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- Title
Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins.
- Authors
Kaji, Hiroyuki; Saito, Haruna; Yamauchi, Yoshio; Shinkawa, Takashi; Taoka, Masato; Hirabayashi, Jun; Kasai, Ken-ichi; Takahashi, Nobuhiro; Isobe, Toshiaki
- 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.
- Publication
Nature biotechnology, 2003, Vol 21, Issue 6, p667
- ISSN
1087-0156
- Publication type
Journal Article
- DOI
10.1038/nbt829