Structural analysis of the GPI glycan

• Published in: PloS one Vol. 16; no. 9; p. e0257435
• Main Authors: Nakano, Miyako, Sabido-Bozo, Susana, Okazaki, Kouta, Aguilera-Romero, Auxiliadora, Rodriguez-Gallardo, Sofia, Cortes-Gomez, Alejandro, Lopez, Sergio, Ikeda, Atsuko, Funato, Kouichi, Muñiz, Manuel
• Format: Journal Article
• Language: English
• Published: San Francisco Public Library of Science 16.09.2021; Public Library of Science (PLoS)
• Subjects: Analysis; Biology and Life Sciences; Cell membranes; Cell surface; Electrophoresis; Endoplasmic reticulum; Enzymes; Eukaryotes; Glycan; Glycolipids; Glycosylphosphatidylinositol; Ions; Lab Protocol; Mass spectrometry; Mass spectroscopy; Peptides; Plasma; Polysaccharides; Post-translation; Proteins; Research and Analysis Methods; Scientific imaging; Structural analysis; Structure; Trypsin; Yeast; Yeasts; United States; Japan; Spain
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0257435

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins.