Proteomic detection of oxidized and reduced thiol proteins in cultured cells.
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.
Cuddihy
Sarah L
SL
Christchurch School of Medicine & Health Sciences, University of Otago, PO Box 4345 Christchurch 8140, New Zealand.
Baty
James W
JW
Brown
Kristin K
KK
Winterbourn
Christine C
CC
Hampton
Mark B
MB
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*
D011506:Proteins; D020543:Proteome; D013438:Sulfhydryl Compounds