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Modification-Specific Proteomics of Plasma Membrane Proteins: Identification and Characterization of Glycosylphosphatidylinositol-Anchored Proteins Released upon Phospholipase D Treatment

Felix Elortza,^† Shabaz Mohammed,^† Jakob Bunkenborg,^† Leonard J. Foster,^† Thomas S. Nühse,^‡ Urs Brodbeck,^§ Scott C. Peck,^‡ and Ole N. Jensen*^†

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark, Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom, and Institute of Biochemistry and Molecular Biology, University of Berne, P.O. Box, CH-3000 Bern 9, Switzerland

J. Proteome Res., 2006, 5 (4), pp 935–943

DOI: 10.1021/pr050419u

Publication Date (Web): February 18, 2006

†

University of Southern Denmark.

Current address: Cooperative Research Centre on Biosciences (CIC bioGUNE), Technology park of Bizkaia, 801 A Building, 48160 Derio, Spain.

Current address: Department of Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

Current address: Department of Biochemistry and Molecular Biology, University of British Columbia, 301-2185 East Mall, Vancouver, BC V6T 1Z4, Canada.

‡

John Innes Centre.

University of Berne.

Corresponding author: Ole Nørregaard Jensen, Ph.D., Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark. Tel., +45 6550 2368; fax, +45 6550 2467; e-mail, jenseno@bmb.sdu.dk. URL: www.protein.sdu.dk.

Abstract

Plasma membrane proteins are displayed through diverse mechanisms, including anchoring in the extracellular leaflet via glycosylphosphatidylinositol (GPI) molecules. GPI-anchored membrane proteins (GPI-APs) are a functionally and structurally diverse protein family, and their importance is well-recognized as they are candidate cell surface biomarker molecules with potential diagnostic and therapeutic applications in molecular medicine. GPI-APs have also attracted interest in plant biotechnology because of their role in root development and cell remodeling. Using a shave-and-conquer concept, we demonstrate that phospholipase D (PLD) treatment of human and plant plasma membrane fractions leads to the release of GPI-anchored proteins that were identified and characterized by capillary liquid chromatography and tandem mass spectrometry. In contrast to phospholipase C, the PLD enzyme is not affected by structural heterogeneity of the GPI moiety, making PLD a generally useful reagent for proteomic investigations of GPI-anchored proteins in a variety of cells, tissues, and organisms. A total of 11 human GPI-APs and 35 Arabidopsis thaliana GPI-APs were identified, representing a significant addition to the number of experimentally detected GPI-APs in both species. Computational GPI-AP sequence analysis tools were investigated for the characterization of the identified GPI-APs, and these demonstrated that there is some discrepancy in their efficiency in classification of GPI-APs and the exact assignment of ω-sites. This study highlights the efficiency of an integrative proteomics approach that combines experimental and computational methods to provide the selectivity, specificity, and sensitivity required for characterization of post-translationally modified membrane proteins.

Keywords: post-translational modification • GPI-anchor • membrane protein • subproteome • modification-specific proteomics • mass spectrometry • glycosylphosphatidylinositol-specific phospholipase D

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