Identification of protein carbonyls after two-dimensional electrophoresis

The oxidative modification of proteins plays a major role in a number of human diseases, but identity of the specific proteins that are most susceptible to oxidation has posed a difficult problem. Protein carbonyls are increased after oxidative stress, and after derivatization with 2,4-dinitrophenyl hydrazine (DNP) they can be detected by various analytical and immunological 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-derivatization process alters the isoelectric points of proteins, and additional procedures must be utilized to remove reaction byproducts. These additional procedures can lead to loss of sample, and poor isoelectric resolution on immobilized pH gradient (IPG) strips. We have developed a method that allows the IPG strips to be derivatized with DNP directly following isoelectric focusing of the proteins. This method allows the visualization of oxidized proteins by 2-DE with high reproducibility.