An automated multidimensional protein identification technology for shotgun proteomics

We describe an automated method for shotgun proteomics named multidimension al protein identification technology (MudPIT, which combines multidimension al liquid chromatography with electrospray ionization tandem mass spectrome try. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Gar vik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that sep arates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chr omatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% betw een two analyses. Furthermore, a dynamic range of 10 000 to 1 between the m ost abundant and least abundant proteins/peptides in a complex peptide mixt ure has been demonstrated. By improving sample preparation along with separ ations, the method improves the overall analysis of proteomes by identifyin g proteins of all functional and physical classes.