Proteome analysis by three-dimensional protein separation: Turnover of cytosolic proteins in hepatocytes

We performed a three-dimensional separation of pulse-chase dual-labelled ra t liver cytosolic proteins using hydrophobic interaction chromatography, is oelectric focusing, and SDS gel electrophoresis. Due to very different expr ession rates but similar size and pi of rat liver cytosolic proteins, we de monstrate the impossibility of successful two-dimensional separations of su ch complex protein mixtures. A pre-fractionation of proteins by hydrophobic interaction chromatography is therefore recommended prior to two-dimension al gel electrophoresis. Our studies confirmed the correlation between prote in turnover rates and surface hydrophobicity.