Presentation

LudgerTag Permethylation of Glycans

Analysis of glycans by MALDI-MS provides mass composition data and is faster than UHPLC techniques (on average one sample can take as little as 1 minute for data acquisition), but sialylated glycans are usually more difficult to analyse because the sialic acid moieties are labile. Permethylation is a popular technique for the derivatisation of carbohydrates for MALDI-MS detection, as it enhances ionization efficiency, stabilizes the sialic acids and aids linkage analysis studies. However, conventional techniques used for permethylation are slow, labour intensive and therefore not suited to high throughput sample analysis.

We have developed a simple to use LudgerTag kit for permethylating glycans, LT-PERMET-VP96. The kit can be used to process 1-96 samples using either a manual method or an automated method that has been adapted to a liquid handling robot. It is suitable for N- or O-glycans and once permethylated, they can be analysed by MALDI-TOF-MS or LC-MS.

We have validated this technology and demonstrated that glycoprofiling data is reliable and comparable to the gold standard HILIC UHPLC method. This technology has been used for a range of high throughput studies at Ludger (and using the automated permethylation sample preparation time is under 5.5 hours for 96 samples).

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Principles - Quick Links:

1. Introduction: Why Permethylate?
2. Ludger Glycan Permethylation Kit
3. Manual/Automated Workflows
4. Examples of how we're using LT-PERMET-96 at Ludger
5. How to start using the Ludger Permethylation Kit

• Downloadable pdf version of presentation

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Data - Quick Links:

1. Overview of a two stage strategy for HT glycoprofiling studies
2. Stage 1: detailed glycan characterisation
3. Stage 2: permethylation and MALDI-TOF-MS
4. Analysis of O glycans
5. QbD studies
6. How to start using the Ludger Permethylation Kit

• Downloadable pdf version of presentation

Posters & Publications

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

Shubhakar A, Kozak RP, Reiding KR, Royle L, Spencer DI, Fernandes DL, Wuhrer M.
Analytical Chemistry. 2016 Sep 6. 88(17):8562-9. doi: 10.1021/acs.analchem.6b01639. Epub 2016 Aug 15.

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

Shubhakar A, Spencer D, Wuhrer M, Fernandes DL
Presented at the 38th Annual Congress of Pharmacology Society of Chile
Castro, Chile. Nov 26-29th 2016

Reliability of a semi-automated, high throughput (HT) MS based glycomics system for discovery of glycan biomarkers and Quality by Design (QbD) studies of glycoprotein therapeutics

Shubhakar A, Spencer DI, Wuhrer M, Fernandes DL
Presented at Gordon Research Conference
Lucca, Italy. March 2015

Drug Glycosylation Analysis using Automated, High Throughput, Orthogonal Methods

Shubhakar A, Kozak RP, Badia-Tortosa C, Gardner RA, Royle L, Wuhrer M, Spencer DI, Fernandes DL
Presented at PEGS 2014: Glycosylation Analysis for Biopharmaceuticals Workshop
Boston, United States. May 2014

Optimisation of drug glycosylation supported by an automated high throughput permethylation MALDI-MS method

Shubhakar A, Spencer DI, Kozak RP, Velez-Suberbie L, Bracewell DG, Fernandes DL, Wuhrer M
Presented at ICS: 27th International Carbohydrate Symposium
Bangalore, India. January 2014

Contacts

Technical Questions

Archana Shubhakar
Business Development Lead
archana.shubhakar@ludger.com

Quotations and Orders

Sales Team
Quotations: info@ludger.com
Orders: sales@ludger.com

View product documentation

Product guide

Product specifications

Application: This kit is used for the permethylation of glycans. Permethylation stabilizes glycan sialic acids for MALDI mass spectrometry analysis and aids linkage analysis studies. The kit can be used with glycans released from glycoproteins.

Description: This kit contains reagents for the permethylation of glycans.Note: This kit does not include methyl iodide / Iodomethane (see shipping section for details)

Number of Samples: Sufficient for up to 96 samples.

Amount of Sample: Up to 1 μg of released glycans.

Suitable Samples: Any unlabelled purified glycans, released from glycoprotein by either PNGaseF, PNGase A, beta-elimination or hydrazinolysis.

Storage Store at 2-8°C.

Shipping: The product should be shipped between 2-8°C. Note: Due to shipping restrictions of methyl iodide/iodomethane, we cannot provide this component with the kit. Therefore, we recommend purchasing iodomethane with purity ≥99.0% (GC) from your local chemical provider. Iodomethane Synonym: methyl iodide CAS Number: 74-88-4

Safety: For research use only. Not for human or drug use. Please read the Safety Data Sheet (SDS) for all chemicals used. All processes involving permethylation reagents should be performed using appropriate personal safety protection – eyeglasses, chemically resistant gloves and where appropriate in a laboratory fume cupboard.

Kit Contents

The kit contains the following items: