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.
Highlights
Glycosylphosphatidylinositol-anchored proteins (GPIAPs) 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
We have integrated protein fractionation methods, mass spectrometry, and bioinformatics techniques into a proteomic body specific for the C terminus of PI-PLC-treated GPI-anchored protein demonstrated that GPI-APs were highly enriched in the aqueous phase after PI-PLC treatment of raftenriched membranes (Fig. 2A, I) and plant membranes (Fig. 2B, III)
As the PI-PLC enzyme used in the present study recognizes a specific GPI anchor structure, probably only a subset of GPI-APs is released from the membrane preparations
Summary
Glycosylphosphatidylinositol-anchored proteins (GPIAPs) 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. Most of the GPI-APs (five of six in HeLa cells and 34 of 44 in A. thaliana cells) were identified based on two or more different peptide tandem mass spectra matching to each individual protein. A total of 11 GPI-APs were each identified based on one peptide sequence obtained by tandem mass spectrometry (one in HeLa cells and 10 in A. thaliana cells).
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