High-throughput quantitative analysis of plant N-glycan using a DNA sequencer

Biochem Biophys Res Commun. 2009 Mar 6;380(2):223-9. doi: 10.1016/j.bbrc.2009.01.070. Epub 2009 Jan 22.

Abstract

High-throughput quantitative analytical method for plant N-glycan has been developed. All steps, including peptide N-glycosidase (PNGase) A treatment, glycan preparation, and exoglycosidase digestion, were optimized for high-throughput applications using 96-well format procedures and automatic analysis on a DNA sequencer. The glycans of horseradish peroxidase with plant-specific core alpha(1,3)-fucose can be distinguished by the comparison of the glycan profiles obtained via PNGase A and F treatments. The peaks of the glycans with (91%) and without (1.2%) alpha(1,3)-fucose could be readily quantified and shown to harbor bisecting beta(1,2)-xylose via simultaneous treatment with alpha(1,3)-mannosidase and beta(1,2)-xylosidase. This optimized method was successfully applied to analyze N-glycans of plant-expressed recombinant antibody, which was engineered to contain a minor amount of glycan harboring beta(1,2)-xylose. These results indicate that our DNA sequencer-based method provides quantitative information for plant-specific N-glycan analysis in a high-throughput manner, which has not previously been achieved by glycan profiling based on mass spectrometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / biosynthesis
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal / genetics
  • Antibodies, Viral / biosynthesis
  • Antibodies, Viral / chemistry
  • Glycoside Hydrolases / chemistry
  • Horseradish Peroxidase / chemistry
  • Mass Spectrometry
  • Plants / chemistry*
  • Polysaccharides / chemistry*
  • Rabies / immunology
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / genetics
  • Sequence Analysis / instrumentation*
  • Sequence Analysis / methods*
  • Sequence Analysis, DNA / instrumentation
  • Tobacco / genetics
  • Tobacco / metabolism

Substances

  • Antibodies, Monoclonal
  • Antibodies, Viral
  • Polysaccharides
  • Recombinant Proteins
  • Horseradish Peroxidase
  • Glycoside Hydrolases