title: Proteomic profile of reversible protein oxidation using PROP, purification of reversibly oxidized proteins.
abstract: Signal transduction pathways that are modulated by thiol oxidation events are beginning to be uncovered, but these discoveries are limited by the availability of relatively few analytical methods to examine protein oxidation compared to other signaling events such as protein phosphorylation. We report here the coupling of PROP, a method to purify reversibly oxidized proteins, with the proteomic identification of the purified mixture using mass spectrometry. A gene ontology (GO), KEGG enrichment and Wikipathways analysis of the identified proteins indicated a significant enrichment in proteins associated with both translation and mRNA splicing. This methodology also enabled the identification of some of the specific cysteine residue targets within identified proteins that are reversibly oxidized by hydrogen peroxide treatment of intact cells. From these identifications, we determined a potential consensus sequence motif associated with oxidized cysteine residues. Furthermore, because we identified proteins and specific sites of oxidation from both abundant proteins and from far less abundant signaling proteins (e.g. hepatoma derived growth factor, prostaglandin E synthase 3), the results suggest that the PROP procedure was efficient. Thus, this PROP-proteomics methodology offers a sensitive means to identify biologically relevant redox signaling events that occur within intact cells.
authors.affiliation: Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia, United States of America. kgv7g@virginia.edu
authors.forename: Ken G
authors.initials: KG
authors.lastname: Victor
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Rady
authors.forename: Janet V
authors.initials: JV
authors.lastname: Cross
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Templeton
mesh_terms: D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D011506:Proteins