Introduction to Mass Spectrometry and Proteomics

Transcription

1 Introduction to Mass Spectrometry and Proteomics Annie Moradian Proteome Exploration Laboratory

2 Contents 1. Basic Principles of Mass Spectrometry Ionization Sources Mass Analyzers Detectors 2. Tandem Mass Analysis Space-based Tandem Time-based Tandem 3. Proteomics Applications and Bioinformatics Tools

3 Birth of Mass Spectrometer JJ Thomson * discovered the electron and isotopes * invented the mass spectrometer * awarded Nobel Prize in Physics 1906 (electron and conduction of electricity in gases)

4 What is mass spectrometry? The smallest scale in the world C 2 H 5 OH? m/z

5 What is mass spectrometry used for in proteomics? Identification of individual proteins and complex protein mixtures (cell lysates, immunoprecipitates) Identification of post-translational modifications (e.g. ubiquitinylation, phosphorylation, glycosylation, disulfide bond determination) Quantification of protein changes

6 What is a mass spectrometer? Single quadrupole Orbitrap QTrap 6500 Orbitrap Fusion Tribrid

7 What is a mass spectrometer? Black box?

8 What is a mass spectrometer? Ionization Source Create ions Mass Analyzer separate the ions based on their m/z Detector measure the quantity of ions of each m/z analyzer Ion source Vacuum 10-4 ~10-10 Torr detector

9 How is an ion generated? Ionization methods (1) EI (Electron Ionization) (2) CI (Chemical Ionization) (3) FAB (Fast Atom Bombardment) (4) MALDI (Matrix Assisted Laser Desorption/Ionization) (5) ESI (Electrospray Ionization) soft Further reading: What can we learn from ambient ionization techniques, doi: /j.jasms

10 Electrospray Ionization & MALDI The Nobel Prize in Chemistry 2002 "for their development of soft desorption ionization methods for mass spectrometric analyses of biological macromolecules" ELECTROSPRAY WINGS FOR MOLECULAR ELEPHANTS Nobel Lecture, December 8, 2002 By JOHN B. FENN

11 4. Matrix-Assisted Laser Desorption/Ionization (MALDI)

12 MALDI Sample plate MH + Analyzer Roles of Matrix 1) Distributing molecules throughout the matrix so that they are completely isolated. 2) Absorbing energy from the laser beam and transfer it to the molecules. 3) Providing protons.

13 MALDI Spectrum Tobacco Mosaic Virus (TMV)-U2 100 [M+H] + Relative Intensity [M+2H] 2+ Stick a piece of infected leaf on the MALDI slide, and add 50% acetic acid before analysis m/z

14 Summary of MALDI Usefulness Ionize non volatile large molecule Non-destructive Highly tolerant to salt content High throughput Robust Limitations Fits to only large mass range analyzer such TOF Universal matrix is not available Not comprehensive, not quantitative

15 5. ElectroSpray Ionization (ESI) Taylor cone Needle Tip Jet Plume

16 5. ElectroSpray Ionization (ESI) Drying Gas Bath Gas Sample Nebulizer Gas Analyte 2~3 kv Charged Droplet Shrink Explosion Coulomb Repulsion increases until Rayleigh Limit Bath Gas Larger Surface Area Less Electrostatic Repulsion + Taylor Cone

17 ESI Mass Spectrum of Protein Relative Intensity m/z 1200

18 Charge state determination I Solving simultaneous equations (n,m) 100 m/z (1) = m/z (2) = Relative Intensity Deconvolution Relative Intensity m/z mass (Da) (1) M+nH n = (2) M+(n-1)H = n-1 n = 19, M = (M, n) H=

19 1.00 EF5, trypsin sh_092905_ (35.756) Sm (Mn, 2x Charge state determination II Using isotope peaks % Da Da m/z

20 Charge state determination II Using isotope peaks Da %

21 Charge state determination II Using isotope peaks 100 % Peptide HLKTEAEMK, C 46 H 79 N 13 O 15 S C 46 H 79 N 13 O 15 S C 12 45C 13 H 79 N 13 O 15 S C 46 H 78 DN 13 O 15 S C 46 H 79 N 12 N 15 O 15 S C 12 44C 13 2H 79 N 13 O 15 S C 46 H 77 D 2 N 13 O 15 S C 45 C 13 H 78 DN 12 O 15 S m/z Monoisotopic mass Average mass

22 Summary of ESI Usefulness Non-destructive Ionize non-volatile large molecules Useful HPLC inlet (HPLC-MS) Multiple charges (low mass range) Concentration dependent -> quantitative Limitations Low tolerance to salt, detergent (Sample clean-up needed; often underestimated) Undersampling in complex mixtures

23 What is a mass spectrometer? Ionization Source Create ions Mass Analyzer separate the ions based on their m/z Detector measure the quantity of ions of each m/z analyzer Ion source Vacuum 10-4 ~10-10 Torr detector

24 How does the analyzer work? (1) Magnetic Sector (2) Quadrupole (3) Ion Trap (4) Time of Flight (5) FT-ICR (6) Orbitrap The performance of mass analyzer Mass range/ Resolution/Scan speed/sensitivity

25 Resolution Resolution = m m 1 Da 50% FWHM 1 Da m 50% 1 Da 0.5 Da Resolution m = = m m = = m

26 Resolution and Accuracy 100 Peptide HLKTEAEMK, C 46 H 79 N 13 O 15 S Monoisotopic mass % m/z % Resolution 5000 Resolution 1000 Sufficient mass resolution to resolve the isotopic distribution is required to measure a monoisotopic molecular weight

27 Resolution Why Is It Important? Enables accurate mass Increases confidence of identification Improves quantitative accuracy Gives access to qualitatively different information

28 1. Quadrupole Analyzer Uses a combination of RF and DC voltages to operate as a mass filter. Ion Source Analyzer Detector Has four parallel metal rods Lets one mass pass through at a time Can scan through all masses or sit at one fixed mass

29 Mass Filtering

30 Mass Filtering Principle

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34 Summary of Quadrupole Usefulness Small size Fast Scan Low cost High sensitivity Limitations Low resolution (3000) Limited mass range (<3000 m/z)

35 2. Linear Ion Trap Analyzer Two Dimensional Quadrupole Analyzer Ions are trapped by barriers Radially: RF potential Axially: DC barriers Linear ion traps in mass spectrometry. Mass Spectrom Rev Jan-Feb;24(1):1-29. Douglas DJ, Frank AJ, Mao D.

36 3. Ion Trap Analyzer Three Dimensional Quadrupole Analyzer End cap electrode Ring electrode Ion trap mass spectrometry: a personal perspective Journal of the American Society for Mass Spectrometry, Volume 13, Issue 6, 2002,

37 Nobel Price in Physics, 1989 Hans Georg Dehmelt Wolfgang Paul 1993 received the Nobel Price in Physics for the development of the ion trap

38 Linear Ion Trap Animation

39 Summary of Ion Trap Usefulness Small size (1 cubic inch) Simple design (low cost) Capability of multiple MS (MS n ) Capability of accumulation for low abundant sample (5,000 ions) Limitations Dynamic range: Limits the total number of ions (Space charge) Relatively low resolution (5,000)

40 4. Time of Flight (TOF) Analyzer Ion Source Analyzer Detector Ions of different mass (accelerated by the same field, V) have different velocities and thus flight times. The larger the mass the slower the ion.

41 Time of Flight source acceleration region field free drift zone detector --20 V V s ~ 20kV l = length of path E mv 2 = 2 = zev t = l v t v = m z 2zeV m E= Initial kinetic energy m= mass v = velocity z= charge V= Extraction pulse potential in Volt l = length of the field free drift zone in meter t = time of flight of the ions in seconds e = magnitude of electron charge in Coulomb = x10-19 C

42 TOF Usefulness High mass range (theoretically unlimited) High sensitivity (non scan type) High resolution with reflectron Limitations m/z dependent resolution

43 5. FT-ICR Analyzer Image current Ion Source Magnetic Field, B Analyzer & Detector

44 Alan G. Marshall and Melvin B. Comisarow invented FT-ICR at the University of British Columbia (1974).

45 Cyclotron Movement Centrifugal force= B Centripetal force magnetic Lorentz force r F=qvB mv 2 r = qvb mv r = qb r: radius of curvature B: magnetic field q:charge v: velocity w c : circulation frequency v r = w c = qb m

46 Multi Components Image current Ion Source Magnetic Field, B Analyzer & Detector

47 Fourier Transform Time Domain FT Frequency Domain w c = zb m (khz) Mass Domain (m/z)

48 41 High Resolution and Accuracy

49 Fourier Transform Animation Xc&feature=player_embedded

50 Summary of FT-ICR MS Usefulness Extremely high resolution (>1,000,000) Non-destructive Improved S/N Capability of MS n Limitations Massive instrumentation High maintenance (superconducting magnet requires regular liquid helium, liquid nitrogen fillings) Sensitivity (space charge effect/ current detection) Expensive

51 Alexander Makarov invented the Orbitrap in 1999.

52 Based on Kingdon trap Kingdon trap (1922) Non magnetic No RF (dynamic) Two end cap electrodes Central wire electrode No way to get ions in No detector

53 Searching for the ideal mass analyzer The resolution of an FT-ICR. The sensitivity of a TOF. The size and capabilities of a Quadrupole Ion Trap (QIT).

54 Electrostatic Fields Earnshaw s Theorem (1842) An ion packet cannot be maintained in a stable stationary equilibrium configuration solely by electrostatic interaction of the charges. But moving ions can be stable!

55 Orbitrap Ions trapped in an electrostatic field Central electrode kept on high voltage Outer electrode is split and able to r pick up an image current induced by ion packets moving inside the trap z φ U ( r, z) k 2 z 2 r 2 / 2 R ln( r / 2 m R m ) A. Makarov, Anal. Chem 2000,

56 Ion Injection and Formation of Ion Rings An ion packet of a selected m/z enters the field Increasing voltage squeezes ions Voltage stabilises and ion trajectories are also stabilized Angular spreading forms a ROTATING RING (r,φ) (r,z) 56

57 Detection of Ions Ion packets enter the analyzer slightly off axis The field inside the trap effects an oscillation of the ion packets/rings The moving ion rings induce an image current on outer electrodes The frequency of harmonic oscillations is proportional to ions m/z k m / z 57

58 Fourier Transform Mathematical operation transforms frequency signal into a time domain spectrum Orbitrap is a Fourier transform-based mass analyzer Baron Joseph Fourier 58 Scigelova et al. Mol. Cellular Proteomics 2011, 10: M

59 Orbitrap Classic

60 Q-Exactive Michalski A et al. Mol Cell Proteomics 2011;10:M by American Society for Biochemistry and Molecular Biology

61 Orbitrap Fusion

62 Animation Orbitrap

63 Summary of Orbitrap MS Usefulness High resolution (240,000/480,000) and speed Non-destructive In newer generation Orbitraps (Elite, Fusion): improved S/N Limitations Dynamic range (<5,000) Sensitivity (space charge effect/ current detection) Expensive No MS n

64 Detector detects emitted ions, electrons, or photons needs to be multiplied 1. Electron Multiplier Tube (EMT) 2. Scintillation Counter (Using PMT) 3. Image current from oscillating signal by orbiting ions (FTICR and Orbitrap only)

65 1. Electron Multiplier Tube (EMT) Ion beam Anode e To Amp Conversion dynode

66 2. Scintillation Counter Conversion Dynode e hv PMT Ion beam Phosphorous Screen or Scintillater

67 Tandem Mass Spectrometry MS1 Fragmentation MS2

68 Tandem MS CID Collisionally Induced Dissociation HCD Higher Collision Dissociation

69 Triple quadrupole scanning modes Domon, B., Aebersold., R., Mass Spectrometry and Protein Analysis, Science 312, 2006.

70 2. Triple Quadrupole: SRM/MRM* SRM/MRM single reaction monitoring/multiple reaction monitoring Further reading: Selected reaction monitoring for quantitative proteomics: a tutorial Vinzenz Lange, Paola Picotti, Bruno Domon, and Ruedi Aebersold, Mol Syst Biol. 2008; 4: 222.

71 Pros and Cons of SRM Q1/Q3 pair =Transition Advantages of SRM: High transmission efficiency High sensitivity, multiplexing Reduction of noise Rapid switch between transitions <2ms Disadvantages of SRM: You need to have prior knowledge Low resolution Unique peptides Differential ionizability of peptides

72 Tandem MS in a Q-TOF Q1 Q2 Mass Filter Collision cell x Analyzer

73 Traditional data-dependent MS/MS Quadrupole Mass Filter Collision cell x Analyzer

74 Traditional data-dependent MS/MS Quadrupole Mass Filter Collision cell x Analyzer

75 Traditional data-dependent MS/MS Quadrupole Mass Filter Collision cell x Analyzer

76 Novel MS E data-independent MS/MS Quadrupole Mass Filter Collision cell x All ion fragmentation (AIF) and SWATH TM are similar techniques on Q-Exactive and TripleTOF instruments. Analyzer

77 Collision Induced Dissociation (CID)-Based Peptide Fragmentation

78 CID-Based Peptide Fragmentation y 3 y 2 y 1 H + R 1 O R 2 O R 3 O R 4 O H N C C N C C N C C N C C OH H H H H H H H H b 1 b 2 b 3 Roepstorff, P. and Fohlman, J. Biomed Mass Spectrom 11: 601 (1984)

79 Charged Ion Formation + b-ions are acylium ions y-ions are ammonium ions +

80 Peptide Identification by Sequencing TIQFVDWCPTGFK m/z b12 b11 b10 b9 b8 b7 b6 b5 b4 b3 b2 b1 TIQFVDWCPTGF TIQFVDWCPTG TIQFVDWCPT TIQFVDWCP TIQFVDWC TIQFVDW TIQFVD TIQFV TIQF TIQ TI T K FK GFK TGFK PTGFK CPTGFK WCPTGFK DWCPTGFK VDWCPTGFK FVDWCPTGFK QFVDWCPTGFK IQFVDWCPTGFK y1 y2 y3 y4 y5 y6 y7 y8 y9 y10 y11 y12 m/z

81 Electron Capture Dissociation/Electron Transfer Dissociation z 2 z 2 z 1 H + R 1 O R 2 O R 3 O R 4 O H N C C N C C N C C N C C OH H H H H H H H H c 1 c 2 c 3 Roepstorff, P. and Fohlman, J. Biomed Mass Spectrom 11: 601 (1984)

82 Proteomics Applications

83 Proteomics Experiments Protein(s) Bottom-Up Middle-Down Top-Down Trypsin Digestion Glu-C Digestion Intact proteins R K K R R K K R K E E E Data Aquisition Data Aquisition Data Aquisition Bioinformatics

84 Shotgun Proteomics vs. Western Blots!

85 Shotgun Proteomics/ Discovery/DDA BioTechniques, Vol 38, No 4, 2005 Global proteome mapping Identification of proteins and PTM s Quantitative analysis by SILAC or isobaric tags

86 Why Targeted Proteomics? Highly reliable quantification of target proteins, that is not possible through Data Dependent/Shotgun proteomics. Nature Methods 10, (2013)

87 Discovery Identification Intact proteins MW PTM Digested peptides Cellular composition Interaction partners PTM Proteomics Pipeline Scientific Hypothesis Question? Metabolic labeling Quantitative ID/quantitation Digested peptides Chemical labeling VS Label free Detecting global protein level changes in different conditions Targeted Known ID/quantitation Digested peptides Internal standards Detecting targeted protein level changes in different conditions Cellular composition Interaction partners PTM

88 Bioinformatics Tools Spectra processing Data storage Data mining Web interface Raw files MS processing Result file Proteomics DB Data processing & mining MS/MS DB search Web interface MaxQuant Skyline MASCOT Proteome Discoverer X!Tandem SCAFFOLD

89 Database search engines m/z MS/MS spectrum Theoretical spectrum Peptide identification Database searching, e.g. MASCOT, SEQUEST, OMSSA, X!Tandem, ROCCIT (roccit.caltech.edu) Protein identification

90 Database search- Typical results

91 Database search-typical results

92 Database search-typical results

93 Visualization tools

94 MRM targeted analysis We developed a multiplex AP-SRM mass spectrometry assay to measure Skp, Cullin, F-box (SCF) containing complex ubiquitin ligase assembly. 69 Different Fbox J. Reitsma et al, Composition and regulation of the cellular repertoire of SCF ubiquitin ligases, Cell, 2017,171 (6), 1326.

95 PEL To provide investigators with state-of-the-art scientific and technical support in proteomics and mass spectrometry by: access to variety of mass spec instrumentation guidance and hands on training promoting research collaboration

96 Reference Material

97 Residual molecular weights of amino acids

98 Residual molecular weights of amino acids

99 Animations Fourier Transform Animation: Orbitrap Elite Q-Exactive

100 References Chhabil Dass, Principles and Practice of Biological Mass Spectrometry, John Wiley & Sons, Inc Douglas A. Skoog, Principles of Instrumental Analysis 5th ed., Brooks Cole 1997 Richard B.Cole, Electrospray Ionization Mass Spectrometry, John Wiley & Sons, Inc Gary Siuzdak Mass Spectrometry for Biotechnology, Academic Press 1996 Michaela Scigelova and Alexander Makarov, Orbitrap Mass Analyzer Overview and Applications in Proteomics, Practical Proteomics 2006, DOI /pmic Richard Chapman: Protein and Peptide Analysis by Mass Spectrometry (Humana Press)

101 Useful websites