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Spectrométrie de masse: tour d’horizon de la technique et applications Pr. Jean-Louis Habib Jiwan UCL - Chemistry Department

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Presentation on theme: "Spectrométrie de masse: tour d’horizon de la technique et applications Pr. Jean-Louis Habib Jiwan UCL - Chemistry Department"— Presentation transcript:

1 Spectrométrie de masse: tour d’horizon de la technique et applications Pr. Jean-Louis Habib Jiwan UCL - Chemistry Department habib@chim.ucl.ac.be

2 Layout  Mass spectrometry  Elemental composition  Radiosterilization of solid metoclopramide  Phytosterols  Glucosamine  Carnitines  Proteomics Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

3 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 - Chemistry DepartmentMay 23 d 2006

4 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 Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 1918

5 EIMS of 2-methyl-3-pentanone (MW = 100) m/z Relative intensity Mass spectrometry Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

6 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 Mass spectrometry Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 http://physics.nist.gov/PhysRefData/Compositions/index.html http://www.webelements.com/

7 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 Mass spectrometry Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

8 HPLC GC Sample Introduction Ion Source Analyser(s) Detector Vacuum Mass spectrometry Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

9 Magnetic and electric sectors Quadrupoles Time of Flight (Linear, Reflectron) Ion Trap ( 3D, Linear, Orbitrap) Fourier Transform Ion Cyclotron Resonance Hybrid instruments Mass spectrometry Analyzers Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

10 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 Mass spectrometry Ion sources Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

11 source mass analyzer detector ions resolved ions sample Mass spectrum intensity m/z Mass spectrometry Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

12 Collision Induced Dissociation detector source Full spectrum MS 2 spectrum Mass spectrometry : tandem mass spec or MS 2 Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

13 Elemental composition Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 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

14 Elemental composition Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 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

15 Elemental composition Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 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

16 Radiosterilization of solid-state metoclopramide Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

17 Radiosterilization of solid-state metoclopramide Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

18 Radiosterilization of solid-state metoclopramide Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Peak 2

19 Radiosterilization of solid-state metoclopramide Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Peak 3

20 Radiosterilization of solid-state metoclopramide Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Peak 4

21 Phytosterols in Spelt Spelt is said to have better nutritive value than winter wheat. Functional properties have traditionally been attributed to spelt. Lack of scientific evidence for these claims. What about the phytosterol contents ?  -sitosterol Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

22 Fine bran ground to 1 mm sieve Saponification in KOH aq (500g/l) / EtOH (1/3), overnight, RT, dark Extraction with diethyl ether Washing with water Filtration (MN 616 wa filter) Evaporation to dryness and redissolution in EtOH SPE (C18, CHCL 3 /MeOH; 95/5) Evaporation to dryness and redissolution in absolute EtOH Filtration on DynaGard TM filters Phytosterols in Spelt Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

23 LC/APCI-MS TIC trace of 19 standard compounds SG FS ASG SE Phytosterols in Spelt Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

24 SG FS ASG ES [M+H-H 2 O] + Phytosterols in Spelt Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

25 LC/APCI-MS TIC trace SG FS Phytosterols in Spelt Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

26 Sat. SterolsUnsat. Sterols sitostanol0.25 (0.02)stigmasterol0.89 (0.02) campestanol0.27 (0.01)cholesterol1.18 (0.03) cholestanol0.31 (0.06)  -sitosterol 1.41 (0.09) campesterol1.42 (0.05) Phytosterols in Spelt Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

27 sterol LOD (ng/µL)LOQ (ng/µL) cholesterol 0.040.08 stigmasterol 0.060.11 campesterol 0.040.07  -sitosterol 0.040.07 cholestanol 0.160.32 campestanol 0.190.37 sitostanol0.200.40 Phytosterols in Spelt Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

28 Combined Steryl Glycoside and Acylated Steryl Glygoside (µg/g) Phytosterols in Spelt Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

29 Combined FS and ES (µg/g) Phytosterols in Spelt Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

30 Glucosamine Stimulates the synthesis of proteoglycans, which, in combination with collagen, form the connective tissue destroyed in osteoarthritis. Anti-inflammatory action ~ 21 millions adults in the USA live with osteoarthritis 25% of visits to primary care physicians and half of all non-steroidal anti-inflammatory prescriptions* * Y. Shao et al J. Pharma. Biomed. Anal. 35 (2004) 625 Dietary supplements as alternative treatment huge business Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

31 Glucosamine

32 Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Glucosamine

33 Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Glucosamine D-Glucosamine chlorhydrate (C6H11O5NH2). HCl MW 215.683.1% of free base D-Glucosamine sulfate (C6H11O5NH2)2. H2SO4. 2KCl MW 605.5 59.2% of free base Difficulties : ► no chromophore ► highly polar ► positively charged at pH 7 HPLC Methods

34 Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Glucosamine H 2 O : CH 3 CN / 50:50 M+1+CH 3 CN M+1 M+Na+CH 3 CN - H 2 O

35 Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Glucosamine H 2 O : MeOH / 50:50 M+1

36 Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Glucosamine NEUTRAL LOSS SCANNING METHOD TRIPLE QUAD INSTRUMENTS Precursor m scan FragmentationFragment detection m-a scan MS1MS2CID

37 Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Glucosamine 180 84

38 Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Glucosamine C18, H 2 O:MeOH / 50:50 200 µl/min 10 µl Y = 56026 + 1571*x R 2 = 1.0000 25 – 25000 ng/ml LOQ ~0.5 ng on column

39 Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Glucosamine 10mg of powder dissolution in 10 ml MeOH:H 2 O / 50:50 dilution 100 x in PBS Results and Reproducibility : 1 capsule, 5 injections : 224 ± 5% mg/g capsule 1 : 233 mg/g capsule 2 : 231 mg/g capsule 3 : 233 mg/g Commercial capsule Commercial solution 10mg of liquide dilution in 10 ml MeOH:H 2 O / 50:50 dilution 10 x in PBS 5 analysis : 0.52 mg/g ± 10%

40 Detection of carnitines in urine Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

41 Detection of carnitines in urine Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

42 Detection of carnitines in urine FABMS spectrum of crude sample Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

43 Detection of carnitines in urine FABMS 2 spectrum of crude sample detection of the precursors of 85 Selective detection at 85 Scanning Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

44 Detection of carnitines in urine : discovery of a new carnitine FABMS 2 spectrum of a crude sample detection of the precursors of 85 ? Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

45 Daughter spectrum of 290 m/z ethylsuccinylcarnitine MS RMN Synthesis Detection of carnitines in urine : discovery of a new carnitine Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

46 Ethylsuccinylc. 2,6-dimethyl- Heptanoylc. Detection of carnitines in urine : discovery of a new carnitine Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

47 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 - Chemistry DepartmentMay 23 d 2006

48 Short introduction to proteomics Protein mixture digestion Peptide mixture separation Proteins PeptidesMS analysis MS data Identification database search digestion Separation: always 2D 2D gel or LC - LC Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

49 Short introduction to proteomics pump SCX column RP column MS system sample injector Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

50 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 - Chemistry DepartmentMay 23 d 2006

51 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 - Chemistry DepartmentMay 23 d 2006

52 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 - Chemistry DepartmentMay 23 d 2006

53 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 cummulative mass b y Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

54 Short introduction to proteomics : peptide sequencing by MS n Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

55 Short introduction to proteomics : peptide sequencing by MS n Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

56 Short introduction to proteomics : peptide sequencing by MS n Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

57 Future ? Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 High throughput analysis Non-covalent association S. Sanglier et al, JASMAS, 14 (2003) 418

58 UCL, SC, Mass Spec. Lab. Pr. Em. Ed de Hoffmann, A. Spôte, R. Rozenberg, R. Libert, G. Petitjean UCL, MD, FARM PMNT Pr. N. Delzenne CHAM Pr. B. Tilquin, A. Maquille, Pr. J. Quetin-Leclercq, UCL, AGRO, BNUT Pr. M. Meurens, Dr. N. Ruibal Moulin de Hollange Dr. D. Delacroix UCL, Saint-Luc Pr. Fr. Van Hoof, Pr. Ph. De Nayer €€€ : Région wallonne, UCL FSR and COSIF Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Acknowledgments

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