Introduction to Mass Spectrometry and Proteomics

Introduction to Mass Spectrometry and Proteomics Annie Moradian Proteome Exploration Laboratory

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

Birth of Mass Spectrometer JJ Thomson http://www.hcc.mnscu.edu/chem * discovered the electron and isotopes * invented the mass spectrometer * awarded Nobel Prize in Physics 1906 (electron and conduction of electricity in gases)

What is mass spectrometry? The smallest scale in the world C 2 H 5 OH? 46.042 m/z

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

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

What is a mass spectrometer? Black box?

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

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:10.1016/j.jasms.2009.07.025

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

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

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.

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 6000 8000 12000 16000 20000 24000 m/z

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

5. ElectroSpray Ionization (ESI) Taylor cone Needle Tip Jet Plume www.newobjective.com/electrospray.index.html

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

ESI Mass Spectrum of Protein + + + + + + + + + + + + + + + 100 20+ 19+ 18+ Relative Intensity 23+ 22+ 21+ 17+ 16+ 15+ 0 800 1000 m/z 1200

Charge state determination I Solving simultaneous equations (n,m) 100 m/z (1) = 893.16 m/z (2) = 942.72 100 16951 Relative Intensity Deconvolution Relative Intensity 0 800 1000 m/z 1200 0 16400 16800 17200 17600 mass (Da) (1) M+nH n = 893.16 (2) M+(n-1)H = 942.72 n-1 n = 19, M = 16951 (M, n) H=1.00784

1.00 EF5, trypsin sh_092905_01 917 (35.756) Sm (Mn, 2x3.0 301.154 69.7 100 Charge state determination II Using isotope peaks % 302.152 1+ 300.15 Da 0.5 2+ 2476.28 Da 0 301 302 303 m/z

Charge state determination II Using isotope peaks 0.336 3+ 2651.1 Da 884.480 884.816 885.151 100 884.771 885.486 885.820 % 677.047 1113.157 1326.649 0 500 600 700 800 900 1000 1100 1200 1300 1400 885

Charge state determination II Using isotope peaks 100 % Peptide HLKTEAEMK, C 46 H 79 N 13 O 15 S 1085.554 1085.554 C 46 H 79 N 13 O 15 S 1086.554 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 1087.554 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 0 200 400 600 800 1000 1200 1400 1600 1800 m/z 1085 1086 1087 1088 1089 Monoisotopic mass 1085.5539 Average mass 1086.2779

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

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

Resolution Resolution = m m 1 Da 50% FWHM 1 Da m 50% 1 Da 0.5 Da 100 100 101 100 101 Resolution m = = m 100 1 m = = m 100 200 0.5

Resolution and Accuracy 100 Peptide HLKTEAEMK, C 46 H 79 N 13 O 15 S 1085.554 Monoisotopic mass 1085.5539 1086.554 % 1087.554 0 200 400 600 800 1000 1200 1400 1600 1800 m/z 1085 1086 1087 1088 1089 100 % Resolution 5000 Resolution 1000 Sufficient mass resolution to resolve the isotopic distribution is required to measure a monoisotopic molecular weight 0 1084 1085 1086 1087 1088 1089

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

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

Mass Filtering

Mass Filtering Principle

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

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. 2005 Jan-Feb;24(1):1-29. Douglas DJ, Frank AJ, Mao D.

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, 589 596

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

Linear Ion Trap Animation https://www.youtube.com/watch?v=4szwtnll4qo https://www.youtube.com/watch?v=_fcxa4k8yss

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)

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.

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 = 1.6022x10-19 C

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

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

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

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

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

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

41 High Resolution and Accuracy

Fourier Transform Animation http://www.youtube.com/watch?v=a5allm9q- Xc&feature=player_embedded

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

Alexander Makarov invented the Orbitrap in 1999.

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

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).

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!

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, 1156-1162

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

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

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: M111.0009431

Orbitrap Classic

Q-Exactive Michalski A et al. Mol Cell Proteomics 2011;10:M111.011015 2011 by American Society for Biochemistry and Molecular Biology

Orbitrap Fusion

Animation Orbitrap http://planetorbitrap.com/orbitrap-elite#tab:animation

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

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)

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

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

Tandem Mass Spectrometry MS1 Fragmentation MS2

Tandem MS CID Collisionally Induced Dissociation HCD Higher Collision Dissociation

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

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.

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

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

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

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

Collision Induced Dissociation (CID)-Based Peptide Fragmentation

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)

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

Peptide Identification by Sequencing TIQFVDWCPTGFK m/z 771.378 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 771.378

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)

Proteomics Applications

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

Shotgun Proteomics vs. Western Blots! https://www.chem.purdue.edu/people/faculty/images/tao%20proteomics-cartoon.jpg

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

Why Targeted Proteomics? Highly reliable quantification of target proteins, that is not possible through Data Dependent/Shotgun proteomics. Nature Methods 10, 19 22 (2013)

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

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

Database search engines 200 400 600 800 1000 1200 m/z MS/MS spectrum Theoretical spectrum Peptide identification Database searching, e.g. MASCOT, SEQUEST, OMSSA, X!Tandem, ROCCIT (roccit.caltech.edu) Protein identification

Database search- Typical results

Database search-typical results

Visualization tools

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.

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

Reference Material

Residual molecular weights of amino acids

Animations Fourier Transform Animation: http://msproteomics.blogspot.com/2007/09/animation-of-ft-icr-ms-by-muddimanlab.html http://www.magnet.fsu.edu/education/tutorials/java/dualsector/index.html Orbitrap Elite http://planetorbitrap.com/orbitrap-elite#tab:animation Q-Exactive http://planetorbitrap.com/q-exactive#tab:animation

References Chhabil Dass, Principles and Practice of Biological Mass Spectrometry, John Wiley & Sons, Inc. 2001 Douglas A. Skoog, Principles of Instrumental Analysis 5th ed., Brooks Cole 1997 Richard B.Cole, Electrospray Ionization Mass Spectrometry, John Wiley & Sons, Inc. 1997 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 10.1002/pmic.200600528 Richard Chapman: Protein and Peptide Analysis by Mass Spectrometry (Humana Press)

Useful websites http://prospector.ucsf.edu http://www.matrixscience.com/ http://prowl.rockefeller.edu http://www.uniprot.org http://maxquant.org/ http://www.proteomecenter.org/ http://chorusproject.org/ http://www.ebi.ac.uk/pride/archive/ http://pel.caltech.edu