Determination of the lipid composition of the GPI anchor

• Published in: PloS one Vol. 16; no. 8; p. e0256184
• Main Authors: Aguilera-Romero, Auxiliadora, Sabido-Bozo, Susana, Lopez, Sergio, Cortes-Gomez, Alejandro, Rodriguez-Gallardo, Sofia, Perez-Linero, Ana Maria, Riezman, Isabelle, Riezman, Howard, Muñiz, Manuel
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 13.08.2021; Public Library of Science (PLoS)
• Subjects: Biochemistry; Biology and Life Sciences; Cell Membrane - chemistry; Cell Membrane - metabolism; Cell surface; Ceramides - chemistry; Ceramides - isolation & purification; Composition; Electrophoresis; Endoplasmic reticulum; Evaluation; Fatty acids; Gas1 protein; Genes, Reporter; Glycosylphosphatidylinositol; Glycosylphosphatidylinositols; Glycosylphosphatidylinositols - chemistry; Glycosylphosphatidylinositols - metabolism; GPI-Linked Proteins - chemistry; GPI-Linked Proteins - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Immunoprecipitation; Ions; Lab Protocol; Lipid composition; Lipid structure; Lipids; Mass Spectrometry; Mass spectroscopy; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Membranes; Methods; Proteins; Protocol; Research and Analysis Methods; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Scientific imaging; Solubilization; Spectroscopy; Supervision; Yeast; Yeasts; United States; Switzerland; Spain
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0256184

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.