Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

Anal Biochem. 1999 May 1;269(2):297-303. doi: 10.1006/abio.1999.4026.


High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carbohydrate Sequence
  • Carbohydrates / analysis*
  • Carbohydrates / chemistry
  • Cattle
  • Chromatography, High Pressure Liquid / methods*
  • Glycoproteins / chemistry*
  • Mass Spectrometry / methods*
  • Molecular Sequence Data
  • Oligosaccharides / analysis
  • Oligosaccharides / chemistry
  • Ribonucleases / chemistry
  • Sugar Alcohols / analysis


  • Carbohydrates
  • Glycoproteins
  • Oligosaccharides
  • Sugar Alcohols
  • Ribonucleases
  • ribonuclease B