title: Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins.
abstract: We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.
authors.affiliation: Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kaji
authors.forename: Haruna
authors.initials: H
authors.lastname: Saito
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Yamauchi
authors.forename: Takashi
authors.initials: T
authors.lastname: Shinkawa
authors.forename: Masato
authors.initials: M
authors.lastname: Taoka
authors.forename: Jun
authors.initials: J
authors.lastname: Hirabayashi
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Kasai
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Takahashi
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Isobe
mesh_terms: D029742:Caenorhabditis elegans Proteins / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017124:Conserved Sequence; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D037102:Lectins / Q000737:chemistry; D046911:Macromolecular Substances; D008358:Mannose / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
chemical_list: D029742:Caenorhabditis elegans Proteins; D006023:Glycoproteins; D037102:Lectins; D046911:Macromolecular Substances; D010455:Peptides; D011134:Polysaccharides; D008358:Mannose