2. Ionization Sources 3. Mass Analyzers 4. Tandem Mass Spectrometry

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1 Dr. Sanjeeva Srivastava 1. Fundamental of Mass Spectrometry Role of MS and basic concepts 2. Ionization Sources 3. Mass Analyzers 4. Tandem Mass Spectrometry 2 1

2 MS basic concepts Mass spectrometry - technique for production of charged molecular species, and their separation by magnetic and electric fields based on mass to charge ratio Abundance (Intensity) (m/z)

3 Molecular specificity Detection sensitivity Versatility and wide applicability Analysis of complex samples 5 Ionization Separation and mass analysis Detection and generation of mass spectrum 6 3

4 Sensitivity, resolution and accuracy vary Sensitivity drops off as mass increases Ion sources generate positive, negative ions & neutrals 7 +1 charge state: [M+H] +2 charge state: [M+2H] charge state: [M+3H]

5 Many charge states in protein many possible proton acceptors in equilibrium with solution Multiple charge states are useful 9 Amino acid 3LC SLC Average Monoisotopic Glycine Gly G Alanine Ala A Serine Ser S Proline Pro P Valine Val V Threonine Thr T Cysteine Cys C Leucine Leu L Isoleucine Ile I Asparagine Asn N Aspartic acid Asp D Glutamine Gln Q Lysine Lys K Glutamic acid Glu E Methionine Met M Histidine His H Phenyalanine Phe F Arginine Arg R Tyrosine Tyr Y Tryptophan Trp W

6 An analytical approach of separating and analyzing intact proteins Top-down involves direct analysis of intact proteins, without previous proteolytic digestion 11 Bottom up - analytical approach of separating & analyzing peptides following proteolytic digestion of a sample digesting a protein mixture into short peptides with a protease analyzing the peptide mixture by MS 12 6

7 Parts of mass spectrometer Sample Inlet Instrument Control Data Processing Ion Source Mass Analyzer Detector Signal Processing Data Out Dass

8 Sample introduction Sample ionization Sample transfer to high vacuum region Ion mass-to-charge filtering Ion detection Data acquisition and analysis 15 HPLC Proteolytic Peptides Protein MS Ionisation Sources ESI OR MALDI MS/MS 8

9 Protein spot Protein band 2D SDS-PAGE Gel Spot/band excision 1D SDS-PAGE gel In-gel trypsin digestion Protein of interest MS Identification Peptide fragments MALDI-TOF-TOF MS Laser Detector SCX columns Q-TOF LC-MS TOF 1 TOF 2 ESI Ion Reflecto source17 Collision r cell Gas phase Solution Phase Solid Phase Electron ionization Chemical ionization (CI) Electrospray Atmospheric- pressure PI Matrix- assisted laser desorption Plasma desorption Photoionization (PI) Atmospheric- pressure CI Fast Atom Bombardment 18 9

10 In MS due to ionization generally large molecules are broken into several random fragments Non-selective fragmentation Very difficult to interpret Therefore, need for soft ionization methods 19 High ionization efficiency Stable ion beam Minimum background ion current Less cross-contamination in successive samples Dass

11 Ionization Sources: Animation Disperses all ions based on mass-to-charge ratio Focuses all mass-resolved ions at a single focal point 22 11

12 Time- of- Flight (TOF) Ion Trap Quadrup ole Magnetic Sector Orbitrap Ion Cyclotron Resonance 23 Mass range: the maximum allowable m/z ratio amenable to analysis Resolution: ability to separate two neighboring mass ions Adaptability Dass

13 Efficiency: transmission multiplied by the duty cycle Mass accuracy: how far is the measured mass from the actual mass Linear dynamic range Speed of spectra acquisition per unit time 25 Sensitivity: minimum concentration of a compound that the instrument can detect Mass Stability 26 13

14 Ability of a mass spectrometer to resolve different molecular species with similar but distinct masses Mass resolution is the dimensionless ratio of m/z value of a peak divided by its width at half maximum intensity 27 Accurate mass measurements Resolve an isotopic cluster when charge state of high-mass compounds is to be determined Enhance the accuracy of quantification 28 14

15 m FWHM: full width, half maximum R = m W 1 / 2 W 1/ m/z 29 Mass accuracy - how close a mass measurement is to its true (theoretical or exact) value Expressed in parts-per-million (ppm) Parts per million = [Mass theor - Mass exp ] (PPM) Mass theor x

16 Mass Analyzers: Animation Two consecutive stages of mass analysis to detect fragment ions Precursor ion 1 st stage: isolate 2nd stage: analyze Abundance Collision Induced Dissociation CID m/z Survey scan (Precursor scan) MS/MS scan (Fragmentation scan) 32 16

17 CID IRMPD ECD ETD EI CI MALDI-TOF Applications PSD ISD CID 33 Trypsin digest MS MS/MS I * I D G F S* Y m/z 34 m/z 17

18 Tandem MS: Animation Fundamental of Mass Spectrometry Role of MS and basic concepts Ionization Sources Mass Analyzers Tandem Mass Spectrometry 36 18

19 Chhabil Dass Fundamentals of Contemporary Mass Spectrometry. DOI: / John Wiley & Sons, Inc Stroobant. V. and de Hoffmann. E. Mass spectrometry: principles and applications John Wiley & sons Ltd. ISBN Ekman. R., Silberring. J., Westman-Brinkmalm. A. and Kraj. A. Mass spectrometry: Instrumentation, Interpretation, and Applications John Wiley & sons Ltd. ISBN Steven A. Carr, Roland S. Annan. Current Protocols in Molecular Biology. UNIT Overview of Peptide and Protein Analysis by Mass Spectrometry. DOI: / Cravatt. B.F., Simon. G.M. and Yates. J.R. The biological impact ofmassspectrometry-based proteomics. Nature 2007, 450, Yates. J.R., Ruse. C.I. and Nakorchevsky. A. Proteomics by Mass Spectrometry: Approaches, Advances, and Applications. Annu. Rev. Biomed. Eng. 2009, 11, Lee, T.A. A beginner s guide to mass spectral interpretation. John Wiley & sons Ltd ISBN: Sascha Sauer & Magdalena Kliem. Nature Reviews Microbiology 8, Mass spectrometry tools for the classification and identification of bacteria. doi: /nrmicro