title: Characterization of dehydroascorbate-mediated modification of glutaredoxin by mass spectrometry.
abstract: Ascorbate is as a potent antioxidant in vivo protecting the organism against oxidative stress. In this process, ascorbate is oxidized in two steps to dehydroascorbate (DHA), which if not efficiently reduced back to ascorbate decomposes irreversibly to a complex mixture of products. We demonstrate that a component of this mixture specifically reacts with the thiol group of cysteine residues at physiological pH to give a protein adduct involving the addition of a 5-carbon fragment of DHA (+112 Da). Incubations of glutaredoxin-1 expressed in Escherichia coli and dehydroascorbate revealed abundant adducts of +112, +224 and +336 Da due to the addition of one, two and three conjugation products of DHA, respectively. ESI-MS of carbamidomethylated glutaredoxin-1 before incubation with DHA, deuterium exchange together with tandem mass spectrometry analysis and LC-ESIMS/MS of modified peptides confirmed structure and sites of modification in the protein. Modification of protein thiols by a DHA-derived product can be involved in oxidative stress-mediated cellular toxicity.
authors.affiliation: Département de Pharmacologie et Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.|École nationale supérieure des ingénieurs en arts chimiques et technologiques, Toulouse, France.
authors.forename: Aurore
authors.initials: A
authors.lastname: Flandrin
authors.affiliation: Département de Pharmacologie et Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.|École nationale supérieure des ingénieurs en arts chimiques et technologiques, Toulouse, France.
authors.forename: Sebastien
authors.initials: S
authors.lastname: Allouche
authors.affiliation: Département de Pharmacologie et Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.|École nationale supérieure des ingénieurs en arts chimiques et technologiques, Toulouse, France.
authors.forename: Yoann
authors.initials: Y
authors.lastname: Rolland
authors.affiliation: Département de Pharmacologie et Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.
authors.forename: François-Olivier
authors.initials: FO
authors.lastname: McDuff
authors.affiliation: Département de Médecine Nucléaire et Radiobiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Richard Wagner
authors.affiliation: Département de Pharmacologie et Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.
authors.forename: Klaus
authors.initials: K
authors.lastname: Klarskov
mesh_terms: D002853:Chromatography, Liquid; D003683:Dehydroascorbic Acid / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000235:genetics; D054477:Glutaredoxins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: C516005:GLRX protein, human; D054477:Glutaredoxins; D011994:Recombinant Proteins; D003683:Dehydroascorbic Acid
keywords: dehydroascorbate; electrospray ionization mass spectrometry; glutaredoxin-1; liquid chromatography; protein S-ascorbylation