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MASS Spectrometry Physical Basis

MASS Spectrometry Physical Basis. Differential deflection of charged species in a magnetic field. Two species, of identical charge and velocity but different mass, will be deflected differently by a magnetic field. The species with the smallest momentum, m, is deflected

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MASS Spectrometry Physical Basis

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  1. MASS Spectrometry Physical Basis Differential deflection of charged species in a magnetic field. Two species, of identical charge and velocity but different mass, will be deflected differently by a magnetic field. The species with the smallest momentum, m, is deflected to a greater extent than that with larger m

  2. MASS Spectrometry Physical Basis Electron soruce To analyser and detector Sample in Charged particles Ionisation chamber Ionisation of lactic acid by electron impact. Latic acid is ionised by electrons forming a molecular ion. This breaks down into a large nmber of fragment ions, each possessing a characteristic mass allowing their identification. The pattern of ions generated is characteristic of the chemical structure of the parent molecule and can therefore be used to calculate this structure Electron Impact ionisation. Sample is bombarded with a high-energy beam of electrons which causes ionisation. These ions are subsequently accelerated and deflected in the analyser

  3. Turbo pumps Diffusion pumps Rough pumps Rotary pumps High Vacuum System Ion source Mass Filter Data System Inlet Detector • Sample plate • Target • HPLC • GC • Solids probe • DCI • MALDI • Electro-spray • (IonSpray) • APCI • FAB • LSIMS • EI • CI • TOF • Quadrupole • Ion Trap • Magnetic • Sector • FTMS • Micro-channel • Plate • Electron • Multiplier • “Hybrid” • PC’s • Sun SPARKStation • DECStation Composition of MASS Spectrometry

  4. Ionisation Modes • EI (Electron ionization) - Filament 등의 thermionic emission에 의해 방출되는 열전자를 이용한 이온화. 이용하기가 매우 간단하고 용이함. 비선택적 이온화 과정에서 fragmentation되는 경향이 강함. • PI (Photoionization) – Lamp나 laser등에서 나오는 빛을 이용한 이온화. 선택적으로서 들뜬 상태에 대한 정보를 필요로 함. 많은 장비가 필요하나 fragmentation이 심하지 않고 다양한 변형이 가능함. • ESI (Electrospray ionization) & TSI (Thermospray ionization) – 응축상 시료에 대한 전기장 또는 열을 이용한 분무방식의 vaporization/ionization. Multiply charged ion이 생성. Fragmentation 이 심하지 않고 액체등의 시료에 매우 적합함.

  5. CI (Chemical ionization) – 이온/분자간 화학반응에 의한 이온화. EI 다음으로 널리 사용되며 양이온뿐만 아니라 음이온도 생성됨. • FI (Field ionization) – 양자역학적 electron tunneling에 의한 이온화. 거의 fragmentation이 없슴. 매우 낮은 감도. • FAB (Fast atom bombardment) ionization - 전하교환을 통해 얻어진 고속 원자를 액체/고체 시료에 충돌시켜 일어나는 분자의 ejection/ionization. 비휘발성 물질 및 쉽게 분해되는 분자들의 질량 분석에 적합. • MALDI (Matrix-assisted laser desorion/ionization) – matrix solution에 laser를 조사할 때 일어나는 탈착 및 이온화. 휘발성이 작거나 잘 녹지 않는 시료에 적합함. Fragmentation이 적음.

  6. FAB Continuous flow fast atom bombardment (FAB) Ionisation. Sample, held in a liquid matrix, is bombarded with a beam Of atoms such as xenon. Ions are formed in a dense gas which may be accelerated towards the analyser and detector. In continuous flow (dynamic) FAB, sample may flow continuously in a capillary passing through the probe as shown PDI Plasma Desorption Ionisation (PDI). 252Cf breaks down to form two particles which travel in opposite directions at the same velocity. One of these activates a time counter (at t0) while the other causes vaporisation and ionisation of the sample. Smaller mass ions travel down the drift tube of the TOF analyser more quickly than larger ones. When ions arrive at detector, time of flight (t) is calculated

  7. MALDI Matrix-assisted laser desorption ionisation (MALDI). A pulse of laser light (UV) is directed at the sample which is Co-crystallised with a suitable matrix component. This matrix component absorbs most of the energy of the laser. Both matrix and sample are vaporised by the laser. In the gas phase, proton exchange occurs between matrix and Sample components producing MH+ ions ESI Electrospary ionisation (ESI). Sample is converted to a fine spray of droplets. Matrix is evaporated from this, leading to Coulombic explosion and the liberation of ions which then are directed to the analyser and detetor. In ion spray ionisation (ISI), the sample is surrounded by a sheath of gas such as N2 which imparts greater energy and hence facilitates faster input of sample to the system

  8. APPI interface의 원리 (atmospheric pressure photoionization ionisation) APCI interface의 원리 (atmospheric pressure chemical ionisation)

  9. MASS Analyzer Magnetic sector Ion trap Ion trap analyser. Ions are either created within or rapidly infected into the trap. The electrostatic field forces them into specific orbits, depending on the m/z ratio. By varying this electrostatic field, ions may be selectively ejected in order of increasing m/z from the trap and detected

  10. Quadrupole EI/MS EI/MS source. Sample (shown as circles) is introduced as vapour into evacuated chamber. Electrons diffuse from the heated filament at an energy of 70 eV. The electron beam ionises sample molecules into anions or cations which are accelerated by an electric potential towards the analyser Quadropole mass analyser. Varying the radiofrequency (RF) component of voltage allows ‘tuning’ for specific m/z ratios. Ions with either larger of smaller m/z collide with rods and are lost

  11. Why interested in MALDI-TOF MS? • High mass range • Speed of analysis • Sensitivity • Simplicity/ease-of-use • New research approach • Robustness/tolerance for sample impurities • Utility as a complement to other MS methods • Low cost • Basic interest in the technique

  12. Useage of MALDI-TOF MS -고분자 합성시료 및 생체물질(단백질, 핵산, 탄수화물등) 분자량 측정 - 단백질 분자량 측정을 통한 post translational modifications 확인, recombinant protein의 QC등 - 합성 펩타이드의 확인, 단백질 분해를 통한 서열분석 및 단백질 ID - 핵산의 분자량 측정을 통한 서열분석, 합성확인 및 SNP 결정 - 핵산 및 단백질 분해에 의한 서열분석 - 미량 생화학, 석유화학 혼합물의 분자량 측정 - SPR bio chip을 이용한 분자 상호작용 연구

  13. MALDI-TOF 주요 응용분야

  14. Sample Prep. of MALDI-TOF MS - 시료는 phosphate buffers, Ammonium salts or organic amines의 유도체, detergent가 포함되었을 경우에는 clean-up 과정이 필요. - 시료의 농도는 peptides와 proteins (0.1 - 10pmol/㎕) Oligonucleotides (10 - 100 pmol/㎕) polymers (100 pmol/㎕) glycoproteins같은 몇몇 단백질은 10 pmol/㎕이상의 농도 필요

  15. MALDI-TOF 질량분석기 • MatrixAssistedLaserDesorptionIonization • Time Of Flight Mass Spectrometry Voyager Voyager DE-STR

  16. Matrix의 화학구조 2,5-dihydroxybenzoic acid (2,5-DHB) 2,4,6-trihydroxy acetophenone (THAP) Sinapinic acid (3,5-Dimethoxy-4-hydroxy cinnamic acid) 2-(4-hydroxyphenylazo)-benzoic acid (HABA) trans-3-indoleacrylic acid 3-hydroxypicolinic acid (3-HPA) -cyano-4-hydroxycinnamic acid Dithranol

  17. Matrix-Assisted Laser Desorption Ionization (MALDI) In Matrix-Assisted Laser Desortion Ionization (MALDI), sample is embedded in a low molecular weight, UV-adsorbing matrix that enhances intact desorption and ionization of the sample. The matrix is present in vast excess of sample, and therefore isolates individual sample molecules.

  18. Flight Tube (0.5 – 4m) Ion Source (4 – 25 KV) Detector KE=1/2mv2 TOF에서 분자 이온들의 분리

  19. Reflector TOF 내 분자이온의 비행 TOF (Time-of-Flight) Flight Tube Detector 4-25 kV Ion Source Reflector (Ion Mirror) Reflector 또는 ion mirror 는 이온발생 부위에서 동일한 질량의 이온들이 초기 에너지 분포(initial energy spread)를 갖는 것을 보정하여 분리능을 증가 시킨다.

  20. Interfacing MS with other methods MS/MS LC/MS GC/MS Electrophoresis/MS

  21. Drug 또는 peptide 혼합물중 특정 분자의 분자만 선택하여 MS/MS 실행 Drug 또는 peptide 혼합물 + + MS MS/MS + + + + Interfacing MS with other methods MS / MS 란? MALDI-TOF 에서는 PSD (post source decay), CID (collision induced dissociation) MS/MS 방법이 있음

  22. Collision-Induced dissociation(CID) in ESI. Three quadropole analysers are used to select for ions with different m/z ratios. Q1 selects intact ions of specific m/z. A colision gas (argon) in introduced into the second analyser (Q2) called the colision cell. Collision with this gas causes fragmentation of the ion into smaller ions. These are analysed in the third quadropole analyser, Q3. Other formats for this kind of experiment are also possible.

  23. LC/MS system suitable for analysis of proteins by electrospray MS. Experimental setup featuring splitting of solvent flow and use of small-diameter HPLC. LC/MS system. The ionspray interface.

  24. Analyte-enrichment interface For GC/MS.

  25. Q-TOF mass spectrometer

  26. MALDI-MS for C-terminal sequence Determination of peptides and proteins degraded by carboxypeptidase Y and P Bernd Thiede, Brigitte Wittmann-Liebold, Michael Bienert, Eberhard Krause FEBS Letters 357 (1995) 65-69

  27. Biochemistry 2004, 43, 13932-13936 Hydrophobic Core around Tyrosine for Human Endothelin-1 Investigated by Photochemically Induced Dynamic Nuclear Polarization Nuclear Magnetic Resonance and Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

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