Mass Spectrometry

1. Mass Spectrometry 시스템생물학과 이선아

2. Mass Spectrometry • Widely used analytical technique • Within an accuracy of 0.01% of total weight of sample and within 5 ppm for small organic molecules • Unequaled sensitivity –Nanomolar range routinely (1 x 10-9M) –Femtomolar range possible (1 x 10-15M) –Attomolar range claimed (1 x 10-18M) • Diversity of applications –Proteins –Oligonucleotides –Oligosaccharides –Lipids –Others • Proteomics –Identification of proteins expressed under specific conditions

3. -3 fundamental parts: Ionization source, the analyzer, the detector -Ionization source 시료분자를 이온화시키고 더 작은 이온으로 쪼갠다. -Mass analyzer 이온들을 mass-to-charge(m/z) ratio에 따라 선택적으로 분리 -Ion detector 이온 흐름을 그 양에 비례하게 전기적인 흐름으로 전환, 증폭시켜 signal을 생성 -Vacuum system

4. Basic components to MS •Ion source –Electrospray(ESI) –Atmospheric Pressure Ionization (APCI) –Chemical Ionization (CI) –Electronic Ionization (EI) –Matrix Assisted Laser DesorptionIonization (MALDI) •Mass Selection –Quadrupole –Time of Flight (TOF) –Magnetic Sector –Ion Trap –Ion Cyclotron •Detector –Phosphor / Photo Diode –Multi-channel Plate (MCP)

5. Ion Source: ESI Electrospray ionization(ESI) -용액 상태의 시료를 이온화(LC-MS) -기존의 방법으로는 얻기 힘들었던 intact 상태의 peptide나 단백질을 이온화 -한 개 이상의 전하를 띤 이온을 생성

6. Ion Source: ESI

7. Ion Source: ESI

8. Ion Source: MALDI Matrix Assisted Laser Desorption Ionization(MALDI)

9. AH+ 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

10. Why MALDI? -Less sensitive to salts -Lower PRACTICAL detection limits -Easier to interpret spectra(less multiple charges) -Quick and easy -Higher mass detection -Higher Throughput(1000>samples per hour)

11. 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

12. The Mass Analyzer: TOF Time Of Flight(TOF) Ion Source Flight Tube 20-25 kV + + 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. + + The Mass Analyzer: TOF Ion Source Flight Tube Detector +

14. + + The Mass Analyzer: TOF Ion Source Flight Tube Detector +

15. The Mass Analyzer: Quadrupole Quadrupole(Mass filter) -4개의 molybdenum 막대로이루어져 있으며, 한쌍(1,2)은 DC voltage, 다른 한쌍(3,4)은 Radio frequency voltage가 가해진다. -가해지는전압의 진폭은 선택된 m/z에 해당되는 ion만 ion source에서 detector까지 통과하게 한다. - quadropole의 전압을 바꾸면서 주어진 mass범위의 이온을 scanning 한다.

16. The Mass Analyzer: Quadrupole

17. Detectors

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

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

20. + 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

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

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

23. 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

24. Tandem MS(MS/MS)

25. Tandem MS(MS/MS)

26. Tandem MS(MS/MS)

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

28. 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

29. 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

30. 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

31. Mass Spectrometry 분석 -base peak -parent peak -radical cations -Isotopes

34. Biomolecule Analysis *과거에는? -Electrophoresis, chromatography, ultracentrifugation -Not very precise *MS이용하면? -Proteins, oligonucleotides, oligosaccharides, lipids -Detect modifications and sequences

35. Post Translational Modifications(PTM’s) • PTM’s are very important in signaling as well as metabolic pathways (e.g. phosphorylation) • Often we want to know not only which modification a protein has undergone, but exactly where in the sequence the modification lies. • Many of the search engines allow for “variable” modifications, but very few at one time (combinatorialy explosive) • There is great opportunity here for robust searches that find PTM’s reliably!

36. Protein sequence Analysis

37. Peptide Sequencing