Determination of N-glycosylation sites and site heterogeneity in glycoproteins

Anal Chem. 2003 Oct 15;75(20):5628-37. doi: 10.1021/ac034414x.


An approach for the characterization of glycosylation sites and oligosaccharide heterogeneity in glycoproteins based on a combination of nonspecific proteolysis, deglycosylation, and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FT MS) is described. Glycoproteins were digested with Pronase yielding primarily glycopeptides and amino acids. Nonglycosylated peptide fragments were susceptible to complete Pronase digestion to their constituent amino acids. Steric hindrance prohibited the digestion of the peptide moiety attached to the glycan. Glycopeptides were desalted and concentrated using solid-phase extraction and analyzed by MALDI MS. The oligosaccharides were also analyzed by MALDI MS after releasing the glycans from glycoproteins using PNGase F. The peptide moiety of the glycopeptides was identified by subtracting the masses of the glycans derived from PNGase F treatment from the masses of the glycopeptides. The experimental strategy was validated using glycoproteins with known oligosaccharide structures, ribonuclease B and chicken ovalbumin. This procedure was then used to determine the N-glycosylation sites and site heterogeneity of a glycoprotein whose glycosylation pattern was unknown, namely, the Xenopus laevis egg cortical granule lectin. This procedure is useful for determining protein site heterogeneity and structural heterogeneities of the oligosaccharide moiety of glycoproteins.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Carbohydrate Sequence
  • Chickens
  • Chromatography, High Pressure Liquid
  • Databases, Protein
  • Fourier Analysis
  • Glycoproteins / chemistry*
  • Glycosylation
  • Lectins / chemistry
  • Lectins / isolation & purification
  • Lectins / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Oligosaccharides / analysis
  • Oligosaccharides / chemistry
  • Oligosaccharides, Branched-Chain / analysis*
  • Oligosaccharides, Branched-Chain / chemistry
  • Ovalbumin / chemistry
  • Ovalbumin / metabolism
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / metabolism
  • Pronase / metabolism
  • Ribonucleases / chemistry
  • Ribonucleases / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods
  • Trypsin / metabolism
  • Xenopus Proteins / chemistry
  • Xenopus Proteins / isolation & purification
  • Xenopus Proteins / metabolism
  • Xenopus laevis


  • Glycoproteins
  • Lectins
  • Oligosaccharides
  • Oligosaccharides, Branched-Chain
  • XL35 protein, Xenopus
  • Xenopus Proteins
  • Ovalbumin
  • Ribonucleases
  • ribonuclease B
  • Trypsin
  • Pronase
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase