title: Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment.
abstract: Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are an important class of glycoproteins that are tethered to the surface of mammalian cells via the lipid GPI. GPI-APs have been implicated in many important cellular functions including cell adhesion, cell signaling, and immune regulation. Proteomic identification of mammalian GPI-APs en masse has been limited technically by poor sensitivity for these low abundance proteins and the use of methods that destroy cell integrity. 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. 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.
authors.affiliation: New England Biolabs, Inc, Ipswich, MA, USA.
authors.forename: Leslie K
authors.initials: LK
authors.lastname: Cortes
authors.forename: Saulius
authors.initials: S
authors.lastname: Vainauskas
authors.forename: Nan
authors.initials: N
authors.lastname: Dai
authors.forename: Colleen M
authors.initials: CM
authors.lastname: McClung
authors.forename: Manesh
authors.initials: M
authors.lastname: Shah
authors.forename: Jack S
authors.initials: JS
authors.lastname: Benner
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Corrêa
authors.forename: Nathan C
authors.initials: NC
authors.lastname: VerBerkmoes
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Taron
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D002853:Chromatography, Liquid; D058851:GPI-Linked Proteins / Q000032:analysis* / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry
chemical_list: D000480:Alkynes; D058851:GPI-Linked Proteins; D011134:Polysaccharides
keywords: Alkyne agarose; Azido sugar analog; Glycoproteins; Lectin affinity; Polarized epithelial cells