Proteomic Analysis of Glycosylphosphatidylinositol-anchored Membrane Proteins

doi:10.1074/mcp.M300079-MCP200

Proteomic Analysis of Glycosylphosphatidylinositol-anchored Membrane Proteins*

From the ^‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark and ^¶Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom

‡‡To whom correspondence should be addressed. Tel.: 45-6550-2368; Fax: 45-6550-2467; E-mail: jenseno{at}bmb.sdu.dk

Abstract

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains unclear, they have attracted attention because they act as enzymes and receptors in cell adhesion, differentiation, and host-pathogen interactions. GPI-APs may represent potential diagnostic and therapeutic targets in humans and are interesting in plant biotechnology because of their key role in root development. We here present a general mass spectrometry-based proteomic “shave-and-conquer” strategy that specifically targets GPI-APs. Using a combination of biochemical methods, mass spectrometry, and computational sequence analysis we identified six GPI-APs in a Homo sapiens lipid raft-enriched fraction and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date.

Footnotes

Published, MCP Papers in Press, September 29, 2003, DOI 10.1074/mcp.M300079-MCP200
↵1 The abbreviations used are: GPI, glycosylphosphatidylinositol; GPI-AP, GPI-anchored protein; CRD, cross-reacting determinant; MS/MS, tandem mass spectrometry; HPLC, high performance liquid chromatography; MPSS, Massively Parallel Signature Sequencing; PI-PLC, phosphatidylinositol phospholipase C; MES, 4-morpholineethanesulfonic acid.
↵2 J. Kronegg and D. Buloz, Detection/prediction of GPI cleavage site (GPI-anchor) in a protein (DGPI) at 129.194.185.165/dgpi/.
↵* This work was supported by a grant from the Danish Natural Sciences Research Council (to O. N. J.) and by funds from the Gatsby Charitable Foundation (to T. S. N. and S. C. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Supported by a post-doctoral fellowship from the Basque Government.
↵‖ Supported by a European Molecular Biology Organization short term fellowship.
↵** Supported by a European Molecular Biology Organization long term fellowship.
- Received August 19, 2003.
- Revision received September 24, 2003.