An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics

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• 作者：Dirk A. Wolters ; Michael P. Washburn ; and John R. Yates ; III
• 刊名：Analytical Chemistry
• 出版年：2001
• 出版时间：December 1, 2001
• 年：2001
• 卷：73
• 期：23
• 页码：5683 - 5690
• 全文大小：164K
• 年卷期：v.73,no.23(December 1, 2001)
• ISSN：1520-6882

文摘

We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandemmass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange(SCX) resin and reversed-phase resin in a biphasiccolumn. We detail the improvements over a systemdescribed by Link et al. (Link, A. J.; Eng, J.; Schieltz, D.M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B.M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that separates and acquires tandem mass spectrafor thousands of peptides. Peptides elute off the SCXphase by increasing pI, and elution off the SCX materialis evenly distributed across an analysis. In addition, wedescribe the chromatographic benchmarks of MudPIT.MudPIT was reproducible within 0.5% between twoanalyses. Furthermore, a dynamic range of 10 000 to 1between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along withseparations, the method improves the overall analysis ofproteomes by identifying proteins of all functional andphysical classes.