Protein carbonyl detection by two-dimensional fluorescence difference gel electrophoresis

SUMMARY Oxidative stress is implicated in a broad variety of chronic and acute diseases, including Alzheimer’s disease, arteriosclerosis and diabetes. In order to study oxidative stress at the proteome level proteins were labeled with Cy2NHS (the N-hydroxyl succinimidyl ester derivative of a cyanine dye reactive with the ε -amino group of lysine residues), or with Cy3- and Cy5HZ (hydrazide derivatives of the dyes reactive with protein carbonyls) and analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Two samples were used for testing the CyHZ labeling; one is low molecular mass marker proteins artificially oxidized by NaOCl and the other renal cortex extracts of 30-week-old diabetes model OLETF (Otsuka Long-Evans Tokushima Fatty) and control LETO (Long-Evans Tokushima Otsuka) rats. Spot volumes V ni and contrasts c ni of 2D-DIGE images were quantified following background subtraction, where the suffices n (=2, 3 or 5) and i (=1, 2, ...) represent the Cy-signal and the Cy2-detected spot identification numbers, respectively. The contrasts c 3 i and c 5 i were on average about 0.53 times as high as c 2 i . Tak-ing the contrast into account, a simple parameter q ni was proposed for judging the quality of spot images. The difference in-gel analysis (DIA) algorithm should be used in combina-tion with the quality parameters to identify statistical outliers as candidates for abnormally carbonylated proteins. Inspection of the background flatness around the candidate spots is also required before making a conjecture as to whether the candidate protein is abnormally oxidized.