Protocol

Glycobiology Protocols

Volume 347 of the series Methods in Molecular Biology pp 57-68

Methods for Analysis of Unusual Forms of O-Glycosylation

  • Aleksandra Nita-LazarAffiliated withDepartment of Biochemistry and Cell Biology, State University of New York at Stony Brook
  • , Robert S. HaltiwangerAffiliated withDepartment of Biochemistry and Cell Biology, State University of New York at Stony Brook

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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.