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Analysis of Carbohydrates on Proteins by Offline Normal-Phase Liquid Chromatography MALDI-TOF/TOF-MS/MS

  • Theodora Tryfona
  • Elaine Stephens
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 658)

Abstract

Glycoproteins typically exist as a diverse population of glycoforms, which can consist of a great number of different glycan structures (including structural isomers) that vary in their degree of occupancy at given glycosylation sites. Hence, the detailed characterization of such glycans can be a very demanding task. Liquid chromatography in combination with mass spectrometric techniques can provide a fast and sensitive tool for the analysis of these structurally complex molecules. Here we describe a sensitive method that employs capillary normal-phase HPLC coupled offline to MALDI-TOF/TOF-MS/MS for the detailed characterization of enzymatically released N-glycans. The normal-phase chromatography allows the separation of some isobaric glycan structures and analysis of the glycans by MALDI high-energy CID gives sequence, linkage and branching information.

Key words

Protein glycosylation N-glycans MALDI-TOF-MS MALDI-CID normal-phase chromatography 

Notes

Acknowledgements

This work was funded by the Food Standards Agency (for TT).

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Theodora Tryfona
    • 1
  • Elaine Stephens
    • 2
  1. 1.Department of BiochemistryUniversity of CambridgeCambridgeUK
  2. 2.MRC Laboratory of Molecular BiologyCambridgeUK

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