Open AccessStructural analysis of the GPI glycan
Author(s) -
Miyako Nakano,
Susana SabidoBozo,
Kouta Okazaki,
Auxiliadora Aguilera-Romero,
Sofia Rodriguez-Gallardo,
Alejandro Cortes-Gomez,
Sergio López,
Atsuko Ikeda,
Kouichi Funato,
Manuel Muñiz
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0257435
Subject(s) - glycan , endoplasmic reticulum , microbiology and biotechnology , biochemistry , membrane protein , chemistry , glycosylation , secretory pathway , proteomics , trypsin , biology , golgi apparatus , enzyme , glycoprotein , gene , membrane
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.