Rapid release of N-linked glycans from glycoproteins by pressure-cycling technology

Anal Chem. 2010 Mar 15;82(6):2588-93. doi: 10.1021/ac100098e.

Abstract

The standard, well-established sample preparation protocol to release N-linked glycans from glycoproteins for downstream analysis requires relatively long deglycosylation times (from several hours to overnight) and relatively high endoglycosidase concentration (from 1:250 to 1:500 enzyme:substrate molar ratio). In this paper, we significantly improve this standard protocol by the use of pressure-cycling technology (PCT) to increase the speed and decrease the relative amount of PNGase F during the release of N-linked glycans from denatured glycoproteins. With the application of pressure cycling from atmospheric to as high as 30 kpsi, >95% release of the asparagine-linked glycans from bovine ribonuclease B, human transferrin, and polyclonal human immunoglobulin was rapidly achieved in a few minutes using as low as 1:2500 enzyme:substrate molar ratio. The deglycosylation rate was first examined by SDS-PAGE at the protein level. The released glycans were then quantitated by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). This new sample preparation protocol readily supports large-scale glycan analysis of biopharmaceuticals with rapid deglycosylation times.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cattle
  • Glycoproteins / metabolism*
  • Glycosylation
  • Humans
  • Immunoglobulin G / metabolism
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / metabolism*
  • Polysaccharides / metabolism*
  • Pressure*

Substances

  • Glycoproteins
  • Immunoglobulin G
  • Polysaccharides
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase