Determination of the lipid composition of the GPI anchor.
Cell Membrane
/ chemistry
Ceramides
/ chemistry
GPI-Linked Proteins
/ chemistry
Genes, Reporter
Glycosylphosphatidylinositols
/ chemistry
Green Fluorescent Proteins
/ genetics
Mass Spectrometry
Membrane Glycoproteins
/ chemistry
Saccharomyces cerevisiae
/ chemistry
Saccharomyces cerevisiae Proteins
/ chemistry
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2021
2021
Historique:
received:
30
05
2021
accepted:
23
07
2021
entrez:
13
8
2021
pubmed:
14
8
2021
medline:
15
12
2021
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 34388214
doi: 10.1371/journal.pone.0256184
pii: PONE-D-21-17892
pmc: PMC8362999
doi:
Substances chimiques
Ceramides
0
GAS1 protein, S cerevisiae
0
GPI-Linked Proteins
0
Glycosylphosphatidylinositols
0
Membrane Glycoproteins
0
Saccharomyces cerevisiae Proteins
0
Green Fluorescent Proteins
147336-22-9
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0256184Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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