometer for Proteomicsfor Proteomics Liquid Chromatography Mass AnalyzerIon Detector Time-of-Flight (TOF) Quadrupole TOF (QTOF) Ion Trap (IT) Fourier Transform- Ion Cyclotron Resonance (FT-ICR) Ion SourceIon Source Chapter 1 Ion Sources and Sample Introduction Unit 1 Sample Introduction methods for sample introduction: *use of insert probe; *capillary infusion; including(in fact): GC,LC,Syringe pump,needle ,etc. Unit 2 Ionization Techniques(1) Most common ionization methods: *electron ionization,EI *fast atom/ion bombardment,FAB *matrix-assisted laser desorption/ionization,MALDI *electrospray ionization,ESI Unit 2 Ionization Techniques(2) 1.Electron Ionization,EI use electron with certain energy impact on the sample and make them ionized.(electron impact) Electron Ionization,EI Unit 2 Ionization Techniques(3) Key notes: 1)Sample is delivered as a gas ;(involving in thermal desorption or “boiling off” the sample from the probe) 2)limited mass range ;(due to the thermal desorption (volatility) requirement) 3)principal problems in analysis of large biomolecules: difficult to volatilize(nonvolatile),thermal decomposition,excessive fragments(in many cases); Unit 2 Ionization Techniques(4) 2.Fast Atom/ion Bombardment(FAB) 1)use a high-energy beam of Xe atoms,Cs ions,or massive glycerol-NH4+ clusters to sputter the sample and matrix from the probe surface into the gas phase. 2)The sample may be already charged or it may become charged through reactions with surrounding molecules or ions. 3)The ionization reaction primarily make sample protonized and form MH+. Unit 2 Ionization Techniques(5) 3、Matrix-Assisted Laser Desorption/Ionization,(MALDI) 1)permit the analysis of high-molecular-weight compounds (and heterogeneous samples) with high sensitivity. 2) uses laser light and a solid matrix. 3)functions of matrix: *absorb the laser light energy to vaporize the matrix and sample; *to minimize sample damage; *facilitate the ionization of samples; Unit 2 Ionization Techniques(6) MALDI matrices 4-hydroxy- cyanocinnamic acid (“alpha- cyano” or 4- HCCA) peptidespeptides 3,5-dimethoxy-4- hydroxycinnamic acid (sinapinic acid) proteinsproteins 2,5-dihydroxybenzoic acid (DHB) peptidespeptides and proteinsproteins matrices for 337 nm irradiation MALDI 337 nm irradiation is provided by a nitrogen (N2) laser The target plate is inserted into the high vacuum region of the source and the sample is irradiated with a laser pulse. The matrix absorbs the laser energy and transfers energy to the analyte molecule. The molecules are desorbed and ionized during this stage of the process. MALDI is most commonly interfaced to a tim