Mass Spectrometry Course Árpád Somogyi Chemistry and Biochemistry MassSpectrometry Facility) University of Debrecen, April 12-23, 2010

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1 Mass Spectrometry Course Árpád Somogyi Chemistry and Biochemistry MassSpectrometry Facility) University of Debrecen, April 12-23, 2010 Introduction, Ionization Methods Mass Analyzers, Ion Activation Methods Small Molecule Analysis: MS and MS/MS Spectrum Interpretation Interpretation of EI and CI mass spectra Sample Preparation, Separation Techniques MS/MS Scan Modes Biological/biochemical Applications Proteomics, Peptide Sequencing, Protein Identification, Library Search and Spectrum Interpretation Mass Spectrometry Introduction, and Ionization Methods University of Debrecen April 12,

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3 UA Chemistry 3

4 Web addresses for protocols, etc.: 4

5 What is Mass Spectrometry? Mass spectrometry is a powerful tool in analytical l chemistry that t provides detailed structural information for a wide variety of compounds (MW: daltons) by using a small amount of sample (μg-ng, g picomol, femtomol) What can we provide by mass spec? MW determination nominal accurate (elemental composition) isotope pattern high resolution Fragmentation fragmentation rules libraries ( fitting ) MS/MS (or MS n ) Thermodynamic parameters Thermodynamic parameters ionization energy (IE) appearance energy (AE) heats of formation (ΔH f ) activation enthalpy (ΔH # ), activation entropy (ΔS # ). 5

6 Sample Preparation Sample Introduction Direct probe/infusion GC HPLC Rough, Turbomolecular, and Cryo pumps Vacuum System Ionization Source Mass Analyzer Detector Computer Electron impact (EI) Electrostatic (ESA) Electron Multiplier Chemical ionization (CI) Atmospheric pressure (API) Electrospray (ESI) Matrix assisted laser Desorption/ionization (MALDI) Surface enhanced LDI (SELDI) Fast atom bombardment (FAB) Magnet (B) Time-of-flight (TOF) Quadrupole (Q) Ion Traps (2D & 3D IT) Ion-Cyclotron Resonance (ICR) Orbitrap (OT) Photomultiplier Faraday cap Array Detectors Multichanel plate (MCP) Difference between single stage MS and tandem MS/MS Ionization Analysis MS: Collide with target to produce fragments MS/MS: Ionization Selection Activation Analysis 6

7 The importance of sample preparation Ionization Methods Neutral species Charged species Removal/addition of electron(s) M + e - (M +. )* + 2e - electron ionization Removal/addition of proton(s) M + (Matrix)-H MH + + (Matrix) - chemical ionization (CI) atmospheric pressure CI (APCI) fast atom bombardment (FAB) electrospray ionization (ESI) matrix assisted laser desorption/ionization (MALDI) desorption electrospray ionization (DESI) direct analysis in real time (DART) 7

8 Electron Impact (EI) Ionization Still widely used in Forensic Environmental Drug Metabolism, etc. 8

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10 Filament: tungsten or rhenium Current: 3-5 A U fil : a few Vs (1-3 V) fil T fil : ca K (ca C in the ion source) Source Block pressure: ca torrs (mean free path ca. 100 m) repeller: 1 V, m/z 100 u, distance 0.1 cm t res = 1.4x10-6 s 10

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12 Chemical Ionization Ion-molecule reaction(s) between a reagent gas and the sample at a relatively high pressure Most common reagent gases methane, isobutane, ammonia Mechanisms CH 4 + e - CH 4 +., CH 3+, CH 2 +., CH 4+ + CH 4 CH 5+ + CH 3 CH 3+ + CH 4 C 2 H 5+ + H 2 CH 5+ + M [M+H] + + CH 4 C 2 H M [M-H] + + C 2 H 6 C 2 H M [M+ C 2 H 5 ] + 12

13 Protonation is one type of ionization M + AH+ MH+ + A CH3CH2NH2 + (NH3)nNH4+ CH3CH2NH3+ + (n-1) NH3 The extent of fragmentation depends on the exothermicity of the reaction Proton affinity (PA): M + H + MH + - ΔH r = PA Proton affinity (PA): M + H+ MH+ - ΔHr = PA PAs of common CI reagents (kcal/mol) methane (131) < water (173) < methanol (185) < CH2=C(CH3)2 (197) < ammonia (205) If the analyte has a much higher PA than that of the unprotonated reagent, the protonation of the analyte will be (very) energetic (fragment rich CI spectra, semi-ci ) 13

14 Sources still being developed DESI- desorption ESI (sample not in solution) 14

15 DART DART Direct Analysis in Real Time Penning Ionization M* + S S+. + M + electron (but also allows MH+, M-H-, etc) 15

16 Can we teach elephants to fly? John Fenn Yes, of course!!! Nobel Price in Chemistry, 2002 John B. Fenn Koichi Tanaka 16

17 H + H + 4/12/2010 Electrospray Ionization ESI Soft Laser Desorption o SLD Matrix Assited Laser Desorption/Ionization MALDI For(very)highm/z 17

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19 The MALDI Plate with Different Samples in Different Matrices 19

20 Manual Zip-Tip Purification (on dry aliquots of HPA matrix) High throughtput With MiniTrack V (Packard Biosciencei Germany) AnchorChip TM, Bruker Daltonics, Germany 20

21 MALDI-TOF spectrum of a pure protein (linear mode) [M+H] Intensity [M+2H] [2M+H] m/z a.i. Exercise 2 This is a MALDI-TOF spectrum of two proteins. Identify the proteins by molecular weights and assign the labeled ions m/z /export/home/tof/data/mslab/091705/pro_6/1lin/pdata/1 unknown Thu Oct 6 14:27:

22 Glycated hemoglobin from a patient with diabetes Chace, D.H., Chem. Rev., 2001, 101,

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24 Identify Protein from Table m/z 24

25 Determination of Charge States and MW of a protein from ESI spectra Calculated Mass Spectrum Re elative Intensity Intensity m/z m/z 25

26 Fragmentation of Big Protein Complexes 26

27 MALDI-TOF nanospray Q-TOF 27

28 a.i Doubly charged [M+2H] 2+ ions Singly charged [M+H] + ions Oligonucleotide MALDI-TOF, Linear Positive Ions Collaboration with Attila Ambrus Non-covalent dimer m/z 28

29 a.i Oligonucleotide MALDI-TOF, Reflectron Positive Ions Singly charged [M+H] Doubly charged [M+2H] m/z m/z a.i Oligonucleotide MALDI-TOF, Reflectron Positive Ions Singly charged [M+H] + region m/z 29

30 7- charge state K] 7- [M-9H+Na a+k] 7- [M-9H+2K] 7- ESI negative mass sepctrum of an oligonucleotide FT-ICR Instrument [M M-7H] 7- [M-8 8H+Na] 7- [M-8H+K 30

31 7- charge state [M-8H H+K] 7- [M-9H+ +Na+K] H+2K] 7- [M DNA mass calculator program: 31

32 Partial Sequencing of Different Products of the Human Cytochrome P450 cyp2d6 Gene of Different Individuals Enzymes bovine spleen phosphodiesterase and snake venom phosphodiasterase Synthetic Polymer Analysis by MS (MALDI-TOF)

33 CH 3 OH exact mass: u 33

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