# Sensitive Fluorescence Quantitation and Efficient Free Radical Characterization of N-Glycans via LC-FLR-HRMS/MS with a Novel Fluorescent Free Radical Tag

## TL;DR

A new fluorescent tag improves glycan analysis by enabling sensitive detection and detailed characterization of complex sugar structures.

## Contribution

A novel fluorescent free radical tag was developed to enhance glycan quantitation and structural analysis.

## Key findings

- The new fluorescent tag provides high sensitivity in liquid chromatography detection.
- The tag enables detailed characterization of branched and isobaric N-glycans.
- The method was successfully tested on standard and biologically relevant glycans.

## Abstract

Glycans are some
of the most difficult biomolecules to analyze
owing to their branching tendencies as well as their regiochemical
and stereochemical diversity. Yet, the correlation between various
pathological states and glycan quantity or structural alterations
has demonstrated the importance and urgency for the development of
a more robust glycan analytical technique. Furthermore, the manufacturing
and regulation of biopharmaceuticals demands a feasible and improved
analytical approach toward the characterization and quantitation of
glycosylations. Unfortunately, multiple commercially available glycan
tags lack, in combination, liquid chromatography detection sensitivity,
chemical stability and, most importantly, optimal glycan characterization
capabilities. Therefore, a novel fluorescent tag coupled with a free
radical approach for glycan characterization was designed and developed
to help address this gap in glycan analysis. The analytical capabilities
of this novel tag were assessed via hydrophilic liquid chromatography–fluorescence
quantitation and ESI/MS free radical-mediated characterization by
using linear glycan standards, branched isobaric glycans lacto-N-difucohexaose I and lacto-N-difucohexaose
II, and N-glycans released from ribonuclease B.