Mass Spectrometry for Chemists and Biochemists

Transcription

1 Erasmus Intensive Program SYNAPS Univ. of Crete - Summer 2007 Mass Spectrometry for Chemists and Biochemists Spiros A. Pergantis Assistant Professor of Analytical Chemistry Department of Chemistry University of Crete spergantis@chemistry.uoc.gr Tel Office Number: A206 1 Module Outline What is Mass Spectrometry (MS) and what is it used for? Ionization Sources Analyzers Detectors Modes of fragmentation in MS Applications: Identification of organic compounds, proteomics, environmental analysis. yphenated techniques (LC-MS and GC-MS) Quantitation with MS Mon Tue Wed Thu Fri Problem Set Take home 2 1

2 Course Material Lecture slides (pdf format) will be available on SYNAPS website and hardcopies will be handed out before each lecture Chapter 9.4 Analytical Mass Spectrometry from the textbook: Analytical Chemistry, Edited by R. Kellner, J.-M. Mermet, M. Otto,.M. Widmer; Wiley-VC, ISBN What can we do with mass spectrometry? 1. Measure mass better than any other technique. 2. Obtain information about chemical structures. What are mass measurements good for? To identify and quantitate, also help characterize: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates and their metabolites, peptides, synthetic organic chemicals, polymers, natural products, organometallics 4 2

3 Some Applications of MS: Pharmaceutical analysis Bioavailability studies Drug metabolism studies, pharmacokinetics Characterization of potential drugs Drug degradation product analysis Screening of drug candidates Identifying drug targets Biomolecule characterization Proteins and peptides Oligonucleotides Environmental analysis Pesticides on foods Soil and groundwater contamination Forensic analysis/clinical 5 Ionization Source Mass Analyzer m/z Detector Intensity Mass Spectrum m/z 6 3

4 Ionization Source Mass Analyzer Detector Ionization Sources Electrospray Ionization (ESI) Nano Electrospray Ionization (NanoESI) Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure Photo Ionization (APPI) Matrix-Assisted Laser Desoprtion/Ionization (MALDI) Desorption/Ionization on Silicon (DIOS) Fast Atom/Ion Bombardment (FAB) Electron Ionization (EI) Chemical Ionization (CI) Mass Analyzers Quadrupoles Quadrupole Ion Trap Linear Ion Trap Ion Trap Limitations Magnetic Sector Quadrupole Time-of-Flight Tandem MS The MALDI with Time-of-Flight Analysis Quadrupole Time-of-Flight MS Fourier Transform Mass Spectrometry (FTMS) Detectors Electron Multiplier Faraday Cup Photomultiplier Conversion Dynode Array Detector Charge(or Inductive) Detector Intensity Mass Spectrum 7 m/z Electron Ionization Mass Spectrometry Filament N 70 V Repeller (V) To mass analyzer 10-6 Torr Collector (V) S Extraction Plate (-V) 8 4

5 Electron Ionization Mass Spectrometry 9 Electron Ionization Mass Spectrometry 10 5

6 Cl N O N C 3 Cl C 3 11 Electron Ionization Mass Spectrometry (low picomole) Advantages Well-Established Fragmentation Libraries* No Suppression Insoluble compounds Interface to GC Non-Polar Samples Disadvantages Molecular ion not always present Need Volatile Sample Need Thermal Stability No Interface to LC Low Mass Compounds (approx. 500 amu) * National Institute of Science and Technology (NIST) database (>100,000 compounds) available to compare fragmentation data 12 6

7 Chemical Ionization Mass Spectrometry Filament N 70 V Repeller (V) To mass analyzer Torr Reagent gas / analyte 1000 / 1 Collector (V) S Extraction Plate (-V) 13 Chemical Ionization Mass Spectrometry i) Acid Base reactions M X (reagent gas ions) M (protonated molecule) X (reagent gas) ii) Complex formation (electrophilic addition) M X (reagent gas ions) MX (pseudomolecular ion) iii) Charge exchange reaction M G. (reagent gas radical ions) M. (pseudomolecular ion) G (reagent gas) Electron capture (highly selective) M e - M -. 7

8 Reagent gases commonly used for CI - MS: methane (C 4 ), ammonia (N 3 ), isobutane (i-c 4 10 ) noble gases for the charge transfer reactions. Methane as ionizing gas C 4 e - 2 e - C. 4 C 4 C. 4 C 5 C. 3 C 4 C 3 C2 5 2 C 3. C % 41% C 4 C 2. C C 4 C 2 3 C % C5 M --> M C4 C25 M --> M C24 C25 M --> [M C25] C35 M --> [M C35] [M1] [M1] [M29] [M41] relative abundance (%) Pseudomolecular ions in Methane CI 25 MW MW MW41 0 m/z M 2 -M 1 =28 amu M 3 -M 1 =40 amu M 3 -M 2 =12 amu M 1-1=MW 16 8

9 Ammonia as ionizing gas N3 (EI ionization) N3 N3 N3 N4 N2 N3 N4 (N 3)2 The reagent ions react with the molecules like Brønsted or like Lewis acids: N4 M M N3 Mr1 N4 M --> [M N4] Mr18 Isobutane chemical ionization C49 M --> M ([m/z]1) or/and [M C49] ([m/z]57) C33 M --> M ([m/z]1) or/and [M C33] ([m/z]39) Negative chemical ionization The reagent gases in negative CI C3O - M --> M - C3O ([m/z]-1) e - R-Cl --> Cl - R Cl - R-Cl --> [RCl2] - ([m/z]35) 9

10 Advantages Parent Ion Interface to GC Insoluble Samples Chemical Ionization (low picomole) Disadvantages No Fragment Library Need Volatile Sample Need Thermal Stability Quantitation Difficult Low Mass Compounds (<1000 amu) Solids Probe Requires Skilled Operator 19 Electron capture (highly selective) M e - M -. The pentafluorobenzyl trimethyl silyl ether derivatives of steroids make them more amenable to high sensitivity measurements using negative chemical ionization

11 21 Electrospray Ionization Mass Spectrometry 3-6 kv Liquid sample Mass Analyzer 2-10 µl/min 11

12 Pneumatically assisted Electrospray MS Nebulization gas 3-6 kv Solution Mass Analyzer µl/min Introducing ions into the gas phase 24 12

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14 Basic groups [M] (-N 2 ) Acidic groups [M-] - (-COO, -O) COOC [M-] - Negative Ions 193 % % 0 O Butyl Paraben m.w. 194 Positive Ions 194 [M] 195 [MNa] m/z [M2] Peptide: Substance P Μr 1347 % [M] Da/e 28 14

15 N-terminal amine 4 M E F R W G K Rel. Inten. 4 ES-MS m/z 2 N 1 N 2 C 2 C 2 C 2 C 2 C COO N N 2 C N C 2 C 2 C 2 C 2 N COO N N C 2 C29 2 N COO 30 15

16 Determination of Protein Relative Molecular Mass Using ESI MS , Παραδείγµατα Ακρίβεια προσδιορισµού µάζας 0,01 % Determination of Protein Relative Molecular Mass Using ESI MS

17 ESI Spectrum of Trypsinogen (MW 23983) M 15 M M 14 M m/z Mass-to-charge ratio 33 Advantages Parent Ion igh Mass Compounds (>100,000 amu) Thermally Labile Compounds (<0º C) Easy to Operate Interface to PLC Zeptomole sensitivity with nanospray ESI - MS (low femtomole to zeptomole) Disadvantages No Fragmentation Need Polar Sample Need Solubility in Polar Solvent (MeO, ACN, 2 O, Acetone are best) Sensitive to Salts Suppression 34 17

18 Matrix Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry

19 The mass spectrum shows the results MALDI TOF spectrum of IgG Relative Abundance (M2) 2 M (M3) Mass (m/z) 37 MALDI MS (low femtomole) Advantages Parent Ion igh Mass Compounds (>100,000 amu) Thermally Labile Compounds (R.T.) Easy to Operate Disadvantages No Fragment Library Wide variety of matrices Quantitation Difficult 38 19