title: Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation.
abstract: Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (∼14%) of gels and proteolytic digestion buffers (∼20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia , Crawley, Western Australia 6009, Australia.
authors.forename: Amber E
authors.identifier: 0000-0002-5940-4531
authors.initials: AE
authors.lastname: Boyatzis
authors.affiliation: Proteomics International , Perth, Western Australia 6009, Australia.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Bringans
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia , Crawley, Western Australia 6009, Australia.
authors.forename: Matthew J
authors.identifier: 0000-0002-5857-7051
authors.initials: MJ
authors.lastname: Piggott
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia , Crawley, Western Australia 6009, Australia.
authors.forename: Marisa N
authors.initials: MN
authors.lastname: Duong
authors.affiliation: Proteomics International , Perth, Western Australia 6009, Australia.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lipscombe
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia , Crawley, Western Australia 6009, Australia.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Arthur
mesh_terms: D000478:Alkylation; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D008301:Maleimides / Q000378:metabolism*; D015336:Molecular Probe Techniques / Q000592:standards*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011506:Proteins / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D008301:Maleimides; D011506:Proteins; D013438:Sulfhydryl Compounds; C043592:maleimide
keywords: gel electrophoresis; mass spectrometry; oxidative stress; proteomics; sample preparation