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Small-Scale Enzymatic Digestion of Glycoproteins and Proteoglycans for Analysis of Oligosaccharides by LC-MS and FACE Gel Electrophoresis

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Glycomics

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 534))

Summary

Structural characterization of oligosaccharides from proteoglycans and other glycoproteins is greatly enhanced through the use of mass spectrometry and gel electrophoresis. Sample preparation for these sensitive techniques often requires enzymatic treatments to produce oligosaccharide sequences for subsequent analysis. This chapter describes several small-scale methods for in-gel, on-blot, and in-solution enzymatic digestions in preparation for graphitized carbon liquid chromatography-mass spectrometry (LC-MS) analysis, with specific applications indicated for glycosaminoglycans (GAGs) and N-linked oligosaccharides. In addition, accompanying procedures for oligosaccharide reduction by sodium borohydride, sample desalting via carbon microcolumn, desialylation by sialidase enzyme treatment, and small-scale oligosaccharide species fractionation are included. Fluorophore-assisted carbohydrate electrophoresis (FACE) is another useful method to isolate derivatized oligosaccharides. Overall, the modularity of these techniques provides ease and flexibility for use in conjunction with mass spectrometric and electrophoretic tools for glycomic research studies.

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Acknowledgments

We thank Dr Neil Davies for the preparation of the FACE gel used in Fig. 2.

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Authors and Affiliations

  1. Graduate School of Biomedical Engineering, The University of New South Wales, Sydney, NSW, Australia

    Ruby P. Estrella

Authors
  1. Ruby P. Estrella
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  2. John M. Whitelock
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  3. Rebecca H. Roubin
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  4. Nicolle H. Packer
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  5. Niclas G. Karlsson
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Corresponding author

Correspondence to Ruby P. Estrella .

Editor information

Editors and Affiliations

  1. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

    Nicolle H. Packer

  2. Centre for Bioanalytical Science, Chemistry Department, NUI Galway, Galway, Ireland

    Niclas G. Karlsson

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Estrella, R.P., Whitelock, J.M., Roubin, R.H., Packer, N.H., Karlsson, N.G. (2009). Small-Scale Enzymatic Digestion of Glycoproteins and Proteoglycans for Analysis of Oligosaccharides by LC-MS and FACE Gel Electrophoresis. In: Packer, N.H., Karlsson, N.G. (eds) Glycomics. Methods in Molecular Biology™, vol 534. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-022-5_13

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  • DOI: https://doi.org/10.1007/978-1-59745-022-5_13

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-774-7

  • Online ISBN: 978-1-59745-022-5

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