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MALDI-TOF Mass Spectrometry

MALDI-TOF Mass Spectrometry

Mass spectrometry is a powerful characterization technique that can identify and separate a wide variety of compounds ranging from chemical to biological entities, based on their mass-to-charge ratio (m/z). The instruments that allow this detection, known as mass spectrometers, have three components: an ion source, a mass analyzer, and a detector. These spectrometers differ based on the nature of their ion source and analyzers.Matrix-assisted laser desorption ionization (MALDI) is a commonly...

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Updated: Mar 17, 2026

Profiling of Permethylated Mucin O-glycans Using Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

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Published on: June 20, 2025

Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS.

Archana Shubhakar^1,2, Radoslaw P Kozak¹, Karli R Reiding³

¹Ludger Ltd. , Culham Science Centre, Abingdon, Oxfordshire, United Kingdom.

Analytical Chemistry

|August 2, 2016

Summary

A new automated kit enables high-throughput glycan permethylation for glycoprotein therapeutics. This method simplifies characterization and quality control, improving drug development efficiency.

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Area of Science:

Biopharmaceutical Analysis
Glycobiology
Analytical Chemistry

Background:

Glycosylation critically impacts glycoprotein therapeutic properties like stability and immunogenicity.
Quality by Design (QbD) frameworks are essential for controlling drug glycosylation.
Current manual glycan analysis methods are time-consuming and not suitable for high-throughput studies.

Purpose of the Study:

To develop and validate a high-throughput (HT) glycan permethylation kit for analyzing glycoprotein therapeutics.
To automate the workflow for glycan release, enrichment, permethylation, and extraction.
To enable efficient characterization and quality control of biopharmaceuticals.

Main Methods:

Development of a 96-well microplate-based glycan permethylation protocol.
Automation of the workflow using a liquid handling robot for N-glycan release, enrichment, permethylation, and liquid-liquid extraction (LLE).
Analysis of permethylated glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

Main Results:

The HT kit was validated according to industry analytical performance guidelines.
Automated sample preparation for 96 glycan samples was completed in under 5 hours.
MALDI-TOF-MS profiles showed good agreement with hydrophilic interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data, with minimal side reactions.
Automated data acquisition on MALDI-TOF-MS averaged less than 1 minute per sample.

Conclusions:

The developed automated HT glycan permethylation kit is convenient, fast, and reliable.
This method facilitates efficient glycan characterization and relative quantitation of glycoprotein therapeutics.
The kit is applicable for drug glycan profiling and clinical glycan biomarker studies.