Carbohydrate release from picomole quantities of glycoprotein and characterisation of glycans by high-performance liquid chromatography and mass spectrometry

J Chromatogr B Biomed Sci Appl. 1999 Oct 29;734(1):169-74. doi: 10.1016/s0378-4347(99)00330-8.

Abstract

Samples of 5 to 20 microg of human IgG were subjected to dithiothreitol treatment to reduce disulphide bridges, followed by tryptic digestion. Glycans released from the tryptic peptide mixture by PNGase F digestion were then derivatised with 2-aminoacridone. Labelled oligosaccharides were separated by normal-phase high-performance liquid chromatography and individual components were collected for matrix-assisted laser desorption ionization time-of-flight and electrospray mass spectrometric analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidohydrolases / metabolism
  • Aminoacridines
  • Chromatography, High Pressure Liquid / methods*
  • Disulfides / metabolism
  • Dithiothreitol / pharmacology
  • Glycoproteins / metabolism*
  • Humans
  • Immunoglobulin G / chemistry
  • Immunoglobulin G / metabolism*
  • Microchemistry
  • Oligosaccharides / analysis
  • Oligosaccharides / isolation & purification
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Polysaccharides / chemistry
  • Polysaccharides / isolation & purification*
  • Polysaccharides / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
  • Trypsin / metabolism

Substances

  • Aminoacridines
  • Disulfides
  • Glycoproteins
  • Immunoglobulin G
  • Oligosaccharides
  • Polysaccharides
  • 2-aminoacridone
  • Trypsin
  • Amidohydrolases
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Dithiothreitol