INTRODUCTIONThis protocol describes the analysis of a proteome using multidimensional protein identification technology (MuDPIT), which couples 2D-LC to MS/MS, to resolve and identify peptides from complex mixtures. In this method, a pulled capillary microcolumn is packed with two independent chromatography phases: a strong cation exchanger and reversed-phase matrix material. Once a complex peptide mixture is loaded onto the system, no additional sample handling is required, because, as the peptides elute from the column, they are directed into the ESI ion-trap mass spectrometer, where they are ionized, mass-selected, and fragmented. Finally, advanced search algorithms match the fragmented peptides to their respective proteins in a database.