Determination of the lipid composition of the GPI anchor.

In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass sp...

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Autores principales:	Auxiliadora Aguilera-Romero, Susana Sabido-Bozo, Sergio Lopez, Alejandro Cortes-Gomez, Sofia Rodriguez-Gallardo, Ana Maria Perez-Linero, Isabelle Riezman, Howard Riezman, Manuel Muñiz
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2907df15772340d8b321990edcd67aef
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spelling	oai:doaj.org-article:2907df15772340d8b321990edcd67aef2021-12-02T20:18:07ZDetermination of the lipid composition of the GPI anchor.1932-620310.1371/journal.pone.0256184https://doaj.org/article/2907df15772340d8b321990edcd67aef2021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0256184https://doaj.org/toc/1932-6203In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation.Auxiliadora Aguilera-RomeroSusana Sabido-BozoSergio LopezAlejandro Cortes-GomezSofia Rodriguez-GallardoAna Maria Perez-LineroIsabelle RiezmanHoward RiezmanManuel MuñizPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 8, p e0256184 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Auxiliadora Aguilera-Romero Susana Sabido-Bozo Sergio Lopez Alejandro Cortes-Gomez Sofia Rodriguez-Gallardo Ana Maria Perez-Linero Isabelle Riezman Howard Riezman Manuel Muñiz Determination of the lipid composition of the GPI anchor.
description	In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation.
format	article
author	Auxiliadora Aguilera-Romero Susana Sabido-Bozo Sergio Lopez Alejandro Cortes-Gomez Sofia Rodriguez-Gallardo Ana Maria Perez-Linero Isabelle Riezman Howard Riezman Manuel Muñiz
author_facet	Auxiliadora Aguilera-Romero Susana Sabido-Bozo Sergio Lopez Alejandro Cortes-Gomez Sofia Rodriguez-Gallardo Ana Maria Perez-Linero Isabelle Riezman Howard Riezman Manuel Muñiz
author_sort	Auxiliadora Aguilera-Romero
title	Determination of the lipid composition of the GPI anchor.
title_short	Determination of the lipid composition of the GPI anchor.
title_full	Determination of the lipid composition of the GPI anchor.
title_fullStr	Determination of the lipid composition of the GPI anchor.
title_full_unstemmed	Determination of the lipid composition of the GPI anchor.
title_sort	determination of the lipid composition of the gpi anchor.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/2907df15772340d8b321990edcd67aef
work_keys_str_mv	AT auxiliadoraaguileraromero determinationofthelipidcompositionofthegpianchor AT susanasabidobozo determinationofthelipidcompositionofthegpianchor AT sergiolopez determinationofthelipidcompositionofthegpianchor AT alejandrocortesgomez determinationofthelipidcompositionofthegpianchor AT sofiarodriguezgallardo determinationofthelipidcompositionofthegpianchor AT anamariaperezlinero determinationofthelipidcompositionofthegpianchor AT isabelleriezman determinationofthelipidcompositionofthegpianchor AT howardriezman determinationofthelipidcompositionofthegpianchor AT manuelmuniz determinationofthelipidcompositionofthegpianchor
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Determination of the lipid composition of the GPI anchor.

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