Characterization of dehydroascorbate‐mediated modification of glutaredoxin by mass spectrometry

• Published in: Journal of mass spectrometry. Vol. 50; no. 12; pp. 1358 - 1366
• Main Authors: Flandrin, Aurore, Allouche, Sebastien, Rolland, Yoann, McDuff, François‐Olivier, Richard Wagner, J, Klarskov, Klaus
• Format: Journal Article
• Language: English
• Published: England Wiley 01.12.2015; Blackwell Publishing Ltd; Wiley Subscription Services, Inc
• Subjects: Adducts; antioxidants; Biocompatibility; Cellular; Chromatography, Liquid; cysteine; cytotoxicity; dehydroascorbate; Dehydroascorbic Acid - chemistry; deuterium; Deuterium Exchange Measurement; electrospray ionization mass spectrometry; Escherichia coli; Escherichia coli - genetics; glutaredoxin-1; Glutaredoxins - chemistry; Glutaredoxins - genetics; Humans; liquid chromatography; Mass spectrometry; oxidative stress; Peptides; protein S-ascorbylation; Proteins; Recombinant Proteins - chemistry; Spectrometry, Mass, Electrospray Ionization - methods; tandem mass spectrometry; Thiols; Toxicity; dehydroascorbate; liquid chromatography; electrospray ionization mass spectrometry; glutaredoxin-1; protein S-ascorbylation
• ISSN: 1076-5174; 1096-9888
• DOI: 10.1002/jms.3706

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. Copyright © 2015 John Wiley & Sons, Ltd.