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Les détecteurs de masse : une révolution en chromatographie 1ère partie : Introduction à

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. Mass spectrometry. Technical realizations sample introduction Ionization methods

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Les détecteurs de masse : une révolution en chromatographie 1ère partie : Introduction à

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

  3. Mass spectrometry 2000 Spectrum of noncovalently associated macromolecules > 106 Da 2005 Orbitrap Makarov 1988 ESI by Fenn First spectra of protein > 20000 Da 1987 MALDI Karas & Hillenkamp 1918 1982 First complete MS spectrum of Insulin and first commercial triple Quad 1974 First LC/MS and FTICR description 1968 First commercial Quad 1958 First commercial TOF 1956 First GC/MS 1953 Quad an Trap description Paul & Steinwedel 1942 First commercial Mass spectrometer 1912 First mass spectrometer Thomson 1897 Thomson discovers e- Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

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

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

  6. Mass spectrometry : fundamentals Ion sources 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 Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  7. 200 000 15 000 MW ESI 1 000 APPI APCI EI/CI Non Polar Highly Polar Mass spectrometry : fundamentals Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

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

  9. Mass spectrometry : fundamentals http://physics.nist.gov/PhysRefData/Compositions/index.html http://www.webelements.com/ Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  10. Mass spectrometry : fundamentals For high molecular weights, the difference between the various masses becomes important : Insulin : C257H383N65O77S6 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 Jiwan UCL – Département de chimie IPL mai 2007

  11. m R = Dm Mass spectrometry : fundamentals Resolution Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

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

  13. Mass spectrometry : tandem mass spec or MS2 Full spectrum source Collision Induced Dissociation MS2 spectrum detector Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  14. Mass spectrometry : scanning mode with a triple quad Precursor selection Fragment detection scan Fragmentation MS1 CID MS2 Fragment ion or product ion scan Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  15. Mass spectrometry : scanning mode with a triple quad Precursor scan Fragment selection detection Fragmentation MS1 CID MS2 Parent ion or precursor ion scan Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

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

  17. Mass spectrometry : scanning mode with a triple quad FABMS2 spectrum of crude sample detection of the precursors of 85 Scanning Selective detection at 85 m/z Detection of carnitines in urine Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  18. Mass spectrometry : scanning mode with a triple quad Precursor m scan Fragment detection m-a scan Fragmentation MS1 CID MS2 Neutral loss scan Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

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

  20. Mass spectrometry : MSn with an ion trap Source : documentation Thermo Finnigan Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  21. Mass spectrometry : MS2 with an ion trap or a triple quad MS2 of glucosamine in ion trap - H20 Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  22. Mass spectrometry : MS2 with an ion trap or a triple quad MS2 of glucosamine in triple quad Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  23. Elemental composition C13H14NO3Cl Masse : 267.0662 M+1 : 268.0740 M+3 : 270.0640 Peak matching results : 268.0735 12C13H1535ClNO3 270.0705 12C13H1537ClNO3 Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

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

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

  26. Short introduction to proteomics Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

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

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

  29. Short introduction to proteomics : mass fingerprinting Digestion Tryptic peptides Peptide mass list … 1529.7348 measured mass … Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  30. Short introduction to proteomics : mass fingerprinting IGGHGAEYGAEALER Mouse Hb alpha 1529.7348 (-0.0048) VGAHAGEYGAEALER Human Hb alpha 1529.7348 (-0.0048) Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  31. Short introduction to proteomics : mass fingerprinting Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  32. Short introduction to proteomics : peptide sequencing by MSn 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 Jiwan UCL – Département de chimie IPL mai 2007

  33. Short introduction to proteomics : peptide sequencing by MSn Source : DC Liebler « Introduction to Proteomics », Humana Press 2002 Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  34. Short introduction to proteomics : peptide sequencing by MSn 1 2 Full scan MS 3 4 time m/z MS2 fragmentation 1 4 m/z m/z 2 3 m/z m/z Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

  35. Short introduction to proteomics : peptide sequencing by MSn Source : DC Liebler « Introduction to Proteomics », Humana Press 2002 Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007

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

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