Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins

Nat Biotechnol. 2003 Jun;21(6):667-72. doi: 10.1038/nbt829. Epub 2003 May 18.


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 types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Caenorhabditis elegans Proteins / analysis
  • Chromatography, High Pressure Liquid / methods*
  • Conserved Sequence
  • Glycoproteins / analysis*
  • Glycoproteins / chemistry
  • Glycoproteins / isolation & purification
  • Glycoproteins / metabolism
  • Isotope Labeling / methods*
  • Lectins / chemistry
  • Macromolecular Substances
  • Mannose / analysis
  • Mannose / chemistry
  • Mass Spectrometry / methods*
  • Peptides / analysis
  • Peptides / chemistry
  • Peptides / metabolism
  • Polysaccharides / analysis
  • Polysaccharides / chemistry
  • Polysaccharides / metabolism
  • Sequence Alignment
  • Sequence Analysis, Protein


  • Caenorhabditis elegans Proteins
  • Glycoproteins
  • Lectins
  • Macromolecular Substances
  • Peptides
  • Polysaccharides
  • Mannose