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Les détecteurs de masse : une révolution en chromatographie 1ère partie : Introduction à la spectrométrie de masse Pr. Jean-Louis Habib Jiwan UCL – Département.

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Presentation on theme: "Les détecteurs de masse : une révolution en chromatographie 1ère partie : Introduction à la spectrométrie de masse Pr. Jean-Louis Habib Jiwan UCL – Département."— Presentation transcript:

1 Les détecteurs de masse : une révolution en chromatographie 1ère partie : Introduction à la spectrométrie de masse Pr. Jean-Louis Habib Jiwan UCL – Département de chimie habib@chim.ucl.ac.be

2 Mass spectrometry Fundamentals ionization processes internal energy time scale of events isotopic distribution isotopic mass Technical realizations sample introduction Ionization methods vacuum systems type of mass analyzers combination of mass analyzers modes of operation coupling of separation device Applications identification quantification Mass spectral interpretation fragmentation pathways characteristic ions rules MS Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

3 Mass spectrometry 1897 Thomson discovers e - 1912 First mass spectrometer Thomson 1942 First commercial Mass spectrometer 1953 Quad an Trap description Paul & Steinwedel 1956 First GC/MS 1958 First commercial TOF 1968 First commercial Quad 1974 First LC/MS and FTICR description 1982 First complete MS spectrum of Insulin and first commercial triple Quad 1987 MALDI Karas & Hillenkamp 1988 ESI by Fenn First spectra of protein > 20000 Da 2000 Spectrum of noncovalently associated macromolecules > 10 6 Da 2005 Orbitrap Makarov 1918 Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

4 EIMS of 2-methyl-3-pentanone (MW = 100) m/z Relative intensity Mass spectrometry : fundamentals Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

5 HPLC GC Sample Introduction Ion Source Analyser(s) Detector Vacuum Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 Mass spectrometry : fundamentals

6 Electron Ionisation Chemical ionisation Fast Atom Bombardment Laser Desorption Ionisation Matrix-Assisted Laser Desorption Ionisation AP-MALDI Electrospray Atmospheric Pressure Chemical Ionisation Atmospheric Pressure Photoionisation DESI, DART Ion sources Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 Mass spectrometry : fundamentals

7 200 000 15 000 1 000 MW Non PolarHighly Polar EI/CI APCI APPI ESI Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 Mass spectrometry : fundamentals

8 Magnetic and electric sectors Quadrupoles Time of Flight (Linear, Reflectron) Ion Trap ( 3D, Linear) Fourier Transform instruments : Ion Cyclotron Resonance, Orbitrap Hybrid instruments Analyzers Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 Mass spectrometry : fundamentals

9 Elémentisotope%Masse isotopique isotope%Masse isotopique isotope%Masse isotopique Masse moyenne C 12 C10012.0000 13 C1.113.003312.011 H 1H1H1001.0078 2H2H0.0152.01401.0079 N 14 N10014.0031 15 N0.3715.000114.0067 O 16 O10015.9949 17 O0.0416.9991 18 O0.2017.999215.9994 S 32 S10031.9721 33 S0.78932.9715 34 S4.4433.967932.066 Cl 35 Cl10034.9688 37 Cl31.9836.965935.453 Br 79 Br10078.9183 81 Br97.2880.916379.904 http://physics.nist.gov/PhysRefData/Compositions/index.html http://www.webelements.com/ Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 Mass spectrometry : fundamentals

10 For high molecular weights, the difference between the various masses becomes important : Insulin : C 257 H 383 N 65 O 77 S 6 Chemical mass 5807.6559 Nominal monoisotopic mass : 5801 Exact monoisotopic mass : 5803.6375 The instrument response will depend on its resolution Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 Mass spectrometry : fundamentals

11 Resolution R = m mm Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 Mass spectrometry : fundamentals

12 source mass analyzer detector ions resolved ions sample Mass spectrum intensity m/z Mass spectrometry : full spectrum Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

13 Collision Induced Dissociation detector source Full spectrum MS 2 spectrum Mass spectrometry : tandem mass spec or MS 2 Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

14 Mass spectrometry : scanning mode with a triple quad MS1 Fragment ion or product ion scan Precursor selection Fragmentation Fragment detection scan CIDMS2 Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

15 Mass spectrometry : scanning mode with a triple quad Parent ion or precursor ion scan Precursor scan Fragmentation Fragment selection detection MS1CIDMS2 Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

16 Mass spectrometry : scanning mode with a triple quad Detection of carnitines in urine Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

17 Mass spectrometry : scanning mode with a triple quad FABMS 2 spectrum of crude sample detection of the precursors of 85 Selective detection at 85 m/z Scanning Detection of carnitines in urine Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

18 Mass spectrometry : scanning mode with a triple quad Neutral loss scan Precursor m scan Fragmentation Fragment detection m-a scan MS1CIDMS2 Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

19 Mass spectrometry : neutral loss scan Acides biliaires dans un échantillon d’urine m scan m-a scan Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

20 Mass spectrometry : MS n with an ion trap Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 Source : documentation Thermo Finnigan

21 Mass spectrometry : MS 2 with an ion trap or a triple quad MS 2 of glucosamine in ion trap - H 2 0 Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

22 Mass spectrometry : MS 2 with an ion trap or a triple quad MS 2 of glucosamine in triple quad Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

23 Elemental composition C 13 H 14 NO 3 Cl Masse : 267.0662 M+1 : 268.0740 M+3 : 270.0640 Peak matching results : 268.0735 12 C 13 H 15 35 ClNO 3 270.0705 12 C 13 H 15 37 ClNO 3 Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

24 Elemental composition Measured : 150.0701 ± 0.010 C : 0 – 30 H : 0 – 60 N : 0 – 10 O : 0 - 15 14 compositions Measured : 150.0701 ± 0.050 60 compositions Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

25 Elemental composition 31 compositions Measured : 322.0151 ± 0.010 C : 0 – 30 H : 0 – 60 N : 0 – 10 O : 0 - 15 Measured : 322.0151 ± 0.010 152 compositions C : 0 – 30 H : 0 – 60 N : 0 – 10 O : 0 – 15 S : 0 - 10 Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

26 Short introduction to proteomics Protein chemistryProteomics Individual proteinComplete mixtures Complete sequence analysisPartial sequence analysis Emphasis structure and function Emphasis on identification by database matching Structural biologySystems biology Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

27 Short introduction to proteomics Protein mixture digestion Peptides mixture separation Proteins PeptidesMS analysis MS data Identification database search digestion Separation: always 2D 2D gel or LC - LC Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

28 + 2+ 3+ 2+ 3+ +2+ 3+ RP - LC SCX column Protonated peptides Short introduction to proteomics 1 st salt pulse 2 nd salt pulse 3 d salt pulse pump SCX column RP column MS system sample injector

29 Short introduction to proteomics : mass fingerprinting DigestionTryptic peptides 1529.5006 1529.6997 1529.7348 1529.9978 1530.2332 1530.4567 1531.0003 Peptide mass list … … 1529.7348 measured mass Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

30 Search m/zMass Tolerance (Da)# Hits 15291478 1529.70.1164 1529.730.0125 1529.7340.0014 1529.73480.00012 IGGHGAEYGAEALERMouse Hb alpha1529.7348 (-0.0048) VGAHAGEYGAEALERHuman Hb alpha1529.7348 (-0.0048) Short introduction to proteomics : mass fingerprinting Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

31 Search m/zMass Tolerance (Da)# Hits 1529.730.1204 1529.73 1252.700.17 1529.73 1252.70 1833.880.11 Short introduction to proteomics : mass fingerprinting Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

32 Short introduction to proteomics : peptide sequencing by MS n A V A G C A G A R 72 171 242 299 402 473 530 601 769 cumulative mass b y Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007

33 Short introduction to proteomics : peptide sequencing by MS n Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 Source : DC Liebler « Introduction to Proteomics », Humana Press 2002

34 Short introduction to proteomics : peptide sequencing by MS n Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 1 2 3 4 time Full scan MS m/z MS 2 fragmentation 1 23 4

35 Short introduction to proteomics : peptide sequencing by MS n Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 Source : DC Liebler « Introduction to Proteomics », Humana Press 2002

36 Further reading Pr. Jean-Louis Habib JiwanUCL – Département de chimieIPL mai 2007 - Ed de Hoffmann and V Stroobant « Mass Spectrometry Principles and Applications » 2nd ed, Wiley 2001 - J H Gross « Mass Spectrometry - A textbook » Springer 2004

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