1. M. Triquigneaux, B. Tuccio, R. Lauricella et L. Charles Laboratoire Chimie Provence, UMR 6264, Equipe Spectrométries Appliquées à la Chimie Structurale, Campus de St Jérôme, 13397 Marseille cedex 20. EPR/MS combination: Mechanism elucidation applied to thiol compounds

2. Electronic Paramagnetic Resonance Specificity paramagnetic species only Sensitivity concentration 10 -7 -10 -8 mol/L Structure g factor, hyperfine coupling constants… Nitroxide EPR/MS Combination 1

3. How to access the global structure of nitroxide ? EPR/MS Combination a N ~ 15.5 G a H ~ 1.8 G aNaN aHaH 2

4. EPR/MS Combination Mass spectrometry Detection of nitroxide radical species Detection of derived redox species Nitroxide Oxoammonium Hydroxylamine m/z m/z – 1 m/z + 1 Tuccio B., Lauricella R., Charles L., Int. J. Mass Spec., 252 (2006), 47-53. El Hassan I., Charles L., Lauricella R., Tuccio B., New J. Chem., 32 (2008), 680-688. H+H+ H+H+ 3

5. Glutathione Aim of the study: reactivity of glutathione toward MNP R.R. antioxidant physiological substrate toxic by-products S-transferase MNP xenobiotics ? MNP 4

6. 1) Glutathione MNP 2) Oxidant S-adduct a N = 18.2 G EPR Study Experimental conditions 5

7. 1) Glutathione MNP Oxidant 2) Photo-irradiation EPR Study O-adduct a N = 27.1 G Experimental conditions 6

8. No Detection of S- and O-adducts as nitroxides Detection of derived redox species MS Study S-adduct O-adduct m/z 393.1m/z 407.1 m/z 409.1 ESI-MS in negative mode 7

9. MS/MS Study S-adduct ESI-MS/MS in negative mode 128.1 143.1 179.1 210.1 254.1 272.1 393.1 25 eV 393.1 m/z Glutathione product ions Dieckhaus C., Fernandez-Metzler C., King R., Krolikowski P., Baillie T., Chem. Res. Toxicol., 18 (2005), 630-638. 8

10. MS/MS Study O-adduct ESI-MS/MS in negative mode 128.1 143.1 179.1 210.1 254.1 272.1 409.1 20 eV 409.1 Glutathione product ions 321.1 304.1 m/z 9

11. MS/MS Study m/z 409.1 m/z 321.1 m/z 304.110

12. MS/MS Study Methylated product of S-adduct ESI-MS/MS in negative mode 128 143254 Glutathione product ions m/z 407.1m/z 272.1 11

13. Inverted spin trapping Forrester-Hepburn -NO + NuH -N(OH)-Nu -N(O. ) -Nu oxidation -NO -.+ NO -N(O. )-Nu oxidation hν NuH Detection of reaction intermediates Distinct experimental conditions Mechanism elucidation 12

14. Mechanism elucidation Forrester-Hepburn Inverted spin trapping MS RPE S-adduct O-adduct 13

15. MNP/GSH/oxidant Stable S-adduct New O-adduct Derivatization methylated product Detailed reactivity of glutathione toward MNP EPR/MS Structural characterization Mechanism elucidation Efficient method for radical study Conclusion 14

16. SACS team Provence University, Laboratory LCP, Marseille SpectropoleFEDER OBJ2142-3341 Organization committeeSMAP 2009 Acknowledgments