Detection of oxidant sensitive thiol proteins by fluorescence labeling and two-dimensional electrophoresis.
Oxidants can activate signaling pathways and modulate a variety of cellular activities. Their action at a molecular level involves the post-translational modification of protein thiols. We have developed a proteomic method to monitor the reduction and oxidation of protein thiols, and identify those thiol proteins most sensitive to oxidation. Cells were disrupted in the presence of N-ethylmaleimide to block the reduced thiol proteins and dithiothreitol was added to reduce the oxidized thiol proteins before labeling with 5-iodoacetamidofluorescein. Two-dimensional (2-D) electrophoresis was used to resolve the labeled samples. We applied the method to Jurkat T lymphocytes and examined the effect of diamide on the oxidized and reduced thiol protein profiles. A small percentage of protein thiols were already oxidized in untreated cells. Exposure of cells to 2 mM diamide for ten minutes led to a dramatic increase in thiol protein oxidation as seen in the oxidized thiol protein map. However, it was difficult to detect any change in the pattern of reduced thiol proteins. Separation of proteins by 2-D electrophoresis revealed approximately 200 thiol proteins that were oxidized by diamide treatment. This method will be valuable in elucidating redox signaling pathways.
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D008956:Models, Chemical; D016877:Oxidants / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005452:Fluoresceins; D016877:Oxidants; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C015296:5-iodoacetamidofluorescein; D005033:Ethylmaleimide; D010100:Oxygen