Ultrahigh throughput, ultrafiltration-based n-glycomics platform for ultraperformance liquid chromatography (ULTRA(3))

Anal Chem. 2015 Aug 18;87(16):8316-22. doi: 10.1021/acs.analchem.5b01463. Epub 2015 Jul 31.

Abstract

Accurate, reproducible, and fast quantification of N-glycans is crucial not only for the development and quality control of modern glycosylated biopharmaceuticals, but also in clinical biomarker discovery. Several methods exist for fluorescent labeling of N-glycans and subsequent chromatographic separation and quantification. However, the methods can be complex, lengthy, and expensive. Here we report an automated ultrafiltration-based N-glycoanalytical workflow combined with a glycan labeling strategy that is based on the reaction of glycosylamines with fluorescent carbamate. The entire protocol is quick, simple, and cost-effective and requires less than 1 μg of protein per sample. As many as 768 affinity purified IgG glycoprotein samples can be prepared in a single run with a liquid handling platform.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminoquinolines / chemistry*
  • Carbamates / chemistry*
  • Chemistry Techniques, Analytical / instrumentation*
  • Chemistry Techniques, Analytical / methods*
  • Chromatography, Liquid / instrumentation*
  • Fluorescent Dyes / chemistry
  • Glycomics*
  • Polysaccharides / chemistry
  • Ultrafiltration

Substances

  • 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
  • Aminoquinolines
  • Carbamates
  • Fluorescent Dyes
  • Polysaccharides