This is a description of a comprehensive two-dimensional liquid chromatography (LC) system for the separation of protein mixtures. This system uses cation-exchange chromatography followed by reversed-phase chromatography (RPLC). The two LC systems are coupled by an eight-port valve equipped with two storage loops and under computer control. The RPLC effluent is sampled by both a UV detector and an electrospray mass spectrometer. In this way, complex mixtures of large biomolecules can be rapidly separated, desalted, and analyzed for molecular weight in less than 2 h. The system's utility is demonstrated with a mixture of standards and an Escherichia coli cell lysate.
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