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Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment.

Cortes LK, Vainauskas S, Dai N, McClung CM, Shah M, Benner JS, Corrêa IR, VerBerkmoes NC, Taron CH - Proteomics (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Inc, Ipswich, MA, USA.

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Related in: MedlinePlus

Schematic of a representative GPI-AP and experimental workflows. (A) Schematic of a representative GPI-AP. The sugar molecules of the GPI anchor, a representative N-glycan appended to asparagine (N) and a representative O-glycan attached to serine or threonine (S/T) are shown. Sites of potential GalNAz incorporation into GPI-AP glycans are indicated with an N3 in the sugar symbol. The site of PI-PLC cleavage resulting in release of the GPI-AP from the cell surface is also indicated. (B) Workflow for the sugar analog capture enrichment. (C) Workflow for the lectin affinity capture enrichment. Neu5Ac: N-acetylneuraminic acid; GlcNAc: N-acetylglucosamine; GalNAc: N-acetylgalactosamine; PI-PLC: phosphatidylinositol-specific phospholipase C; GalNAz: N-azidoacetylgalactosamine; WGA: wheat germ agglutinin; α-MM: methyl α-d-mannopyranoside.
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fig01: Schematic of a representative GPI-AP and experimental workflows. (A) Schematic of a representative GPI-AP. The sugar molecules of the GPI anchor, a representative N-glycan appended to asparagine (N) and a representative O-glycan attached to serine or threonine (S/T) are shown. Sites of potential GalNAz incorporation into GPI-AP glycans are indicated with an N3 in the sugar symbol. The site of PI-PLC cleavage resulting in release of the GPI-AP from the cell surface is also indicated. (B) Workflow for the sugar analog capture enrichment. (C) Workflow for the lectin affinity capture enrichment. Neu5Ac: N-acetylneuraminic acid; GlcNAc: N-acetylglucosamine; GalNAc: N-acetylgalactosamine; PI-PLC: phosphatidylinositol-specific phospholipase C; GalNAz: N-azidoacetylgalactosamine; WGA: wheat germ agglutinin; α-MM: methyl α-d-mannopyranoside.

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.


Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment.

Cortes LK, Vainauskas S, Dai N, McClung CM, Shah M, Benner JS, Corrêa IR, VerBerkmoes NC, Taron CH - Proteomics (2014)

Schematic of a representative GPI-AP and experimental workflows. (A) Schematic of a representative GPI-AP. The sugar molecules of the GPI anchor, a representative N-glycan appended to asparagine (N) and a representative O-glycan attached to serine or threonine (S/T) are shown. Sites of potential GalNAz incorporation into GPI-AP glycans are indicated with an N3 in the sugar symbol. The site of PI-PLC cleavage resulting in release of the GPI-AP from the cell surface is also indicated. (B) Workflow for the sugar analog capture enrichment. (C) Workflow for the lectin affinity capture enrichment. Neu5Ac: N-acetylneuraminic acid; GlcNAc: N-acetylglucosamine; GalNAc: N-acetylgalactosamine; PI-PLC: phosphatidylinositol-specific phospholipase C; GalNAz: N-azidoacetylgalactosamine; WGA: wheat germ agglutinin; α-MM: methyl α-d-mannopyranoside.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260145&req=5

fig01: Schematic of a representative GPI-AP and experimental workflows. (A) Schematic of a representative GPI-AP. The sugar molecules of the GPI anchor, a representative N-glycan appended to asparagine (N) and a representative O-glycan attached to serine or threonine (S/T) are shown. Sites of potential GalNAz incorporation into GPI-AP glycans are indicated with an N3 in the sugar symbol. The site of PI-PLC cleavage resulting in release of the GPI-AP from the cell surface is also indicated. (B) Workflow for the sugar analog capture enrichment. (C) Workflow for the lectin affinity capture enrichment. Neu5Ac: N-acetylneuraminic acid; GlcNAc: N-acetylglucosamine; GalNAc: N-acetylgalactosamine; PI-PLC: phosphatidylinositol-specific phospholipase C; GalNAz: N-azidoacetylgalactosamine; WGA: wheat germ agglutinin; α-MM: methyl α-d-mannopyranoside.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Inc, Ipswich, MA, USA.

Show MeSH
Related in: MedlinePlus