Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment.
Bottom Line: Here, we present methodology that permits identification of GPI-APs liberated directly from the surface of intact mammalian cells through exploitation of their appended glycans to enrich for these proteins ahead of LC-MS/MS analyses.We validate our approach in HeLa cells, identifying a greater number of GPI-APs from intact cells than has been previously identified from isolated HeLa membranes and a lipid raft preparation.Our approach provides a new method to achieve greater sensitivity in the identification of low abundance GPI-APs from the surface of live cells and the nondestructive nature of the method provides new opportunities for the temporal or spatial analysis of cellular GPI-AP expression and dynamics.
Affiliation: New England Biolabs, Inc, Ipswich, MA, USA.Show MeSH
Related in: MedlinePlus
Mentions: Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a class of proteins tethered to the plasma membrane of cells that perform or mediate a variety of critical cellular functions including signal transduction, immune recognition, complement regulation, and cell adhesion. The GPI anchor consists of a conserved core glycan linked on its reducing end to the lipid phosphatidylinositol and covalently attached to protein via phosphoethanolamine on its nonreducing end (Fig. 1A). GPIs are assembled and transferred en bloc to the C-termini of various secretory glycoproteins in the ER. GPI-APs are then transported to the cell surface via the secretory pathway. In addition to its GPI anchor, most characterized GPI-APs also possess additional carbohydrate modifications such as N- and/or O-linked glycans (Fig. 1A) 1–4.