Identification of Oxidized Proteins Based on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis, Immunochemical Detection, Isoelectric Focusing, and Microsequencing

Abstract

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.