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An automated multidimensional protein identification technology for shotgun proteomics

Academic Article
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Overview

authors

  • Wolters, D. A.
  • Washburn, M. P.
  • Yates III, John

publication date

  • December 2001

journal

  • Analytical Chemistry  Journal

abstract

  • 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 10000 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.

subject areas

  • Autoanalysis
  • Chromatography, Liquid
  • Proteins
  • Proteome
  • Reproducibility of Results
  • Saccharomyces cerevisiae
  • Spectrometry, Mass, Electrospray Ionization
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Identity

International Standard Serial Number (ISSN)

  • 0003-2700

Digital Object Identifier (DOI)

  • 10.1021/ac010617e

PubMed ID

  • 11774908
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Additional Document Info

start page

  • 5683

end page

  • 5690

volume

  • 73

issue

  • 23

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