Summary
The analysis of the N-glycan portions of glycoproteins has become important for the detection of various diseases involving altered glycosylation patterns, including cancer and congenital disorders of glycosylation, and in the quality control of therapeutic recombinant glycoproteins. Here in this chapter we give detailed analysis procedures necessary for the complete structural elucidation of any N-glycan mixture found in naturally occurring and recombinant glycoproteins. The protocols include monosaccharide composition analysis on the intact glycoprotein, the enzymatic release of N-glycans from glycoprotein and their separation from O-glycopeptides and peptides, and the structural characterization of the glycans by exoglycosidase digestions, methylation (“linkage”) analysis, and mass spectrometry/tandem mass spectrometry of permethylated glycans using matrix-assisted laser desorption and electrospray ionization methods.
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References
Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G. & Marth, J., eds. (1999) Essentials of Glycobiology (Cold Spring Harbor Laboratory Press, New York, NY).
Freeze, H. H. & Aebi, M. (2005) Altered glycan structures: the molecular basis of congenital disorders of glycosylation.Current Opinion in Structural Biology 15, 490–498.
Ohtsubo, K. & Marth, J. D. (2006). Glycosylation in Cellular Mechanisms of Health and Disease. Cell 126, 855–867.
Sethuramana, N. & Stadheim, T. A. (2006) Challenges in therapeutic glycoprotein production. Current Opinion in Biotechnology 17, 341–346.
Dell, A. & Morris, H. R. (2001) Glycoprotein Structure Determination by Mass Spectrometry. Science 291, 2351–2356
Medzihradszky, K. F. (2005) Characterization of Protein N-Glycosylation. Methods in Enzymology 405, 116–138
Maley, F., Trimble, R. B., Tarentino, A. L. Thomas, H. & Plummer, J. (1989) Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Analytical Biochemistry 180, 195–204
Kobata, A. (1979) Use of endo- and exoglycosidases for structural studies of glycoconjugates. Analytical Biochemistry 100, 1–14
Prime, S., Dearnley, J., Ventom, A. M., Parekh, R. B. & Edge, C. J. (1996) Oligosaccharide sequencing based on exo- and endoglycosidase digestion and liquid chromatographic analysis of the products. Journal of Chromatography A. 720, 263–274
York, W. S., Darvill, A. G., McNeil, M., Stevenson, T. T. & Albersheim, P. (1985) Isolation and characterization of plant cell walls and cell wall components. Methods in Enzymology 118, 3–40
Anumula, K. R. & Taylor, P. B. (1992) A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates. Anal Biochem 203, 101–108
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Azadi, P., Heiss, C. (2009). Mass Spectrometry of N-Linked Glycans. 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_3
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DOI: https://doi.org/10.1007/978-1-59745-022-5_3
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