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PROTEIN IDENTIFICATION BY MASS SPECTROMETRY

PROTEIN IDENTIFICATION BY MASS SPECTROMETRY. 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

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PROTEIN IDENTIFICATION BY MASS SPECTROMETRY

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  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. What is mass spectrometry ? Ion Source Mass Analyzer Detector Simple schematic of a mass spectrometer

  5. 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

  6. 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

  7. Terminology • Isotopes • Ionization • Resolution

  8. + + + + + + + + + + + + n n n n n n n n n n n n n ISOTOPES • Atoms of an element with differing numbers of neutrons in their nuclei 13C 12C 99% abundance 1% abundance

  9. 10 carbons C10 10/10/2003 10:44:00 AM C10 Simulation 120.00 10 x 12C C10 Profile 0.85 Resolution: 0.80 Daltons 0.25 at 5% height 0.75 Charges 1 Chrg dist 0 0.70 Ions 11 0.65 Min Ion Ab. 1e-020 Min Ions 5000 0.60 Max Ions. 20000 0.55 0.50 Abundance 0.45 9 x 12C + 1 x 13C 0.40 0.35 0.30 8 x 12C + 2 x 13C 0.25 0.20 0.15 121.00 0.10 0.05 122.00 123.01 0.00 119.0 119.5 120.0 120.5 121.0 121.5 122.0 122.5 123.0 123.5 m/z

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

  11. KISSME 10/10/2003 11:25:02 AM C28 H50 N7 O10 S1 Simulation 676.33 KISSME +H 0.65 Profile Res=2705 Resolution: 0.60 Daltons 0.25 at 5% height 0.55 Charges 1 Chrg dist 0 0.50 Ions 24120 Min Ion Ab. 1e-020 0.45 Min Ions 5000 Max Ions. 20000 0.40 0.35 Abundance 0.30 0.25 677.33 0.20 0.15 0.10 678.33 0.05 679.33 680.33 681.33 0.00 676 677 678 679 680 681 m/z

  12. KISSME 10/10/2003 11:25:45 AM C28 H50 N7 O10 S1 Simulation 676.33 KISSME +H 0.65 Profile Res=901 Resolution: 0.60 Daltons 0.75 at 5% height 0.55 Charges 1 Chrg dist 0 0.50 Ions 24120 Min Ion Ab. 1e-020 0.45 Min Ions 5000 Max Ions. 20000 0.40 0.35 Abundance 0.30 0.25 677.33 0.20 0.15 0.10 678.34 0.05 679.33 680.33 681.34 0.00 676 677 678 679 680 681 m/z

  13. KISSME 10/10/2003 11:26:20 AM C28 H50 N7 O10 S1 Simulation 676.33 KISSME +H 0.65 Profile Res=520 Resolution: 0.60 Daltons 1.3 at 5% height 0.55 Charges 1 Chrg dist 0 0.50 Ions 24120 Min Ion Ab. 1e-020 0.45 Min Ions 5000 Max Ions. 20000 0.40 0.35 Abundance 0.30 0.25 677.33 0.20 0.15 0.10 678.33 0.05 679.33 680.33 681.33 0.00 676 677 678 679 680 681 m/z

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

  15. Soft-ionization methodsfor 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

  16. MALDI = Matrix-AssistedLaser 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

  17. MALDI = Matrix-AssistedLaser Desorption / Ionization laser M A M A M M A M M M M A M M A M A M M M M A A A M M M A A M M M M M M A M A M (ions and neutrals) A = analyte M = matrix

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

  19. 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

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

  21. MALDI-TOF mass spectrometers

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

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

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

  25. Laser fires, clock starts Mirror 12 9 3 6 Laser fires…Clock Starts Detector Laser Analyte Sample plate

  26. 12 Peptide ions are accelerated towards the mirror 9 3 6

  27. 12 9 3 6 Peptide ions enter the mirror

  28. 12 9 3 6 Peptide ions are reflected towards the detector

  29. 12 9 3 6 Larger ions take longer to “fly” from the sample plate to the detector

  30. 12 9 3 6 Peptide ions approach the detector

  31. 12 9 3 6 Smallest peptide ion reaches the detector Intensity scale Mass scale

  32. 12 9 3 6 intensity mass

  33. 12 “Mass Spectrum” shows distribution and intensity of peptides in the sample 9 3 6 intensity mass

  34. Mass Spectrumgraph showing all peptides in the sample 4700 Reflector Spec #1 MC[BP = 842.5, 682] 842.5100 100 682.0 90 80 1475.7573 927.4976 70 1179.6012 1235.5361 60 % intensity 1479.7865 50 1045.5690 1791.7343 40 2211.1003 1277.7130 1567.7380 866.0283 30 1439.8096 1127.5532 1774.8944 797.3553 1066.5088 1249.6176 768.9401 20 1512.7854 1740.8289 1083.5294 849.5275 1305.7148 2284.1841 2705.1484 1769.7886 2510.1404 1994.9866 10 1393.7023 2330.1951 3312.2693 2561.1523 0 2687.4 3350.2 4013.0 2024.6 1361.8 699.0 Mass (m/z) Small peptides Large peptides

  35. Mass (daltons) TOF (nanoseconds) How Time-Of-Flight works

  36. How Time-Of-Flight works Mass (daltons) TOF (nanoseconds)

  37. How Time-Of-Flight works Mass (daltons) TOF (nanoseconds)

  38. How Time-Of-Flight works 5nsec = 1000 daltons Mass (daltons) TOF (nanoseconds)

  39. How Time-Of-Flight works 5nsec = 1000 daltons Mass (daltons) TOF (nanoseconds)

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

  41. Generate mass list of all peptides • Computer program used to convert graph data (from mass spectrum) to table data (list of peptide masses)

  42. Simple Mass Spectrum 771.9 1165.2 361.4 Intensity 607.7 992.1 320.3 681.7 200 400 600 800 1000 1200 1400 1600 Mass (Da) One protein, digested with trypsin, peptides extracted, analyzed by MALDI

  43. Database search using computer algorithm PLEASEMAKEMANYKRISPYKREMETREATSTHEYAREGREAT e.g. MASCOT Sequence matches List of masses

  44. QUESTIONS?

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