Oxidative modification of proteins by reactive oxygen species (ROS) is implicated in a variety of chronic and acute diseases. One of popular methods for detecting protein oxidation is hydrazone derivatization of protein carbonyls with appropriate hydrazine reagents. In order to study protein carbonyls, we developed a new proteomic method for detecting protein carbonyls using a Cy-dye labeled-hydrazide (CHz). Application of the present method to detecting protein carbonyls in a renal cortex of diabetes model OLETF rat is also reported.
The use of CHz is an improvement of the previous method based on the use of biotin-hydrazide (BHz) (Oh-Ishi et al, Free Radic Biol Med. 34, 11-22, 2003), which was not particularly good at quantifying the carbonyls because it relied on the use of Western blotting (Figure:Right) with avidin alkaline phosphatase after agarose two-dimensional gel electrophoresis (2-DE). The new method has advantages over the previous one by directly detecting protein carbonyls by fluorescence in 2-DE gels (Figure:Left). The present method is much easier and has greater potentialities than the previous one toward clarifying oxidative modifications of proteins.
