Adaptation of Protein Carbonyl Detection to the Requirements of Proteome Analysis Demonstrated for Hypoxia/Reoxygenation in Isolated Rat Liver Mitochondria

The key technique in proteome analysis is high-resolution two-dimensional (2D) electrophoretic separation of proteins from biological samples. This method combines isoelectric focusing (IEF) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Derivatization of protein carbonyls with 2,4-dinitrophenylhydrazine (DNPH) and subsequent anti-dinitrophenyl (DNP) immunoblotting is widely used for the detection of oxidatively modified proteins. In previous studies on adapting this method to 2D electrophoresis the derivatization step was carried out before and after the 2D procedure, resulting in an altered spot pattern and high background staining, respectively. The aim of the present experiments was to develop a method for protein derivatization with DNPH between the IEF and the SDS–PAGE steps. Mitochondria were exposed to 10 min hypoxia and 5 min reoxygenation. After IEF using immobilized pH gradients the gel strips were incubated in DNPH/trifluoroacetic acid/SDS for 20 min and neutralized, and SDS–PAGE was performed. Proteins were either stained with Coomassie dye or subjected to Western blotting using anti-DNP IgG. Gels and blots were scanned and matched to a master gel, and the relative carbonyl content of each spot was calculated and compared for five experiments. Importantly, the spot patterns in DNPH-treated and untreated gels were not different. Protein carbonyls could be detected in 59 of the 125 matched spots. Although there was no significant increase in the total protein carbonyl content after hypoxia/reoxygenation, eighteen 2D spots exhibited an increase in carbonyl content. However, most protein spots did not show a change or even a decline (4 spots) in protein carbonyls.