Abstract
In contrast to the linear sequences of protein and DNA, oligosaccharides are branched structures. In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants. Glycan analysis therefore requires high-resolution separation techniques that can provide detailed structural analysis, including both monosaccharide sequence and linkage information. This chapter describes how a combination of high-performance liquid chromatography (HPLC) and exoglycosidase enzyme array digestions can deliver quantitative glycan analysis of sugars released from glycoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel bands by matching HPLC elution positions with a database of standard glycans.
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References
Parekh, R. B., Dwek, R. A., Sutton, B. J., et al. (1985) Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 316, 452–457.
Block, T. M., Comunale, M. A., Lowman, M., et al. (2005) Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans. Proc. Natl. Acad. Sci. USA 102, 779–784.
Butler, M., Quelhas, D., Critchley, A. J., et al. (2003) Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis. Glycobiology 13, 601–622.
Kuhn, P., Tarentino, A. L., Plummer, T. H., Jr., and Van Roey, P. (1994) Crystal structure of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F at 2.2-A resolution. Biochemistry 33, 11,699–11,706.
Navazio, L., Miuzzo, M., Royle, L., et al. (2002) Monitoring endoplasmic reticulumto-Golgi traffic of a plant calreticulin by protein glycosylation analysis. Biochemistry 41, 14,141–14,149.
Tekoah, Y., Ko, K., Koprowski, H., et al. (2004) Controlled glycosylation of therapeutic antibodies in plants. Arch. Biochem. Biophys. 426, 266–278.
Wormald, M. R., Rudd, P. M., Harvey, D. J., Chang, S. C., Scragg, I. G., and Dwek, R. A. (1997) Variations in oligosaccharide-protein interactions in immunoglobulin G determine the site-specific glycosylation profiles and modulate the dynamic motion of the Fc oligosaccharides. Biochemistry 36, 1370–1380.
Takahashi, N., Ishii, I., Ishihara, H., et al. (1987) Comparative structural study of the N-linked oligosaccharides of human normal and pathological immunoglobulin G. Biochemistry 26, 1137–1144.
Homans, S. W., Ferguson, M. A., Dwek, R. A., Rademacher, T. W., Anand, R., and Williams, A. F. (1988) Complete structure of the glycosyl phosphatidylinositol membrane anchor of rat brain Thy-1 glycoprotein. Nature 333, 269–272.
Parekh, R. B., Dwek, R. A., Rudd, P. M., et al. (1989) N-glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line. Biochemistry 28, 7670–7679.
Küster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A., and Harvey, D. J. (1997) Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography. Anal. Biochem. 250, 82–101.
Radcliffe, C. M., Diedrich, G., Harvey, D. J., Dwek, R. A., Cresswell, P., and Rudd, P. M. (2002) Identification of specific glycoforms of major histocompatibility complex class I heavy chains suggests that class I peptide loading is an adaptation of the quality control pathway involving calreticulin and ERp57. J. Biol. Chem. 277, 46,415–46,423.
Guile, G. R., Rudd, P. M., Wing, D. R., Prime, S. B., and Dwek, R. A. (1996) A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem. 240, 210–226.
Royle, L., Roos, A., Harvey, D. J., et al. (2003) Secretory IgA N-and O-glycans provide a link between the innate and adaptive immune systems. J. Biol. Chem. 278, 20,140–20,153.
Zamze, S., Harvey, D. J., Chen, Y. J., Guile, G. R., Dwek, R. A., and Wing, D. R. (1998) Sialylated N-glycans in adult rat brain tissue—a widespread distribution of disialylated antennae in complex and hybrid structures. Eur. J. Biochem. 258, 243–270.
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Royle, L., Radcliffe, C.M., Dwek, R.A., Rudd, P.M. (2006). Detailed Structural Analysis of N-Glycans Released From Glycoproteins in SDS-PAGE Gel Bands Using HPLC Combined With Exoglycosidase Array Digestions. In: Brockhausen, I. (eds) Glycobiology Protocols. Methods in Molecular Biology, vol 347. Humana Press. https://doi.org/10.1385/1-59745-167-3:125
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DOI: https://doi.org/10.1385/1-59745-167-3:125
Publisher Name: Humana Press
Print ISBN: 978-1-58829-553-8
Online ISBN: 978-1-59745-167-3
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