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Glycome mapping on DNA sequencing equipment.

Wouter Laroy¹, Roland Contreras, Nico Callewaert

¹Unit for Molecular Glycobiology, Department for Molecular Biomedical Research, Ghent University, and VIB, Technologiepark 927, B-9052 Gent-Zwijnaarde, Belgium. natprotcontact@dmbr.ugent.be

Nature Protocols

|April 5, 2007

Summary

This study presents a novel protocol for analyzing protein-linked glycans using DNA sequencing technology. The method offers high throughput, sensitivity, and resolves complex glycan structures, advancing glycomics research.

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Targeted DNA Methylation Analysis by Next-generation Sequencing

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Published on: February 24, 2015

Area of Science:

Analytical Chemistry
Biochemistry
Glycobiology

Background:

Glycan analysis is crucial for understanding biological processes.
Existing methods like mass spectrometry face challenges in resolving glycan stereoisomers.
High-throughput, sensitive, and quantitative glycan analysis is essential for glycomics.

Purpose of the Study:

To adapt and optimize a protocol for protein-linked glycan analysis using modern DNA sequencing equipment.
To provide a robust and high-throughput method for glycomics studies.
To enable the resolution of isobaric glycan stereoisomers, surpassing mass spectrometry limitations.

Main Methods:

Adaptation of a polyacrylamide gel-based glycan analysis technique to multicapillary DNA sequencers.
Integration of High-Performance Liquid Chromatography (HPLC) for fractionation of 8-amino-1,3,6-pyrenetrisulfonic acid (APTS)-labeled glycans.
Capillary electrophoretic profiling for rapid analysis of glycans.

Main Results:

The developed protocol demonstrates routine resolution of isobaric glycan stereoisomers.
The method achieves high throughput, high sensitivity, and reliable quantification.
An optional HPLC fractionation step allows for two-dimensional profiling and targeted mass spectrometry analysis.

Conclusions:

The protocol offers a powerful new tool for glycomics, leveraging DNA sequencing technology.
This approach overcomes limitations of traditional mass spectrometry for glycan stereoisomer resolution.
The method provides a robust platform for comprehensive glycan analysis and sequencing.