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 me thod combines isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacr ylamide 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 modifie d proteins. In previous studies on adapting this method to 2D electrophores is the derivatization step was carried out before and after the 2D procedur e, resulting in an altered spot pattern and high background staining, respe ctively. The aim of the present experiments was to develop a method for pro tein derivatization with DNPH between the IEF and the SDS-PAGE steps, Mitoc hondria were exposed to 10 min hypoxia and 5 min reoxygenation, After IEF u sing immobilized pH gradients the gel strips were incubated in DNPH/trifluo ro-acetic acid/SDS for 20 min and neutralized, and SDS-PAGE was performed. Proteins were either stained with Coomassie dye or subjected to Western blo tting using anti-DNP IgG, Gels and blots were scanned and matched to a mast er gel, and the relative carbonyl content of each spot was calculated and c ompared for five experiments. Importantly, the spot patterns in DNPH-treate d and untreated gels were not different. Protein carbonyls could be detecte d in 59 of the 125 matched spots. Although there was no significant increas e in the total protein carbonyl content after hypoxia/reoxygenation, eighte en 2D spots exhibited an increase in carbonyl content. However, most protei n spots did not show a change or even a decline (4 spots) in protein carbon yls. (C) 2000 Academic Press.