title: 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.
authors.affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, Research Triangle Park, North Carolina 27709, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Perdivara
authors.forename: Leesa J
authors.initials: LJ
authors.lastname: Deterding
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Tomer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D016477:Artifacts; D002853:Chromatography, Liquid; D004229:Dithiothreitol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007737:Kynurenine / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007737:Kynurenine; D014364:Tryptophan; D014357:Trypsin; D004229:Dithiothreitol