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Analysis of Ser/Thr-Linked Sugar Chains

  • Kalyan R. Anumula
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1934)

Abstract

Analysis of carbohydrate structures is an integral part of understanding the structure-function relationship of glycans as well as whole glycoproteins. Glycan profiling by HPLC with fluorescence detection is a powerful technique that sheds considerable light into understanding glycan structures. Profiling of N-linked glycans by HPLC and mass spectrometry is well established. However procedures for profiling Ser/Thr-linked sugar chains are still a challenge since there is no enzyme capable of releasing the intact glycans. Simplistic profiling of O-linked sugar chains is possible only by the virtue of anthranilic acid (AA, 2-aminobenzoic acid, 2-AA) labeling chemistry (Anumula, Anal Biochem 457:31–37, 2014), which eliminates the need for intermediary isolation steps, e.g., desalting and chromatographic purification, for labeling. O-linked sugar chains were released by hydrazinolysis at 60 °C for 6 h. Hydrazine was evaporated, and sugar chains were N-acetylated and derivatized with 2-AA in the same reaction mixture and separated on an Amide-80 column. Such simple hydrazinolysis protocols should benefit not only the biotechnology industry but also academic laboratories for characterization of glycoproteins. Detailed structure analysis is possible with AA-labeled glycans using mass spectrometry and NMR.

Key words

Monoclonal Antibodies MAbs Recombinant IgG N-linked Oligosaccharides Glycans HPLC Fluorescence Anthranilic acid Mapping 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kalyan R. Anumula
    • 1
  1. 1.Glycan TechnologiesNorth WalesUSA

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