Identification of oxidized proteins based on sodium dodecyl sulfate- polyacrylamide gel electrophoresis, immunochemical detection, isoelectric focusing, and microsequencing

It has been demonstrated that an age-associated increase in protein oxidation is a highly selective rather than random phenomenon. Addition of carbonyl groups is a widely used marker for the detection of oxidative damage to the proteins. Carbonyls can be detected immunochemically using antibodies against dinitrophenylhydrazine (DNPH), a reagent that specifically reacts with protein carbonyl groups. In this paper, a procedure for the identification of oxidized proteins during aging or under pathological conditions is described. Protein samples were treated with DNPH and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), followed by immunochemical detection. Proteins exhibiting positive immunostain were then purified by SDS-PAGE followed by isoelectric focusing. The focused protein band was further concentrated by another run of SDS-PAGE and transferred onto Immobilon-P membrane. The identity of the protein was revealed by automated Edman microsequencing and a computer database search. This method is successfully demonstrated for the identification of housefly arginine kinase, a cytosolic protein that is involved in the energy metabolism of insect muscle cells.