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.

Ascorbic acid metabolism and functions: A comparison of plants and mammals

“…DHA reacts with cysteine, lysine and arginine residues to form dehydroascorbylated proteins [43] , [44] , [45] , [46] , [47] and also reacts with guanosine [48] . In the case of the eye, which has high ascorbate concentration, DHA and its breakdown products cause glycation of eye lens proteins such as crystallin [49] and a GSH-DHA adduct forms in Jurkat cells fed with DHA [50] .…”

Ascorbylation of a Reactive Cysteine in the Major Apple Allergen Mal d 1

“…We next probed the reactivity of Mal d 1 toward L-ascorbate using ESI FT-ICR mass spectrometry (Figure 1C), which indicated the formation of an adduct between Mal d 1 and a five-carbon fragment of ascorbate, corresponding to a mass shift of +112 Da (C 5 H 4 O 3 ). This mass shift has previously been observed for a model protein, where it was attributed to the attachment of a six-membered ring structure to the side chain sulfur atom of cysteine that contains five carbon atoms from ascorbate [14]. To characterize the adduct between Mal d 1 and ascorbate in detail, we employed heteronuclear NMR spectroscopy.…”

“…Interestingly, recent studies established a distinct reactivity of peptides towards ascorbate [13,14]. It was shown that the oxidized form of ascorbate, dehydroascorbate, has a propensity to spontaneously react with the reactive thiol group of cysteines under acidic and neutral conditions, leading to S-ascorbylation of the cysteine side chain.…”