1. 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 -9 M) –Femtomolar range possible (1 x 10 -15 M) –Attomolar range claimed (1 x 10 -18 M) Diversity of applications –Proteins –Oligonucleotides –Oligosaccharides –Lipids –Others Proteomics –Identification of proteins expressed under specific conditions

2. - 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) ratio 에 따라 선택적으로 분 리 -Ion detector 이온 흐름을 그 양에 비례하게 전기적인 흐름으로 전환, 증 폭시켜 signal 을 생성 -Vacuum system

3. 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)

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

5. Ion Source: ESI

7. Ion Source: MALDI Matrix Assisted Laser Desorption Ionization(MALDI) Laser matrix + analyte Sample support a m m m m m m m m a a a a++ + + m a m +

8. Matrix

9. h Laser +20 kV Variable Ground Grid Grid AH + Sample plate 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 +.

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

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

13. 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

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

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

16. The Mass Analyzer: Quadrupole Quadrupole(Mass filter) -4 개의 molybdenum 막대로 이루어져 있으며, 한쌍 (1,2) 은 DC voltage, 다른 한쌍 (3,4) 은 Radio frequency voltage 가 가해진다. - 가해지는 전압의 진폭은 선택된 m/z 에 해당 되는 ion 만 ion source 에서 detector 까지 통 과하게 한다. - quadropole 의 전압을 바꾸면서 주어진 mass 범위의 이온을 scanning 한다.

17. The Mass Analyzer: Quadrupole

18. Detectors

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

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

22. + -1000 V-100 V e-e- L D= 6-25 u Ions are detected with a Microchannel Plate Multification by secondary emission Secondary emissive materials: Beryllium oxide, magnesium oxide etc

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

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

25. + -1000 V-100 V e-e- e-e- e-e- e-e- L D= 6-25 u e-e- e-e- e-e- e-e- e-e- e-e- e-e- e-e- ~10 3 Amplification Ions are detected with a Microchannel Plate

26. Tandem MS(MS/MS)

29. Laser Sample plate Analyte molecules in matrix Acceleration grids Drift tube Ion detector Mass spectrum Vacuum system Vacuum lock MALDI TOF MS

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

31. Daughter ion mass spectrum Ion detector Ion reflector MALDI ion source CID MS/MS spectrum of daughter ions is measured in a single acquisition; no pasting of segments; low sample consumption, high speed, high sensitivity LID Laser TOF/TOF-MS/MS with Parent ion selector

32. 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 데이터 가 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 plate 에 떨어뜨려 용액을 말리는 시간 ( 약 5~10 분 ), MALDI-TOF 로 분석하는 시간 (1 분 이내 )  기기 사용 및 유지하기 위한 비용이 저렴 : LC/MS, GC/MS 처럼 질소 또는 아르곤 가스를 사용하지 않고, 미 량의 Matrix 와 ACN, TFA 등을 이용함으로 시약 비용도 저렴함

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

36. Peptide Sequencing

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

40. Peptide Mass Fingerprinting Analytical technique for protein identification (protein sequence) Unknown protein of interest cleaved into peptide by protease Collection of peptides resulting from this cleavage comprise a unique identifier of the unknown protein Mass measured with MALDI-TOF and ESI-TOF in silico compared to the genome

41. Computer programs translate the known genome of the organism into proteins Theoretically cut the proteins into peptides with the same protease (ex.Trypsin: K or R) Calculate the absolute masses of the peptides from each protein the masses of the peptides of the unknown protein vs the theoretical peptide masses of each protein encoded in the genome Results statistically analyzed to find the best match

42. In Gel Digestion & Mass Spectrometry

43. Trypsin Digest Cut out 2D-Gel Spot ProteinPeptides

44. Peptide Mass Fingerprinting Trypsin NK K K K K K R R R R C N C K K K K K K R R R R Protein Tryptic peptide mixture. Masses measured by MS. Every peptide has a basic C- terminus. A protein can be identified in a database by matching masses of a subset of the tryptic peptides against calculated values.

45. MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT intact protein enzyme peptide fragments

46. Peptide Mass Fingerprinting 2D-Gel In Gel Digestion MS 848.1 1272.5 492.6 883.2 2978.9 812.6 1432.3 3127.1 996.8 702.4 164.9 2748.2 848.3 1272.7 493.2 882.6 2978.3 364.1 948.9 3128.8 Database 3514.2 2837.1 263.9 147.4 1429.7 199.6 142.3 640.8 Is identical to In Silico Digestion “ Spot removal ”

47. 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!

48. Protein sequence Analysis

49. For sequencing of an entire Protein…?? Divide and Conquer !!!

50. Deduction of Full Amino Acid Sequence of a Protein by Overlapping the Sequences Obtained from individual Peptides

51. Edman Degradation Sequentially Removes One Residue at a Time from the Amino End of a Peptide up to 50 times Each round can be complete within 1 hr and the Edman degradation can be repeated up to 50 cycles in Practice.

52. Lymphomas and Leukemias

53. Regulatory Mutations Chromosome 17 Messenger RNA Her2 geneHer2 gene amplification Overexpression Her2 protein Normal expression