title: Proteomic detection of oxidized and reduced thiol proteins in cultured cells.
abstract: The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.
authors.affiliation: Christchurch School of Medicine & Health Sciences, University of Otago, PO Box 4345 Christchurch 8140, New Zealand.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Cuddihy
authors.forename: James W
authors.initials: JW
authors.lastname: Baty
authors.forename: Kristin K
authors.initials: KK
authors.lastname: Brown
authors.forename: Christine C
authors.initials: CC
authors.lastname: Winterbourn
authors.forename: Mark B
authors.initials: MB
authors.lastname: Hampton
mesh_terms: D002478:Cells, Cultured / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D011506:Proteins; D020543:Proteome; D013438:Sulfhydryl Compounds