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Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment
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. Pr...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BlackWell Publishing Ltd
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260145/ https://www.ncbi.nlm.nih.gov/pubmed/25262930 http://dx.doi.org/10.1002/pmic.201400148 |
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author | Cortes, Leslie K Vainauskas, Saulius Dai, Nan McClung, Colleen M Shah, Manesh Benner, Jack S Corrêa, Ivan R VerBerkmoes, Nathan C Taron, Christopher H |
author_facet | Cortes, Leslie K Vainauskas, Saulius Dai, Nan McClung, Colleen M Shah, Manesh Benner, Jack S Corrêa, Ivan R VerBerkmoes, Nathan C Taron, Christopher H |
author_sort | Cortes, Leslie K |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4260145 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BlackWell Publishing Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-42601452014-12-11 Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment Cortes, Leslie K Vainauskas, Saulius Dai, Nan McClung, Colleen M Shah, Manesh Benner, Jack S Corrêa, Ivan R VerBerkmoes, Nathan C Taron, Christopher H Proteomics Glycoproteomics 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. BlackWell Publishing Ltd 2014-11 2014-11-12 /pmc/articles/PMC4260145/ /pubmed/25262930 http://dx.doi.org/10.1002/pmic.201400148 Text en © 2014 The Authors. PROTEOMICS published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://creativecommons.org/licenses/by/3.0/ This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Glycoproteomics Cortes, Leslie K Vainauskas, Saulius Dai, Nan McClung, Colleen M Shah, Manesh Benner, Jack S Corrêa, Ivan R VerBerkmoes, Nathan C Taron, Christopher H Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment |
title | Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment |
title_full | Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment |
title_fullStr | Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment |
title_full_unstemmed | Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment |
title_short | Proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment |
title_sort | proteomic identification of mammalian cell surface derived glycosylphosphatidylinositol-anchored proteins through selective glycan enrichment |
topic | Glycoproteomics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260145/ https://www.ncbi.nlm.nih.gov/pubmed/25262930 http://dx.doi.org/10.1002/pmic.201400148 |
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