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An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics

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Torrey Mesa Research Institute, 3115 Merryfield Row, San Diego, California 92121, and Department of Cell Biology SR11, 10550 North Torrey Pines Road, The Scripps Research Institute, La Jolla, California 92037
Cite this: Anal. Chem. 2001, 73, 23, 5683–5690
Publication Date (Web):October 25, 2001
Copyright © 2001 American Chemical Society

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    We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. 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.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676−682) that separates 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 chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10 000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.

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     Torrey Mesa Research Institute.


     Corresponding author:  (e-mail) [email protected]; (phone) (858) 784-8862.

     The Scripps Research Institute.

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