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

We performed a three-dimensional separation of pulse-chase dual-labelled rat liver cytosolic proteins using hydrophobic interaction chromatography, isoelectric focusing, and SDS gel electrophoresis. Due to very different expression rates but similar size and pI of rat liver cytosolic proteins, we demonstrate the impossibility of successful two-dimensional separations of such complex protein mixtures. A pre-fractionation of proteins by hydrophobic interaction chromatography is therefore recommended prior to two-dimensional gel electrophoresis. Our studies confirmed the correlation between protein turnover rates and surface hydrophobicity.