Nakano, MiyakoSabido-Bozo, SusanaOkazaki, KoutaAguilera-Romero, AuxiliadoraRodriguez-Gallardo, SofiaCortes-Gomez, AlejandroLopez, SergioIkeda, AtsukoFunato, KouichiMuñiz, Manuel2022-11-252022-11-252021-09-16Nakano M, Sabido-Bozo S, Okazaki K, Aguilera-Romero A, Rodriguez-Gallardo S, Cortes-Gomez A, et al. Structural analysis of the GPI glycan. PLoS One. 2021 Sep 16;16(9):e0257435http://hdl.handle.net/10668/4410Glycosylphosphatidylinositol (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.enAtribución 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/GlycosylphosphatidylinositolTrypsinEndoplasmic reticulumMass spectrometryElectrophoresisDigestionPeptidesGlicosilfosfatidilinositolesTripsinaRetículo endoplásmicoEspectrometría de masasElectroforesisDigestiónPéptidosMedical Subject Headings::Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Endoplasmic ReticulumMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Glycoproteins::Membrane Glycoproteins::GPI-Linked ProteinsMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::PeptidesMedical Subject Headings::Organisms::Eukaryota::Fungi::Ascomycota::Saccharomycetales::Saccharomyces::Saccharomyces cerevisiaeMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Mass Spectrometry::Tandem Mass SpectrometryMedical Subject Headings::Chemicals and Drugs::Carbohydrates::Glycoconjugates::Glycolipids::GlycosylphosphatidylinositolsMedical Subject Headings::Chemicals and Drugs::Enzymes and Coenzymes::Enzymes::Hydrolases::Peptide Hydrolases::Endopeptidases::Serine Endopeptidases::TrypsinMedical Subject Headings::Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Biosynthetic PathwaysMedical Subject Headings::Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Protein Modification, Translational::Protein Processing, Post-TranslationalMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Mass SpectrometryMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::ElectrophoresisMedical Subject Headings::Phenomena and Processes::Digestive System and Oral Physiological Phenomena::Digestive System Physiological Phenomena::Digestive System Processes::Digestionresearch article34529709open access10.1371/journal.pone.02574351932-6203PMC8445438