Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography

We describe an approach for fractionating complex protein samples prior to two-dimensional gel electrophoresis using reversed-phase high-performance l iquid chromatography. Whole lysates of cells and tissue were prefractionate d by reversed-phase chromatography and elution with a five-step gradient of increasing acetonitrile concentrations. The proteins obtained at each step were subsequently separated by high-resolution two-dimensional gel electro phoresis (2-DE). The reproducibility of this prefractionation technique pro ved to be optimal for comparing 2-DE gels from two different cell states. I n addition, this method is suitable for enriching low-abundance proteins ba rely detectable by silver staining to amounts that can be detected by Cooma ssie blue and further analyzed by mass spectrometry.