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Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

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Glycan analysis via derivatization with a fluorogenic pyrylium dye.

Sine A Johannesen¹, Sophie R Beeren, Dennis Blank

¹Carlsberg Laboratory, Gamle Carlsberg Vej 10, Copenhagen V DK-1799, Denmark.

Carbohydrate Research

|March 20, 2012

Summary

A new method uses a fluorogenic pyrylium dye to label oligosaccharides for glycomics analysis. This technique simplifies carbohydrate characterization using capillary electrophoresis and mass spectrometry without excess dye removal.

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Glycan Node Analysis: A Bottom-up Approach to Glycomics

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Published on: May 22, 2016

Area of Science:

Glycomics
Analytical Chemistry
Biochemistry

Background:

Glycomics analysis requires robust carbohydrate characterization methods.
Developing simple derivatization protocols for sugars is crucial for analysis.
Existing methods often require complex steps like excess dye removal.

Purpose of the Study:

To develop a simplified labeling method for oligosaccharide mixtures.
To enable analysis of carbohydrates using capillary electrophoresis (CE) and MALDI-ToF-MS.
To introduce a fluorogenic pyrylium dye for enhanced detection.

Main Methods:

Oligosaccharide mixtures were labeled with a fluorogenic pyrylium dye (Py-1).
A two-step process involved reductive amination with ethylenediamine, followed by reaction with the pyrylium dye.
Analysis was performed using CE and MALDI-ToF-MS.

Main Results:

The pyrylium dye (Py-1) is non-fluorescent until conjugated, then fluoresces at 600-640 nm.
Excess dye removal was not necessary before analysis.
Good peak separation was observed in CE analysis of maltooligosaccharides.
MALDI-ToF-MS enabled sensitive detection of oligosaccharides up to 58 glucose units.

Conclusions:

This pyrylium dye labeling method offers a simplified approach to oligosaccharide characterization.
The method is compatible with both CE and MALDI-ToF-MS, enhancing glycomics analysis.
The protocol's efficiency and sensitivity facilitate the study of complex carbohydrate structures.