This chapter presents methods to analyze proteins that are potentially N-glycosylated or O-mannozylated. The type and extent of glycosylation can be assessed by perturbing glycosylation in various ways that affect the apparent size of the protein. For example, apparent protein size can be decreased by treatment of yeast cells with the antibiotic tunicamycin, which inhibits N-glycosylation, or by treatment of the protein with enzymes such as endogtycosidase H (Endo- H), which specifically cleaves N-linked carbohydrate chains. The carbohydrate moieties of glycoproteins can also be analyzed. One approach involves labeling yeast glycoproteins with radioactive sugars. The labeled oligosaccharides can then be cleaved from the protein and analyzed by chromatography. These techniques are illustrated using invertase and chitinase as model glycoproteins in the chapter. Analysis of chitinase suggested the presence of O-linked mannose, and this possibility is supported by the fact that the protein could be radiolabeled with [2-3H]mannose.
ASJC Scopus subject areas
- Molecular Biology