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.We further apply our approach to define the cohort of endogenous GPI-APs that populate the distinct apical and basolateral membrane surfaces of polarized epithelial cell monolayers.
Affiliation: New England Biolabs, Inc, Ipswich, MA, USA.Show MeSH
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Mentions: In a prior study, we established polarized ARPE-19 cells as a model system for in vivo incorporation of GalNAz into GPI anchors and N-glycans 35. Thus, we used GalNAz-labeled ARPE-19 cells to concurrently determine the apical and BL GPI proteomes of a polarized monolayer. In this experiment, ARPE-19 cells were grown as a polarized monolayer on a permeable membrane and labeled with GalNAz (Fig. 2A). Apical and BL cell surfaces were separately treated with PI-PLC, and labeled GPI-APs were captured by the SAE method. The intactness of tight junctions of the polarized monolayer during the PI-PLC treatment has been verified previously by the measurement of protein diffusion across the cell monolayer 35. We identified 29 GPI-APs from the apical surface and 24 GPI-APs from the BL surface (Table2, Supporting Information Tables 7, 8). Protein identifications were highly reproducible with 73% of apically identified GPI-APs and 71% of GPI-APs on the BL surface being observed in two or more biological replicates (Table2, Supporting Information Fig. 1). Notably, 24 of the 29 proteins were observed on both membranes, with only five observed exclusively on the apical surface and no GPI-APs exclusively found on the BL surface (Fig. 2B). While relatively little is known about the polarized distribution of GPI-APs in ARPE-19 cells, the presence of CD73 on the apical and BL membrane domains correlated with its previously reported localization determined by Western blotting, immunofluorescence, and enzymatic activity 35,51. These results successfully demonstrate our ability to identify GPI-APs on discrete membrane domains of live polarized cells.