Publication:
Structural analysis of the GPI glycan

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Abstract
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.
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MeSH Terms
Medical Subject Headings::Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Endoplasmic Reticulum
Medical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Glycoproteins::Membrane Glycoproteins::GPI-Linked Proteins
Medical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Peptides
Medical Subject Headings::Organisms::Eukaryota::Fungi::Ascomycota::Saccharomycetales::Saccharomyces::Saccharomyces cerevisiae
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Mass Spectrometry::Tandem Mass Spectrometry
Medical Subject Headings::Chemicals and Drugs::Carbohydrates::Glycoconjugates::Glycolipids::Glycosylphosphatidylinositols
Medical Subject Headings::Chemicals and Drugs::Enzymes and Coenzymes::Enzymes::Hydrolases::Peptide Hydrolases::Endopeptidases::Serine Endopeptidases::Trypsin
Medical Subject Headings::Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Biosynthetic Pathways
Medical Subject Headings::Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Protein Modification, Translational::Protein Processing, Post-Translational
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Mass Spectrometry
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Electrophoresis
Medical Subject Headings::Phenomena and Processes::Digestive System and Oral Physiological Phenomena::Digestive System Physiological Phenomena::Digestive System Processes::Digestion
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Keywords
Glycosylphosphatidylinositol, Trypsin, Endoplasmic reticulum, Mass spectrometry, Electrophoresis, Digestion, Peptides, Glicosilfosfatidilinositoles, Tripsina, Retículo endoplásmico, Espectrometría de masas, Electroforesis, Digestión, Péptidos
Citation
Nakano 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):e0257435