Abstract
N-linked oligosaccharides are complex non-template-derived structures that are attached to the side chains of asparagine, via the nitrogen atom. Specific changes in the N-glycans of serum glycoproteins have been associated with the pathogenesis of many diseases. The oligosaccharides present on the CH2 domain of immunoglobulins are known to modulate the effector functions of the molecule. These glycans provoke various biological effects, necessitating the development of robust high-throughput technology in order to fully characterize the N-glycosylation profile. This chapter describes in detail four methods to release N-glycans from the glycoprotein of interest. Two of these protocols, referred to as the “In-Gel Block” and “1D sodium dodecyl sulfate-polyacrylamide gel electrophoresis” methods, require immobilization of the glycoprotein prior to analysis. An automated method is also described, involving the purification of immunoglobulins directly from fermentation media, and, finally, an “In-solution method” is detailed, which directly releases the N-glycans into solution. HILIC and WAX-HPLC are used to analyze the N-glycan profile. Exoglycosidase enzymes digestion arrays, in combination with computer-assisted data analysis, are used to determine both the sequence and linkage of the N-glycans present.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Varki A, Cummings RD, Esko JD, Freeze H, Hart G, Marth J (2009) Essentials of glycobiology. Cold Spring Harbour Laboratory, Cold Spring Harbour, NY
Alavi A, Axford JS (2008) Sweet and sour: the impact of sugars on disease. Rheumatology (Oxford) 47:760–770
Rodriguez J et al. (2010) High productivity of human recombinant beta-interferon from a low-temperature perfusion culture. J Biotechnol 150:509–518
Chung CH et al. (2008) Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. N Engl J Med 358:1109–1117
Pacis E et al. (2011) Effects of cell culture conditions on antibody N-linked glycosylation—what affects high mannose 5 glycoform. Biotechnol Bioeng 108(10):2348–2358
Wong NS et al. (2010) An investigation of intracellular glycosylation activities in CHO cells: effects of nucleotide sugar precursor feeding. Biotechnol Bioeng 107:321–336
Ferrara C et al. (2006) The carbohydrate at Fc gamma RIIIa Asn-162. An element required for high affinity binding to non-fucosylated IgG glycoforms. J Biol Chem 281:5032–5036
Royle L et al. (2006) Detailed structural analysis of N-glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions. Methods Mol Biol 347:125–143
Gabius H-J (2009) The sugar code: fundamentals of glycosciences. Blackwell, London
Royle L et al. (2008) HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software. Anal Biochem 376:1–12
Campbell MP et al. (2008) GlycoBase and autoGU: tools for HPLC-based glycan analysis. Bioinformatics 24:1214–1216
Marino K et al. (2010) A systematic approach to protein glycosylation analysis: a path through the maze. Nat Chem Biol 6:713–723
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Doherty, M., McManus, C.A., Duke, R., Rudd, P.M. (2012). High-Throughput Quantitative N-Glycan Analysis of Glycoproteins. In: Voynov, V., Caravella, J. (eds) Therapeutic Proteins. Methods in Molecular Biology, vol 899. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-921-1_19
Download citation
DOI: https://doi.org/10.1007/978-1-61779-921-1_19
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-920-4
Online ISBN: 978-1-61779-921-1
eBook Packages: Springer Protocols