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

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

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

Mass spectrometry 1897 Thomson discovers e 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 > Da 2000 Spectrum of noncovalently associated macromolecules > 10 6 Da 2005 Orbitrap Makarov Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d

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

Elémentisotope%Masse isotopique isotope%Masse isotopique isotope%Masse isotopique Masse moyenne C 12 C C H 1H1H H2H N 14 N N O 16 O O O S 32 S S S Cl 35 Cl Cl Br 79 Br Br Mass spectrometry Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d

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 Nominal monoisotopic mass : 5801 Exact monoisotopic mass : The instrument response will depend on its resolution Mass spectrometry Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

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

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

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

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

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

Elemental composition Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 C 13 H 14 NO 3 Cl Masse : M+1 : M+3 : Peak matching results : C 13 H ClNO C 13 H ClNO 3

Elemental composition Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006 Measured : ± C : 0 – 30 H : 0 – 60 N : 0 – 10 O : compositions Measured : ± compositions

Elemental composition Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d compositions Measured : ± C : 0 – 30 H : 0 – 60 N : 0 – 10 O : Measured : ± compositions C : 0 – 30 H : 0 – 60 N : 0 – 10 O : 0 – 15 S :

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

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

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

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

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

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

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

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

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

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

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

sterol LOD (ng/µL)LOQ (ng/µL) cholesterol stigmasterol campesterol  -sitosterol cholestanol campestanol sitostanol Phytosterols in Spelt Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

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

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

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

Glucosamine

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

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

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

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

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

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

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

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%

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

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

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

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

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

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

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

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

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

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

Short introduction to proteomics : mass fingerprinting DigestionTryptic peptides Peptide mass list … … measured mass Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

Search m/zMass Tolerance (Da)# Hits IGGHGAEYGAEALERMouse Hb alpha ( ) VGAHAGEYGAEALERHuman Hb alpha ( ) Short introduction to proteomics : mass fingerprinting Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

Search m/zMass Tolerance (Da)# Hits Short introduction to proteomics : mass fingerprinting Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

Short introduction to proteomics : peptide sequencing by MS n A V A G C A G A R cummulative mass b y Pr. Jean-Louis Habib JiwanUCL - Chemistry DepartmentMay 23 d 2006

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

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

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

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

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