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Primary Ionization Techniques for Molecules

Electrospray Ionization (ESI). Matrix Assisted Laser Desorption Ionization (MALDI). Primary Ionization Techniques for Molecules. +. +. Laser. +. m. m. m. a. +. a. a. m. m. m. m. +. m. a. a. a. m. matrix + analyte. m. Sample support.

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Primary Ionization Techniques for Molecules

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  1. Electrospray Ionization (ESI) Matrix Assisted Laser Desorption Ionization (MALDI) Primary Ionization Techniques for Molecules

  2. + + Laser + m m m a + a a m m m m + m a a a m matrix + analyte m Sample support Matrix Assisted Laser Desorption Ionization (MALDI)

  3. AH+ Matrix Assisted Laser Desorption Ionization (MALDI) Laser • 1. Sample (A) is mixed with excess matrix (M) and dried • on a MALDI plate. • 2. Laser flash ionizes matrix molecules. • 3. Sample molecules are ionized by proton transfer from matrix: • MH+ + A  M + AH+. Sample plate hn Variable Ground Grid Grid +20 kV

  4. 40000 30000 20000 10000 0 MALDI/TOF Mass Spectrum (M+H)+ Relative Abundance (M+2H)2+ (M+3H)3+ 50000 100000 150000 200000 m/z

  5. Matrix

  6. Dilute samples to the concentrations shown in the table below. • If the sample concentration is unknown a dilution series may be needed • to produce a good spot on the MALDI plate. Sample Dilution/Concentration Note: highly dilute samples can be concentrated by Speed-Vac or Solid Phase Extraction.

  7. Time of Flight (TOF)

  8. Calibration of the mass scale The mass-to-charge ratio of an ion is proportional to the square of its time of flight in the analyzer (“drift time”). t = Drift time L = Drift length m = Mass K = Kinetic energy of ion z = Number of charges on ion

  9. MALDI: Matrix Assisted Laser Desorption Ionization Sample plate hn • Sample (M) is mixed with excess matrix (X) and dried on a MALDI plate. • The plate is loaded onto the sample stage in the Ion Source to Mass Analyzer Sample & Matrix GroundGrid

  10. MALDI: Matrix Assisted Laser Desorption Ionization Laser hn • Laser flash produces matrix neutrals (X), matrix ions (XH)+, (X-H)- , and sample neutrals (M). • 3. Sample molecules are ionized by proton transfer from matrix ions: • XH+ + M X + MH+. • X-H- + M X + M-H-

  11. MALDI: Matrix Assisted Laser Desorption Ionization hn • Ion Extraction: High voltage is applied to the sample plate, accelerating ions out of the Ion Source into the Flight Tube. MH+ +20 kV

  12. Time-of-Flight Mass Analyzer Ion Source Flight Tube 20-25 kV Detector + + Principle: If ions are accelerated with the same potential at a fixed point and a fixed initial time and are allowed to drift, the ions will separate according to their mass to charge ratios.

  13. Time-of-Flight Mass Analyzer + + Ion Source Flight Tube Detector + The ions enter the flight tube with the lighter ions travelling faster than the heavier ions to the detector

  14. Time-of-Flight Mass Analyzer + + Ion Source Flight Tube Detector + The lighter ions strike the detector before the heavier ions. This “time of flight” (TOF) can be converted to mass

  15. Analyte molecules in matrix Principle of MALDI-TOF-MS Vacuumlock Laser Vacuum system Ion detector Sampleplate Acceleration grids Drift tube Mass spectrum

  16. Laser High resolution TOF-MS with ion reflector MALDI ionsource Ion reflector Iondetector HiRes mass spectrum The reflector focuses ion of same mass but different velocity on detector; high resolution is obtained

  17. LID Laser TOF/TOF-MS/MS with CID MALDI ionsource Parent ionselector Ion reflector Iondetector MS/MS spectrum of daughter ionsis measured in a single acquisition;no pasting of segments;low sample consumption, high speed, high sensitivity Daughter ion mass spectrum

  18. Mass analyzers

  19. + Ions are detected with a Microchannel Plate -1000 V -100 V D= 6-25 u Primary Ion from Flight Tube L

  20. + Ions are detected with a Microchannel Plate -1000 V -100 V D= 6-25 u L

  21. + Ions are detected with a Microchannel Plate -1000 V -100 V D= 6-25 u Multification by secondary emission e- Secondary emissive materials: Beryllium oxide, magnesium oxide etc L

  22. + Ions are detected with a Microchannel Plate -1000 V -100 V D= 6-25 u e- e- e- e- L

  23. + Ions are detected with a Microchannel Plate -1000 V -100 V D= 6-25 u e- e- e- e- L

  24. e- e- e- e- e- e- e- e- + Ions are detected with a Microchannel Plate -1000 V -100 V D= 6-25 u e- e- e- e- ~103 Amplification L

  25. Why interested in MALDI-TOF MS • 분자량 측정 • 큰분자량 물질 분석 • 혼합물 분석 : 한 종류의 성분이 아닌 몇 종류가 혼재해 있어도 분석이 가능함 • 미량분석 : 매우 민감하여 미량의 시료도 분석 가능 함 : 펩타이드 경우 fmol 분석 가능 • 데이터 분석이 쉬움 : 분자 구조가 깨어 지지 않고, 보통 다 전하를(multiple charging)띠지 않으므로 데이터가 다른 질량 분석기에서 보다 단순하여 해석이 용이함 • 염의 영향이 적음 : 생체단백질 분리에 이용되는 완충용액, 염 등에 LC/MS 보다 영향을 덜 받음 • 분석이 신속함 : 시료와 Matrix 섞어 sample plate에 떨어뜨려 용액을 말리는 시간(약 5~10 분), MALDI-TOF 로 분석하는 시간 (1분 이내) • 기기 사용 및 유지하기 위한 비용이 저렴 : LC/MS, GC/MS 처럼 질소 또는 아르곤 가스를 사용하지 않고, 미량의 Matrix와 ACN, TFA등을 이용함으로 시약 비용도 저렴함

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