Mass spectrometric identification of oxidative modifications of tryptophan residues in proteins: Chemical artifact or post-translational modification?

Abstract

Oxidative modification of tryptophan to kynurenine (KYN) and N-formyl kynurenine (NFK) has been described in mitochondrial proteins associated with redox metabolism, and in human cataract lenses. To a large extent, however, previously reported identifications of these modifications were performed using peptide mass fingerprinting and/or tandem-MS data of proteins separated by gel electrophoresis. To date, it is uncertain whether NFK and KYN may represent sample handling artifacts or exclusively post-translational events. To address the problem of the origin of tryptophan oxidation, we characterized several antibodies by liquid chromatography-tandem mass spectrometry, with and without the use of electrophoretic separation of heavy and light chains. Antibodies are not normally expected to undergo oxidative modifications, however, several tryptophan (Trp) residues on both heavy and light chains were found extensively modified to both doubly oxidized Trp and KYN following SDS-PAGE separation and in-gel digestion. In contrast, those residues were observed as non-modified upon in-solution digestion. These results indicate that Trp oxidation may occur as an artifact in proteins separated by SDS-PAGE, and their presence should be carefully interpreted, especially when gel electrophoretic separation methods are employed. (J Am Soc

Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis

“…For His-containing peptides R4-1 and P5-1 that exhibit multiple charge states, the dominant species were triply charged and quantification was based on only these species. Peak area analysis of the Trp-bearing peptide (P1-2) and its various oxidized forms (e.g., kynurenine, hydroxytryptophan, and dihydroxytryptophan; see also Perdivara et al, 2010) revealed that ;98% of P1-2 remained unoxidized.…”

Post-translational Modifications and Mass Spectrometry Detection

“…Additionally, it may be difficult to ascertain whether the oxidation of highly reactive residues, such as methionine (181) or cysteine, represent artifacts upon sample handling, or true PTMs. Tomer and co-workers, have shown that tryptophan oxidation may occurs following to protein purification and isolation, particularly with the use of gel electrophoresis (182); and electrospray ionization has also been reported to induce oxidation of methionine, tryptophan or tyrosine residues (183).…”

Advances in purification and separation of posttranslationally modified proteins

“…Further, it might be difficult to distinguish between biologically relevant PTMs and artifacts introduced during an analysis. For example, tryptophan oxidation is considered to be a natural irreversible PTM, but it has been recently found that kynurenine and N-formyl kynurenine tryptophan oxidation products can be introduced into a protein during SDS-PAGE separation [132].…”