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Mass Spectrometry Overview and Mass Spectrometry of Proteins and Glycoproteins

Mass Spectrometry Overview and Mass Spectrometry of Proteins and Glycoproteins

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Mass Spectrometry Overview and Mass Spectrometry of Proteins and Glycoproteins

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  1. Mass Spectrometry Overview and Mass Spectrometry of Proteins and Glycoproteins David Graham, Ph.D. Assistant Professor, Department of Molecular and Comparative Pathobiology School of Medicine Director for The Center for Resources in Integrative Biology

  2. Goals • Better Understanding of Mass Spectrometry • Basic introduction • Components of MS • Basic Principles • Types of instruments • MS as applied to proteins, peptides and glycopeptides • ECD/ETD • Analyzing MS data • Software tools • Workflows • Extracting Biological Meaning

  3. Sources: • Agard lab • • Cobb lab •‎ • ME 330.804: Mass Spectrometry in an “Omics” World • Johns Hopkins – multiple faculty contributers • MAMSLAB: Slides from the late Robert Cotter • Books: • Mass Spectrometry of Glycoproteins : Methods and Protocols • Editor(s): Jennifer J. Kohler1, Steven M. Patrie2 • Mass Spectrometry of Proteins and Peptides : Mass Spectrometry of Proteins and Peptides • Editor(s): John R. Chapman1 • Both available through welch medical library online

  4. More.. • The Expanding Role of Mass Spectrometry in Biotechnology,GarySiuzdak (2nd edition 2006) ISBN 0-9742451-0-0 • Mass Spectrometry Desk Reference, O. David Sparkman (2000, 1st edition) ISBN 0-9660813-2-3 • MassSpectrometry of BiologicalMaterials, Barbara S. Larsen& Charles N. McEwen (2nd. Edition 1998) ISBN 978-0824701574 • ProteinsandProteomics: A LaboratoryManual, editedby Richard Simpson (2003) ISBN 0-87969-554-4 • MassSpectrometryin Biophysics: Conformationand Dynamics of Biomolecules, Igor A. KaltashovandStephen J. Eyles (2005) ISBN 0-471-45602-0 • Time-of-Flight MassSpectrometry: Instrumentation andApplications in BiologicalResearch, Robert J. Cotter (1997) ISBN 0-8412-3474-4 • Disclaimer– besteffort has beenmadetoreferenceoriginalsources. Pleasecontactdgraham@jhmi.eduforcorrection of anyerrorsorommisions.

  5. Mass spectrometry is applied physics • Magnetism • Newtons laws of motion • Basic tennants are dealing with charged molecules • Two laws: • Lorenz force law: • If a particle of charge q moves with velocity v in the presence of an electric field E and a magnetic field B, then it will experience a force (F) • Newtons second law (non-relatavistic motion): • F=ma • The terms F can be related and the equation derived: • (m/q)a= E + v x B

  6. Basic equations governing mass spectrometry Ion’s kinetic E function of accelerating voltage (V) and charge (z). Centrifugal force Applied magnetic field balance as ion goes through flight tube Combine equations to obtain: Fundamental equation of mass spectrometry Change ‘mass-to-charge’ (m/z) ratio by changing V or changing B. NOTE: if B, V, z constant, then: Cobb lab

  7. What is the take home point? • We can control our voltages • We know our distances • We know our field strengths • Thus: • A simple set of equations can be used to calculate the m/z for all different types of mass spectrometers

  8. Ion source:makes ions Basic components of a mass spectrometer Sample Mass analyzer: separates ions Detector:presents information Modified from Agard lab

  9. Mass Spectrometer Block Diagram High Vacuum System Ion source Mass Analyzer Data System Inlet Detector Modified from Agard lab

  10. Mass Spectrometer Block Diagram Turbo pumps High Vacuum System Ion source Mass Analyzer Data System Inlet Detector Modified from Agard lab

  11. Sample Introduction High Vacuum System Ion Source Mass Analyzer Data System Inlet Detector HPLC Flow injection Sample plate Modified from Agard lab

  12. Ion Source High Vacuum System Ion Source Mass Analyzer Data System Inlet Detector MALDI ESI FAB SIMS EI CI Modified from Agard lab

  13. Ion Sources make ions from sample molecules(Ionization is required to move and detect molecules.) Partialvacuum Sample Inlet Nozzle (Lower Voltage) Pressure = 1 atmInner tube diam. = 100 um MH+ N2 + + + + + + + + + + + + MH2+ + + + + + + + + + + + + Sample in solution + + + + + + + + + + + + + + + + + N2 gas + + + + + MH3+ High voltage applied to metal sheath (~4 kV) Charged droplets Electrospray ionization: Introduced by John Fenn (Nobel Prize 2002): Yamashita, M.; Fenn, J.B., J. Phys. Chem. 88 (1984) 4451. Whitehouse, C.M.; Dreyer, R.N.; Yamashita, M.; Fenn, J.B., Anal. Chem. 57 (1985) 675. Fenn, J.B.; Mann, M.; Meng, C.K.; Wong, S.F.; Whitehouse, C.M., Science 246 (1989) 64. Sources: Agard lab and MAMSLAB

  14. Favors ejection of multiply charged Ions Based on an ion evaporation model: Iribarne, J.V.; Thomson, B.A., J. Chem. Phys. 64 (1976) 2287. Thomson, B.A.; Iribarne, J.V., J. Chem. Phys. 71 (1979) 4451. Sources: Agard lab and MAMSLAB

  15. Low Voltage (0.5 kv) High Voltage (5 kv) Low Voltage (0.1 kv) MS LC Column Flow Drying Gas Nebulizing Gas Assisted Electrospray

  16. MALDI: Matrix Assisted Laser Desorption Ionization Sample plate Laser • Sample is mixed with matrix (X) and dried on plate. • Matrix absorbs UV or IR energy from laser • Matrix ionizes and dissociates; undergoes a phase change to supercompressed gas • Some analytes are ionized by proton transfer: XH+ + M  MH+ + X. • Matrix expands supersonically and ions are entrained in the plume • Koichi Tanaka (Nobel Prize 2002) hn MH+ Grid (0 V) +/- 20 kV Modified from Agard lab and Cotter lab (MAMSLAB)

  17. Common MALDI Matrices Source: MAMSLAB

  18. Mass Analyzer High Vacuum System Ion source Mass Analyzer Data System Inlet Detector Time of flight (TOF) Quadrupole Ion Trap Orbitrap Magnetic Sector FTMS Modified from Agard lab

  19. Mass analyzers • Mass analyzers separate ions based on their mass-to-charge ratio (m/z) • Operate under high vacuum (keeps ions from bumping into gas molecules) • Actually measure mass-to-charge ratio of ions (m/z) • Key specifications are resolution, mass measurement accuracy, and sensitivity. • Several kinds exist: for bioanalysis, quadrupole,time-of-flight and ion traps are most used. Modified from Agard lab

  20. Quadrupole Mass Analyzer Uses a combination of RF and DC voltages to operate as a mass filter. • Has four parallel metal rods. • Lets one mass pass through at a time. • Can scan through all masses or sit at one fixed mass. Modified from Agard lab

  21. m1 m2 m4 m3 m2 m1 m4 m3 mass scanning mode m1 m2 m2 m2 m2 m2 m4 m3 single mass transmission mode Quadrupoles have variable ion transmission modes Modified from Agard lab

  22. Time-of-flight (TOF) Mass Analyzer Source Drift region (flight tube) + + detector + + V • Ions are formed in pulses. • The drift region is field free. • Measures the time for ions to reach the detector. • Small ions reach the detector before large ones. Modified from Agard lab

  23. Time of Flight Equation ME 330.804

  24. Ion Trap Mass Analyzer (Developed in the 20’s) Top View Cut away side view ^ Kingdon KH (1923). "A Method for the Neutralization of Electron Space Charge by Positive Ionization at Very Low Gas Pressures”. Physical Review 21 (4): 408. Bibcode:1923PhRv...21..408K. doi:10.1103/PhysRev.21.408.

  25. Quadropole ion trap mass spectrometers (ITMS)

  26. Ion Trap Design modified by Alexander Makarov • Uses a combination of electrostatic attraction (charge) and centripetal forces • Image current is detected as ions orbit central electrode (detected on outer electrode) • Data is processed in a similar manner to FTICR data (Fourrier Transformed) Centrifugal force Makarov A. (2000). "Electrostatic axially harmonic orbital trapping: A high-performance technique of mass analysis". Analytical Chemistry : AC 72 (6): 1156–62. doi:10.1021/ac991131p.

  27. Detectors High Vacuum System Ion source Mass Analyzer Data System Inlet Detector Microchannel Plate Electron Multiplier Hybrid with photomultiplier Modified from Agard lab

  28. Microchannel plate detector primary ion - 1000V + - e - e L - e - e - 100V D L >> D Modified from Agard lab

  29. Data System High Vacuum System Ion source Mass Analyzer Data System Inlet Detector Controller software (VENDOR specific) Modified from Agard lab

  30. Summary: acquiring a mass spectrum Ionization Mass Sorting (filtering) Detection Ion Source Ion Detector Mass Analyzer • Form ions • (charged molecules) Sort Ions by Mass (m/z) • Detect ions 100 75 Inlet • Solid • Liquid • Vapor 50 25 0 1330 1340 1350 Mass Spectrum Modified from Agard lab

  31. 40000 30000 20000 10000 0 The mass spectrum shows the results MALDI TOF spectrum of IgG MH+ Relative Abundance (M+2H)2+ (M+3H)3+ 50000 100000 150000 200000 Mass (m/z) Modified from Agard lab

  32. ESI Spectrum of Trypsinogen (MW 23983) M + 15 H+ 1599.8 M + 16 H+ M + 14 H+ 1499.9 1714.1 M + 13 H+ 1845.9 1411.9 1999.6 2181.6 m/z Mass-to-charge ratio Modified from Agard lab

  33. Atomic Mass Units • Despite being called a Dalton after John Dalton in 1803 who suggested 1H, the discovery of naturally occurring isotopes in 1912 eventually lead to one AMU or Dalton (Da) as being based upon using carbon 12, 12C, as a reference • One Dalton is defined as 1/12 the mass of a single carbon-12 atom • Thus, one 12C atom has a mass of 12.0000 Da.

  34. Stable isotopes of peptide elements ME 330.804

  35. 1981.84 1982.84 1983.84 Isotopes • We use isotopes to resolve the charge state of peaks since most element has more than one stable isotope “Monoisotopic mass” No 13C atoms (all 12C) One 13C atom Mass difference of 1 Da indicates a singly charged Peptide z=2 delta=0.5 z=3 delta=0.333 z=4 delta=0.25 Etc. Two 13C atoms Mass spectrum of peptide with 94 C-atoms (19 amino acid residues) Modified from Agard lab

  36. 4361.45 4360.45 m/z Isotope pattern for a larger peptide (207 C-atoms) Modified from Agard lab

  37. Mass spectrum of insulin 2 x 13C 13C 12C: 5730.61 Insulin has 257 C-atoms. Above this mass, the monoisotopic peak is too small to be very useful, and the average mass is usually used. Modified from Agard lab

  38. Monoisotopic mass When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement. Modified from Agard lab

  39. Average mass Average mass corresponds to the centroid of the unresolved peak cluster When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the isotope peaks in the cluster, which is the same as the average or chemical mass. Modified from Agard lab

  40. What if the resolution is not so good? At lower resolution, the mass measured is the average mass. Better resolution Poorer resolution 6130 6140 6150 6160 6170 Mass Modified from Agard lab

  41. Resolution =18100 8000 6000 Resolution = 14200 Counts 4000 Resolution = 4500 2000 0 2840 2845 2850 2855 Mass (m/z) Mass accuracy depends on resolution 15 ppm error 24 ppm error 55 ppm error Modified from Agard lab

  42. How is resolution calculated? • Resolution is the ratio of the mass divided by full width at half maximum. Also known as resolving power • R = m/Δmwhere • Δm= peak width (FWHM definition) • Δm= mass difference between two • peaks (valley definition) • What mass resolution is required to separate m/z 88 and 89? m/Δm = 88/1 = 88 Modified from Agard lab / ME 330.804

  43. Resolution and Accuracy of Mass Analyzers ME 330.804

  44. With high resolution mass spectrometry it is possibleto do “Top Down” Proteomics ME 330.804

  45. Usually in combination with ECD or ETD ME 330.804

  46. Typically we perform “bottom up” proteomics approaches • Proteins are either chemically cleaved or digested with endopeptidases (most commonly trypsin) ME 330.804

  47. yn-i xn-i ai bi low energy high energy Since resulting peptides follow a repeating pattern.. zn-i vn-i wn-i -HN--CH--CO--NH--CH--CO--NH- Ri CH-R’ R” ci di+1