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Article

Integration of Multidimensional Chromatographic Protein Separations with a Combined “Top-Down” and “Bottom-Up” Proteomic Strategy

Supporting Info

Kevin M. Millea,^† Ira S. Krull,^† Steven A. Cohen,^‡ John C. Gebler,^‡ and Scott J. Berger*^‡

Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, and Life Sciences R&D, Waters Corporation, Milford, Massachusetts 01757

J. Proteome Res., 2006, 5 (1), pp 135–146

DOI: 10.1021/pr050278w

Publication Date (Web): December 3, 2005

†

Northeastern University.

‡

Waters Corporation.

To whom all correspondence should be addressed. Waters Corporation, 34 Maple Street, Mail Stop: TG, Milford, Massachusetts 01757. Phone: (508) 482-3592. Fax: (508) 482-3625. E-mail: Scott_Berger@Waters.com.

Abstract

In this paper, we present a combined top-down/bottom-up proteomic analysis workflow for the characterization of proteomic samples. This workflow combines protein fractionation (multidimensional chromatographic separation) with parallel online ESI-TOF−MS intact protein analysis, and fraction collection. Collected fractions were digested and protein identifications were produced using MALDI Q-TOF−MS analysis. These identifications were then linked with corresponding ESI-TOF−MS intact protein mass data to permit full protein characterization. This methodology was applied to an E. coli cytosolic protein fraction, and enabled the identification and characterization of proteins exhibiting co-translational processing, post-translational modification, and proteolytic processing events. The approach also provided the ability to distinguish between closely related protein isoforms. The summary of results from this study indicated that roughly one-third of all detected components generated corresponding data from both top-down and bottom-up analyses, and that significant and novel information can be derived from this application of the hybrid analytical methodology.

Keywords: proteomic analysis • multidimensional chromatography • intact protein • LC−MS • top-down • bottom-up • protein modification • protein processing

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