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O O HO AcNH Mass Spectrometry Overview and Mass Spectrometry of Proteins and Glycoproteins David Graham, Ph.D. Assistant Professor, Department of Molecular.

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Presentation on theme: "O O HO AcNH Mass Spectrometry Overview and Mass Spectrometry of Proteins and Glycoproteins David Graham, Ph.D. Assistant Professor, Department of Molecular."— Presentation transcript:

1 O O HO AcNH 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 O O HO AcNH 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 O O HO AcNH Sources: Agard lab – Cobb lab – ec.ppt ‎ 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. Kohler 1, Steven M. Patrie 2 Mass Spectrometry of Proteins and Peptides : Mass Spectrometry of Proteins and Peptides Editor(s): John R. Chapman 1 – Both available through welch medical library online

4 O O HO AcNH More.. The Expanding Role of Mass Spectrometry in Biotechnology,Gary Siuzdak (2nd edition 2006) ISBN 0-9742451-0-0 Mass Spectrometry Desk Reference, O. David Sparkman (2000, 1st edition) ISBN 0-9660813-2-3 Mass Spectrometry of Biological Materials, Barbara S. Larsen & Charles N. McEwen (2nd. Edition 1998) ISBN 978-0824701574 Proteins and Proteomics: A Laboratory Manual, edited by Richard Simpson (2003) ISBN 0-87969-554-4 Mass Spectrometry in Biophysics: Conformation and Dynamics of Biomolecules, Igor A. Kaltashov and Stephen J. Eyles (2005) ISBN 0-471-45602-0 Time-of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research, Robert J. Cotter (1997) ISBN 0- 8412-3474-4 Disclaimer – best effort has been made to reference original sources. Please contact for correction of any errors or

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

7 O O HO AcNH 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 O O HO AcNH Ion source: makes ions Mass analyzer: separates ions Detector: presents information Sample Basic components of a mass spectrometer Modified from Agard lab

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

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

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

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

13 O O HO AcNH High voltage applied to metal sheath (~4 kV) Sample Inlet Nozzle (Lower Voltage) Charged droplets + + + + + + + + ++ + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + MH + MH 3 + MH 2 + Pressure = 1 atm Inner tube diam. = 100 um Sample in solution N2N2 N 2 gas Partial vacuum Electrospray ionization: Ion Sources make ions from sample molecules (Ionization is required to move and detect molecules.) Sources: Agard lab and MAMSLAB 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.

14 O O HO AcNH 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 O O HO AcNH Assisted Electrospray Nebulizing Gas LC Column Flow High Voltage (5 kv)Low Voltage (0.5 kv) MS Drying Gas Low Voltage (0.1 kv)

16 O O HO AcNH h Laser 1.Sample is mixed with matrix (X) and dried on plate. 2.Matrix absorbs UV or IR energy from laser 3.Matrix ionizes and dissociates; undergoes a phase change to supercompressed gas 4.Some analytes are ionized by proton transfer: XH + + M  MH + + X. 5.Matrix expands supersonically and ions are entrained in the plume Koichi Tanaka (Nobel Prize 2002) MH + MALDI: Matrix Assisted Laser Desorption Ionization +/- 20 kV Grid (0 V) Sample plate Modified from Agard lab and Cotter lab (MAMSLAB)

17 O O HO AcNH Common MALDI Matrices Source: MAMSLAB

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

19 O O HO AcNH ¤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. Mass analyzers Modified from Agard lab

20 O O HO AcNH 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 O O HO AcNH mass scanning mode m1 m3 m4 m2 m3 m1 m4 m2 single mass transmission mode m2 m3 m1 m4 m2 Quadrupoles have variable ion transmission modes Modified from Agard lab

22 O O HO AcNH 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 O O HO AcNH Time of Flight Equation ME 330.804

24 O O HO AcNH 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 O O HO AcNH

26 O O HO AcNH Quadropole ion trap mass spectrometers (ITMS)

27 O O HO AcNH

28 O O HO AcNH

29 O O HO AcNH 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) 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. Centrifugal force

30 O O HO AcNH Inlet Ion source Mass Analyzer Detector Data System High Vacuum System Microchannel Plate Electron Multiplier Hybrid with photomultiplier Detectors Modified from Agard lab

31 O O HO AcNH + e - primary ion e - e - e - L D -1000V -100V L >> D Microchannel plate detector Modified from Agard lab

32 O O HO AcNH Inlet Ion source Mass Analyzer Detector Data System High Vacuum System Controller software (VENDOR specific) Data System Modified from Agard lab

33 O O HO AcNH Inlet Ionization Mass Analyzer Mass Sorting (filtering) Ion Detector Detection Ion Source Solid Liquid Vapor Detect ions Form ions (charged molecules) Sort Ions by Mass (m/z) 133013401350 100 75 50 25 0 Mass Spectrum Summary: acquiring a mass spectrum Modified from Agard lab

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

35 O O HO AcNH ESI Spectrum of Trypsinogen (MW 23983) 1599.8 1499.9 1714.1 1845.9 1411.9 1999.6 2181.6 M + 15 H + M + 13 H + M + 14 H + M + 16 H + m/zMass-to-charge ratio Modified from Agard lab

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

37 O O HO AcNH Stable isotopes of peptide elements ME 330.804

38 O O HO AcNH Isotopes We use isotopes to resolve the charge state of peaks since most element has more than one stable isotope 1981.84 1982.84 1983.84 Mass spectrum of peptide with 94 C-atoms (19 amino acid residues) No 13 C atoms (all 12 C) One 13 C atom Two 13 C atoms “ Monoisotopic mass ” 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. Modified from Agard lab

39 O O HO AcNH m/z 4360.45 4361.45 Isotope pattern for a larger peptide (207 C-atoms) Modified from Agard lab

40 O O HO AcNH Mass spectrum of insulin 12 C : 5730.61 13 C 2 x 13 C 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

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

42 O O HO AcNH 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

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

44 O O HO AcNH Mass 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 Modified from Agard lab

45 O O HO AcNH 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 /Δ m where 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

46 O O HO AcNH Resolution and Accuracy of Mass Analyzers ME 330.804

47 O O HO AcNH With high resolution mass spectrometry it is possible to do “Top Down” Proteomics ME 330.804

48 O O HO AcNH Usually in combination with ECD or ETD ME 330.804

49 O O HO AcNH Typically we perform “bottom up” proteomics approaches Proteins are either chemically cleaved or digested with endopeptidases (most commonly trypsin) ME 330.804

50 O O HO AcNH Since resulting peptides follow a repeating pattern.. -HN--CH--CO--NH--CH--CO--NH- RiRi CH-R ’ cici z n-i R”R” d i+1 v n-i w n-i low energy high energy aiai x n-i bibi y n-i

51 O O HO AcNH We can deduce the sequence of the peptide by subtracting one fragment ion from the next Can “read” the sequence N->C using the b-ions and C->N using the y ions Proteome software

52 O O HO AcNH “Bottom up” Schema ME 330.804

53 O O HO AcNH “Bottom Up” Strategies ME 330.804

54 O O HO AcNH Need to use “Tandem Mass Spectrometry” or MS/MS ME 330.804

55 O O HO AcNH Examples of tandem (and hybrid) instruments: Tandem in time: Ion trap mass spectrometer (ITMS) Fourier transform mass spectrometer (FTMS) Linear ion trap/FTMS (LTQ-FT) Tandem in space: Triple quadrupoles Quadrupole/time-of-flight (QTOF) Time-of-flight/time-of-flight (TOF/TOF) Ion trap/time-of-flight (trapTOF, Qit/TOF) ME 330.804

56 O O HO AcNH Ion Traps perform separate experiments in the time domain ME 330.804

57 O O HO AcNH At JHU Orbitrap Elite and Velos Mass Range m/z 50 - 2,000, m/z 200 - 4,000 Resolution 60,000 at m/z 400 at a scan (FWHM) rate of 4 Hz Minimum resolution 15,000 Maximum resolution > 240,000 at m/z 400 Dynamic Range > 5,000 within a single scan guaranteeing specified mass accuracy MSn, for n = 1 through 10 ETD Option

58 O O HO AcNH LTQ Orbitrap – where does it happen? ME 330.804

59 O O HO AcNH TOF/TOF instruments conceptually easier ME 330.804

60 O O HO AcNH High versus low energy collisions ME 330.804

61 O O HO AcNH At JHU ME 330.804

62 O O HO AcNH Commercially available TOF/TOF instruments ME 330.804

63 O O HO AcNH At JHU ME 330.804

64 O O HO AcNH For intact glycopeptides Higher energy fragmentation can be used for unambigous identification of sites of N-linked glycan utilization – Overcomes the problems associated with PNGaseF and deamidation – HCD feature on Orbitrap instrumentation (C-TRAP) High Energy CID by MALDI TOF/TOF – Uses Argon as a collision gas

65 O O HO AcNH For OGlcNAc ECD and ETD are recommended ME 330.804

66 O O HO AcNH How Does ECD Work? ME 330.804

67 O O HO AcNH In contrast ETD does not use free electrons. ME 330.804

68 O O HO AcNH Velos with ETD option allows for HCD or ETD and for glycopeptides ME 330.804

69 O O HO AcNH Triple Quadrupole Instruments are Best for Quantitation of Peptides ME 330.804

70 O O HO AcNH Tripple Quadrupole Instruments are best for quantitation ME 330.804

71 O O HO AcNH Last steps: Bioinformatics. Step 1. Data extraction Mancuso et al., Data extraction from proteomics raw data: An evaluation of nine tandem MS tools using a large Orbitrap data set: JPR: 2012

72 O O HO AcNH Courtesy R. Gundry Next: Choose Database

73 O O HO AcNH Courtesy R. Gundry

74 O O HO AcNH Courtesy R. Gundry

75 O O HO AcNH Database size affects sensitivity Large databases: – Unrestricted search (e.g. no-enzyme) – Large number of entries Algorithms lose sensitivity as search space is increased (more peptides have to be queried) For both Mascot and Sequest, more correct peptide IDs when used IPI (56,000 entries) vs. NR (1.5 million entries) Mascot is more affected than Sequest – In large database searches, Mascot will list the peptides in the top 10, but not list them first (when compare to smaller DB) – Sequest better able to rank poorer quality peptides, especially when large database used and unconstrained searches done Kapp, et. al., Proteomics, 5(13),3475-90 Courtesy R. Gundry

76 O O HO AcNH Beware redundancy..

77 O O HO AcNH Database on Demand

78 O O HO AcNH There are a myriad of search tools out there..

79 O O HO AcNH Some perform better than others

80 O O HO AcNH At JHU – recommend MASCOT (NHLBI maintained) $8.00/hr of search

81 O O HO AcNH Mascot is best paired with Proteome Discoverer (Proteomics Core Facility)

82 O O HO AcNH

83 O O HO AcNH Making sense of it all Highly recommend Scaffold and Scaffold PTM Can export results from Proteome Discoverer Easy view of experimental findings

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