Mass spectrometry work-flow Liquid chromatography In-gel digestion Ionization source Mass analyzers Tandem mass spectrometry 12/11/12

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1 Dr. Sanjeeva Srivastava Mass spectrometry work-flow Liquid chromatography In-gel digestion Ionization source Mass analyzers Tandem mass spectrometry 2 1

2 HPLC Proteolytic Peptides Protein 5 MS Ionisation Sources ESI 4 OR MALDI MS/MS 4 2

3 Spot excision 2D SDS-PAGE Gel MALDI-TOF-TOF MS LASER He He He He Protein of interest He He He TOF 1 He TOF 2 Reflector Collision Cell Detector Trypsin digestion 5 6 3

4 7 Separate mixture components on basis of differences in affinity for stationary & mobile phase Removes undesired impurities Increased sensitivity, detection of low level proteins Separates peptide mixture 8 4

5 Based upon hydrophobic binding interaction between peptides/proteins (mobile phase) immobilized hydrophobic ligand (stationary phase) 9 Mobile phases A B HPLC pump Injector A buffer (0.1% Formic acid, 5% ACN) B buffer (0.1% Formic acid, 80% ACN) RP column Desalt and separate MS 10 5

6 RP is used with ESI due to compatibility of RP s acidic aqueous & polar mobile with ESI In-line RP-HPLC is useful desalting peptides before ESI no need for off-line desalting 11 Silica based cation exchange stationary phase Sulfonic acid cation-based exchange ligand Ligand covalently bound to polymer coated silica Retention Elution 12 6

7 Microcapillary HPLC s low flow rate is more sensitive than standard RP-HPLC Microcapillary HPLC columns prepared using fused silica capillary Column packing 13 Multidimensional separations Size exclusion chromatography (SEC) Ion exchange chromatography (IEX) Capillary electrophoresis (CE) Reversed-phase (RP) Affinity chromatography 14 7

8 I. SEC RP II. RP SEC CE CE III. IMAC Avidin RP RP IV. SCX RP 15 SCX separation by charge RP (C18) separation by hydrophobicity 16 8

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10 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 19 ESI requires sample of interest to be in solution To ionize samples high voltage is applied to high conductively coated needle Distinguishing feature of ESI its ability to produce multiply charged ions 20 10

11 MS Sample Orifice Protein/Peptide ( mm) Flow: nl/ min Ion current ~2.2 kv 2-10 mm Capillary tip 21 Desolvation of ions occurs at atmospheric pressure and mass analyzer is maintained at lower pressure During movement, evaporation reduces droplet size Ions when enter into MS, droplets are dried using a stream of inert gas 22 11

12 Drop Ground Screen Wire Power Supply High Voltage High Voltage High Voltage 23 12

13 Time- of- Flight (TOF) Ion Trap Quadrup ole Magnetic Sector Orbitrap Ion Cyclotron Resonance 25 Scanning MS Ion- beam MS Trapping MS TOF Quadrupole IT, Orbitrap, and FT-ICR MALDI ESI 26 13

14 Time of Flight (TOF) LASER Sample Ionisation Ion Source Source TOF Ion Current Reflector Detector 28 14

15 Quadrupole Quadrupole (Q) set of 4 parallel metallic rods Radio frequency mode Scanning mode Neutral loss scan and precursor ion scanning mode 30 15

16 TQ 3 arrangements similar to quadrupole Q1 Q2 Q3 31 Ion Trap 32 16

17 Consist of a chamber surrounded by a ring electrode and two end-cap electrodes Voltage applied to ring electrode determines which ion remain in the trap Ring electrode Fragment ions End cap electrode

18 Uses cyclotron motion (cyclotron frequency) to resolve ions Most complex, difficult to operate Highest resolution, mass accuracy and sensitivity Multiple tandem experiments feasible MS/MS of very large ions feasible

19 MALDI can be coupled to tandem TOF-TOF or hybrid Q-TOF analyzers, separated by collision cell Much higher sensitivity than TQ and single TOF Source Ionisation LASER Ion Source Detector Ion Current Reflector TOF 1 TOF 2 Collision Cell RF Ion guide RF ion guide Detector Ion Source Quadrupole Collision Cell TOF Combines front part of a TQ with a TOF analyzer to measure the mass of the ions Reflectron 19

20 MS: concepts review Animation Instrument Resolution LIT/LTQ (Linear Ion Trap) TQ (Triple Quadrupole) Mass Accuracy Sensitivity Scan Rate ppm Femtomole Fast ppm Attomole Moderate LTQ-Orbitrap 100,000 2 ppm Femtomole Moderate LTQ-FTICR 500,000 < 2 ppm Femtomole Slow Q-TOF 10, ppm Attomole Moderate, Fast Ref: Annu. Rev. Biomed. Eng :

21 Mass Spectrometry work-flow In-gel digestion Liquid chromatography Ionization source Mass analyzers Tandem mass spectrometry 41 Lee, T.A. A beginner s guide to mass spectral interpretation. John Wiley & sons Ltd ISBN: Stroobant. V. and de Hoffmann. E. Mass spectrometry: principles and applications John Wiley & sons Ltd. ISBN Chhabil Dass Fundamentals of Contemporary Mass Spectrometry. DOI: / John Wiley & Sons, Inc Medzihradszky KF, Campbell JM, Baldwin MA, Falick AM, Juhasz P, Vestal ML, Burlingame AL. The characteristics of peptide collisioninduced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer. Anal Chem. 2000, 72, Yates JR, Ruse CI, Nakorchevsky A. Proteomics by mass spectrometry: approaches, advances, and applications. Annu Rev Biomed Eng. 2009, 11,

22 Yates JR, Ruse CI, Nakorchevsky A. Proteomics by mass spectrometry: approaches, advances, and applications. Annu Rev Biomed Eng. 2009;11: Tomas Bergman, Ella Cederlund, Hans Jörnvall, Elizabeth Fowler. DOI: / ps1108s31. Current Protocols in Protein Science. Liquid Chromatography-Mass Spectrometry, Third Edition. Wilfried M. A. Niessen. CRC Press Pages ISBN: Liquid Chromatography-Mass Spectrometry, Third Edition. Wilfried M. A. Niessen. CRC Press Pages ISBN: Timothy D. Veenstra, John R. Yates. Proteomics for Biological Discovery. Chapter 1. Mass Spectrometry: The Foundation of. Proteomics DOI: / ch1 Agilent Technologies: Andrew J Link. CSHL Proteomics Course