Identification of protein carbonyls after two-dimensional electrophoresis

The oxidative modification of proteins plays a major role in a number of hu man diseases, but identity of the specific proteins that are most susceptib le to oxidation has posed a difficult problem. Protein carbonyls are increa sed after oxidative stress, and after derivatization with 2,4-dinitrophenyl hydrazine (DNP) they can be detected by various analytical and immunologic al methods. Although high resolution two-dimensional electrophoresis (2-DE) can resolve virtually all proteins present in a cell or tissue it has been difficult to determine the oxidized proteins because the DNP-derivatizatio n process alters the isoelectric points of proteins, and additional procedu res must be utilized to remove reaction byproducts. These additional proced ures can lead to loss of sample, and poor isoelectric resolution on immobil ized pH gradient (IPG) strips. We have developed a method that allows the I PG strips to be derivatized with DNP directly following isoelectric focusin g of the proteins. This method allows the visualization of oxidized protein s by 2-DE with high reproducibility.