1. PROTEIN IDENTIFICATION BY MASS SPECTROMETRY

2. OBJECTIVES To become familiar with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) To become familiar with contemporary protein identification approaches To identify unknown proteins from collected samples by peptide mass fingerprinting, using mass spectral data of tryptic peptides for database querying

3. What is mass spectrometry ? Most accurate technique available to determine the mass of an analyte (proteins, peptides, small molecules)

4. Simple schematic of a mass spectrometer Mass AnalyzerIon Source Detector What is mass spectrometry ?

6. Basics of mass spectrometry Determination of mass to charge ratio (m/z) – note in MALDI-TOF MS, the charge is 1 so we essentially determine mass (i.e. m/1) Components of MS - ionization and entrance to gas phase - ion (peptide) separation - mass analysis

7. Output from MS - mass-to-charge ratio (m/z) ratio of exact mass to net charge For example: 802 m/z ion (z=1; +1 charged) = 802 amu 500 m/z ion (z=2; +2 charged) = 1000 amu 327 m/z ion (z=3; +3 charged) = ???? amu

8. Terminology Isotopes Ionization Resolution

9. ISOTOPES Atoms of an element with differing numbers of neutrons in their nuclei + n + + + + + n n n n n n + n + + + + + n n n n n 12 C 13 C 99% abundance1% abundance

10. 10 carbons 10 x 12 C 9 x 12 C + 1 x 13 C 8 x 12 C + 2 x 13 C C1010/10/2003 10:44:00 AM C10 Simulation C10 Profile Resolution: Daltons 0.25 at 5% height Charges 1 Chrg dist 0 Ions 11 Min Ion Ab. 1e-020 Min Ions 5000 Max Ions. 20000 119.0119.5120.0120.5121.0121.5122.0122.5123.0123.5 m/z 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 Abundance 120.00 121.00 122.00 123.01

11. RESOLUTION How well two ions can be separated Good resolution essential to distinguishing isotopes Resolution usually decreases as the mass of the analyte increases

12. Res=2705 KISSME10/10/2003 11:25:02 AM C28 H50 N7 O10 S1 Simulation KISSME+H Profile Resolution: Daltons 0.25 at 5% height Charges 1 Chrg dist 0 Ions 24120 Min Ion Ab. 1e-020 Min Ions 5000 Max Ions. 20000 676677678679680681 m/z 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 Abundance 676.33 677.33 678.33 679.33 680.33 681.33

13. Res=901 KISSME10/10/2003 11:25:45 AM C28 H50 N7 O10 S1 Simulation KISSME+H Profile Resolution: Daltons 0.75 at 5% height Charges 1 Chrg dist 0 Ions 24120 Min Ion Ab. 1e-020 Min Ions 5000 Max Ions. 20000 676677678679680681 m/z 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 Abundance 676.33 677.33 678.34 679.33 680.33 681.34

14. Res=520 KISSME10/10/2003 11:26:20 AM C28 H50 N7 O10 S1 Simulation KISSME+H Profile Resolution: Daltons 1.3 at 5% height Charges 1 Chrg dist 0 Ions 24120 Min Ion Ab. 1e-020 Min Ions 5000 Max Ions. 20000 676677678679680681 m/z 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 Abundance 676.33 677.33 678.33 679.33 680.33 681.33

15. IONIZATION A + H + = (A+H) + A + Na + = (A+Na) + A - H = (A-H) - Giving an electric charge to a neutral species

16. Soft-ionization methods for proteomics MALDI –In MALDI-TOF MS, ionization occurs when the laser hits the sample Electrospray –In electrospray, ionization occurs in the liquid as it exits a spray needle

17. MALDI = Matrix-Assisted Laser Desorption / Ionization analyte is dried with matrix on target –matrix: CHCA, DHB, sinapinic acid –matrix absorbs laser energy and transfers to analyte generates predominantly singly-charged ions

18. MALDI = Matrix-Assisted Laser Desorption / Ionization A A A A A M M M M M M M M M M M M MM M M M M M M M M M M M M A A A A A A A A (ions and neutrals) laser A = analyte M = matrix

19. Mass Analyzers Time-Of-Flight (TOF) Quadrupole Ion Trap Ion Cyclotron Resonance

20. Mass to Charge Ratio (m/z) Measure m/z not mass –C7H7 mass is 91 Da –C7H7+1; m/z = 91 –C7H7+2; m/z = 45.5

21. Mass Analyzers: Reflectron Time-Of-Flight Ion Source Detector Electrostatic Mirror +++ + + +

22. MALDI-TOF mass spectrometers

23. MALDI-TOF MS matrix-assisted laser desorption / ionization- time of flight mass spectrometer Voyager DE-PRO Peptides put on to plate surface

24. Laser Sample plate Detector Mirror MALDI-TOF MS Design

25. Laser Sample plate Detector Mirror MALDI-TOF MS Design

26. 12 39 6 Laser fires, clock starts Laser fires…Clock Starts Laser Analyte Sample plate Detector Mirror

27. 12 39 6 Peptide ions are accelerated towards the mirror

28. 12 39 6 Peptide ions enter the mirror

29. 12 39 6 Peptide ions are reflected towards the detector

30. 12 39 6 Larger ions take longer to “fly” from the sample plate to the detector

31. 12 39 6 Peptide ions approach the detector

32. 12 39 6 Mass scale Intensity scale Smallest peptide ion reaches the detector

33. mass intensity 12 39 6

34. mass intensity 12 39 6 “Mass Spectrum” shows distribution and intensity of peptides in the sample

35. Mass (m/z) % intensity Mass Spectrum graph showing all peptides in the sample Small peptidesLarge peptides 699.0 1361.82024.62687.43350.24013.0 682.0 0 10 20 30 40 50 60 70 80 90 100 4700 Reflector Spec #1 MC[BP = 842.5, 682] 842.5100 1475.7573 927.4976 1179.6012 1235.5361 1479.7865 1045.5690 1791.7343 2211.1003 1277.7130 1567.7380 866.0283 1439.8096 1127.5532 1774.8944 797.3553 1066.50881249.6176 768.9401 1512.7854 1740.8289 1083.5294 849.5275 1305.7148 2284.1841 2705.1484 1769.7886 2510.1404 1994.9866 1393.7023 2330.1951 3312.2693 2561.1523

36. Time of Flight Standard Mass 4.5904 6.51296 7.91570 10.52093 12.32465 18.33657 Time of FlightMass? 5 6 9 10 15 Mass (daltons) TOF (nanoseconds) How Time-Of-Flight works

37. Time of Flight Standard Mass 4.5904 6.51296 7.91570 10.52093 12.32465 18.33657 Time of FlightMass? 5 6 9 10 15 How Time-Of-Flight works Mass (daltons) TOF (nanoseconds)

38. Time of Flight Standard Mass 4.5904 6.51296 7.91570 10.52093 12.32465 18.33657 Time of FlightMass? 5 6 9 10 15 How Time-Of-Flight works Mass (daltons) TOF (nanoseconds)

39. Mass (daltons) TOF (nanoseconds) Time of Flight Standard Mass 4.5904 6.51296 7.91570 10.52093 12.32465 18.33657 Time of FlightMass? 51000 6 9 10 15 How Time-Of-Flight works 5nsec = 1000 daltons

40. Time of Flight Standard Mass 4.5904 6.51296 7.91570 10.52093 12.32465 18.33657 Time of FlightMass? 51000 61250 91750 102000 153000 How Time-Of-Flight works Mass (daltons) TOF (nanoseconds) 5nsec = 1000 daltons

41. Peptide mass fingerprinting (PMF) digestMS Search HIT SCORE Protein X 1000 Protein Y 50 Protein Z 5 Protein X theoretical digestProtein Y theoretical digestProtein Z theoretical digest Spectrum processing 1554.25 2055.39 1942.44 1755.67 987.55 855.34 677.68 Report Protein (gel band or spot) peptides Mass spectrum Peak list

42. Computer program used to convert graph data (from mass spectrum) to table data (list of peptide masses) Generate mass list of all peptides 1007.467 1023.462 1036.481 1066.493 1082.488 1105.601 1153.524 1172.581 972.5954 973.4202 975.6104 977.4627 982.4603 983.4991 998.4552

43. 2004006008001000120014001600 Mass (Da) Intensity 681.7 Simple Mass Spectrum One protein, digested with trypsin, peptides extracted, analyzed by MALDI 771.9 992.1 1165.2 320.3 361.4 607.7

44. PLEASEMAKEMANYKRISPYKREMETREATSTHEYAREGREAT 320.3 361.4 607.7 681.7 771.9 992.1 1165.2 Database search using computer algorithm List of masses e.g. MASCOT Sequence matches EAT EGR ISPYK EMETR EMANYK PLEASEMAK EATSTHEYAR

45. QUESTIONS?