Screening for N-glycosylated proteins by liquid chromatography mass spectrometry

Proteomics. 2004 Feb;4(2):454-65. doi: 10.1002/pmic.200300556.


In the last few years mass spectrometry has become the method of choice for characterization of post-translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N-glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys-C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line reversed-phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified.

MeSH terms

  • Amino Acid Sequence
  • Blood Proteins / analysis*
  • Blood Proteins / chemistry
  • Chromatography, Liquid
  • Concanavalin A / chemistry*
  • Glycoproteins / analysis*
  • Glycoproteins / blood
  • Glycoproteins / chemistry
  • Glycosylation
  • Humans
  • Mass Spectrometry
  • Molecular Sequence Data
  • Oligosaccharides / analysis*
  • Oligosaccharides / blood
  • Oligosaccharides / chemistry
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / metabolism
  • Wheat Germ Agglutinins / chemistry*


  • Blood Proteins
  • Glycoproteins
  • Oligosaccharides
  • Wheat Germ Agglutinins
  • Concanavalin A
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase