1. Yonsei Proteome Research Center Peptide Mass Finger-Printing Part II. MALDI-TOF 2013 생화학 실험 (1) 6 주차 자료 임종선 조교 limjs@proteomix.org 내선 6625

2. Yonsei Proteome Research Center Concept of Mass Spectrometry Mass Spectrometer? Instrument measuring molecular weight (MW) of sample Only picomolar concentrations is required Accuracy of 0.01% of total weight of sample Able to detect amino acid substitution/post-translational modifications

3. Yonsei Proteome Research Center History of Mass spectrometry 1953 : Quadrupole and the ion trap(W. Paul at H.S. Steinwedel). Nobel Prize to Paul in 1989. 1956 : First GC-MS 1968 : First commercial quadrupole 1975 : First commercial GC-MS 1990s : Explosive growth in biological MS, due to ESI & MALDI 2002 : Nobel Prize to Fenn & Tanaka for ESI & MALDI 2005 : Commercialization of Orbitrap MS Concept of Mass Spectrometry

4. Yonsei Proteome Research Center How does Mass Spectrometer work? Concept of Mass Spectrometry Inlet Ion Source Mass Analyzer Mass Analyzer Detector

5. Yonsei Proteome Research Center 1. Inlet 2. Ion Source 3. Mass Analyze r 4. Detecto r 5. Data System High Vacuum System MALDI ESI FAB LSIMS EI CI HPLC Flow injection Sample plate Time of flight (TOF) Quadrupole Ion Trap Magnetic Sector FTMS Microchannel plate Photomultiplier Electron multiplier Types of Machines / Techniques Concept of Mass Spectrometry

6. Yonsei Proteome Research Center “Sample Preparation” Concept of MALDI-TOF Peptide Sample Preparation Excise Wash Extract Dry gel Reconstruction Inlet Digest

7. Yonsei Proteome Research Center “1. Inlet: MALDI-TOF matrix” Concept of MALDI-TOF

8. Yonsei Proteome Research Center “2. Ion Source: MALDI” (Matrix Assisted Laser Desorption Ionization) Concept of MALDI-TOF h Laser AH + +20 kV Sample plate Ionization is triggered by a laser beam A matrix is used to protect the biomolecule from being destroyed by direct laser beam and to facilitate vaporization and ionization

9. Yonsei Proteome Research Center “2. Ion Source: MALDI” (Matrix Assisted Laser Desorption Ionization) Concept of MALDI-TOF

10. Yonsei Proteome Research Center “2. Ion Source: MALDI” (Matrix Assisted Laser Desorption Ionization) Question Which ion will strike the detector faster? The ions enter the flight tube with the lighter ions traveling faster than the heavier ions. SO, GREEN ION WILL STRIKE THE DETECTOR FASTER! Laser

11. Yonsei Proteome Research Center “3. Mass Analyzer: TOF” (Time Of Flight) Concept of MALDI-TOF amp comp L+L+ M+M+ H+H+ +20 kV +22 kV 0 kV 1) Ions enter source region, accelerated toward reflectron. 2) Ions separate in space based on their relative mass-to-charge (m/z). 3) Ions reverse path in reflectron. 4) Ions impact detector. Flight time Signal [Molecular weight] Reflectron TOF

12. Yonsei Proteome Research Center “3. Mass Analyzer: TOF” (Time Of Flight) Concept of MALDI-TOF Linear TOF Linear TOF is used in larger molecules. *we are going to use reflectron TOF.

13. Yonsei Proteome Research Center Concept of MALDI-TOF Mass accuracy : How accurate is the mass measurement? Resolution : How well separated are the peaks from each other? Sensitivity : How small an amount can be detected / analyzed? “4. Detection”

14. Yonsei Proteome Research Center Mass measurement accuracy depends on resolution Mass measurement accuracy depends on Resolution 0 2000 4000 6000 8000 Counts 2840 2845 2850 2855 Mass (m/z) Resolution = 14200 Resolution = 4500 Resolution =18100 15 ppm error 24 ppm error 55 ppm error High resolution means better mass accuracy “4. Detection” Concept of MALDI-TOF

15. Yonsei Proteome Research Center Relative Abundance Mass (m/z) 0 10000 20000 30000 40000 50000 100000 150000 200000 (M+2H) 2+ (M+3H) 3+ Theoretical MALDI TOF SPECTRUM Of ONE PEPTIDE MH+ “4. Detection” Concept of MALDI-TOF 150000 149876

16. Yonsei Proteome Research Center “4. Detection” Concept of MALDI-TOF 150000 149876 No 13 C atoms (all 12 C) One 13 C atom Two 13 C atoms “Monoisotopic mass” Three 13 C atoms We calculate resolution and accuracy with these peaks. Annotations on spectra will be for the monoisotopic peaks only. m/z

17. Yonsei Proteome Research Center “4. Detection” Concept of MALDI-TOF How to calculate mass? m/z of monoisotopic peak = 431.73 m/z = 431.73 m/4 = 431.73 m=431.73 x 4=1726.92 You must subtract mass of H+ (1) [ 1726.92] – 4 = 1722.92 Assume these are peaks found at [M+4H] 4+

18. Yonsei Proteome Research Center Relative Intensity(%) 02500500010000 2001 2501 3334 5001 10001 “4. Detection” Question Theoretical Results Assume this is a result of MALDI-TOF for ONE SINGLE PEPTIDE. Please calculate the mass of this peptide. (M+2H) 2+ MH + (M+3H) 3+ (M+4H) 4+ (M+5H) 5+ Question

19. Yonsei Proteome Research Center “4. Detection” Solution H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ [M + 5H] 5+ [M + 4H] 4+ [M + 3H] 3+ [M + 2H] 2+ [MH] + M/Z = 10,003 / 3 M/Z = 3334 M/Z = 10,005 / 5 M/Z = 2001 M/Z = 10,004 / 4 M/Z = 2501 M/Z = 10,002 / 2 M/Z = 5001 M/Z = 10,001 / 1 M/Z = 10,001 Relative Intensity(%) 02500500010000 (M+2H) 2+ MH + The same protein with a molecular weight of 10,000 contains 5, 4, 3, 2, and 1 charges (M+3H) 3+ (M+4H) 4+ (M+5H) 5+ 2001 2501 3334 5001 10001

20. Yonsei Proteome Research Center 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. “5. Database” Concepts of MALDI-TOF

21. Yonsei Proteome Research Center MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT intact protein enzyme peptide fragments “5. Database” Concepts of MALDI-TOF

22. Yonsei Proteome Research Center Gel In Gel Digestion 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 “5. Database” Concepts of MALDI-TOF

23. Yonsei Proteome Research Center “5. Database” Concepts of MALDI-TOF

24. Yonsei Proteome Research Center 실험 방법 준비물 : Desalting : Poros buffer(C18 resin in 70% ACN), Zip tip, 100% ACN, 2% Formic Acid, MATRIX buffer(CHCA 8~10mg in 70% ACN) 순서 : 1. zip tip 의 끝 0.3~0.5cm 정도를 구부린다. 2. Poros buffer 를 3~4ul 넣어 실린지로 밀어준다. Zip tip 끝에 3~5mm 정도 충진되도록 3. 100% CAN 으로 (10ul) C18 resin 을 wash 해준다. 4. 2% F.A 로 (20ul) C18 resin 을 activation 시킨다. 5. ingel-digested sample 을 흘려준다. 6. 2% F.A 로 (10ul) peptide 외의 chemical 을 wash 해준다. 7. Matrix buffer 로 Matrix 와 함께 펩타이드를 회수한다. Plate 위에 buffer 를 loading (1ul)

25. Yonsei Proteome Research Center 실험 방법 MALDI-TOF analysis 1. Plate 를 MALDI 기기 안으로 injection 한다. 2. 기기의 laser 를 켜고 약 10-30 분간 laser 를 안정화 시킨다. 3. plating 한 Standard sample 을 큰 오차범위에서 작은 오차범위로 서서히 좁혀 가며 찍어본다. - Calibration : 기기의 오차범위를 줄여줌 Resolution / peak intensity 를 확인하 며, laser 의 강도 / mirror 의 위치를 조정하여 분석에 최적화시킨다. 4. Strandard sample 을 분석한 후 분석하고자 하는 원래의 시료를 분석한다. 5. 분석 후 결과 spectrum 을 추출 프로그램 (Data explorer) 을 사용하여, spectrum list 를 작성한다. 6. Data search engine (profound ; Mascot; MS-fit) 에 입력한다. 이 떄 시료에 사용한 효소, 시약으로 인한 modify 를 지정하고, 시료의 종 (taxonomy) 등을 설 정하여 준다. 7. 분석된 결과를 토대로 유의성 여부를 검토한다.