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Methods for Analysis of Unusual Forms of O-Glycosylation

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Glycobiology Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 347))

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Abstract

The identification of the novel forms of O-linked glycosylation, O-fucose, and O-glucose requires the development of new methods for their analysis. Here we describe approaches to analyze these novel O-glycans. The major method involves metabolic radiolabeling of recombinant glycoproteins expressed in Lec1 Chinese hamster ovary (CHO) cells. The glycoproteins are purified from the media and the stoichiometry of modification is determined by comparing protein levels (by immunoblot) and incorporated radioactivity (by fluorography). The O-glycans are subsequently released by alkali-induced β-elimination, and released saccharides are analyzed using a combination of chromatography and exoglycosidase digestion. With these methods, we can determine both stoichiometry and the structure of the glycans on the expressed proteins. We have begun to utilize mass spectrometry in addition to metabolic radiolabeling methods to analyze these structures.

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Authors and Affiliations

  1. Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY

    Aleksandra Nita-Lazar PhD & Robert S. Haltiwanger PhD

Authors
  1. Aleksandra Nita-Lazar PhD
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  2. Robert S. Haltiwanger PhD
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Editor information

Editors and Affiliations

  1. Department of Medicine, Queen’s University, Kingston, Ontario, Canada

    Inka Brockhausen

  2. Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada

    Inka Brockhausen

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© 2006 Humana Press Inc.

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Nita-Lazar, A., Haltiwanger, R.S. (2006). Methods for Analysis of Unusual Forms of O-Glycosylation. In: Brockhausen, I. (eds) Glycobiology Protocols. Methods in Molecular Biology, vol 347. Humana Press. https://doi.org/10.1385/1-59745-167-3:57

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  • DOI: https://doi.org/10.1385/1-59745-167-3:57

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-553-8

  • Online ISBN: 978-1-59745-167-3

  • eBook Packages: Springer Protocols

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