Abstract
Enzymatic sequencing of oligosaccharides provides structural information on sequence of monosaccharides and type of linkage within the oligosaccharide chain. This data can be obtained by stepwise enzymatic digestion of a single, isolated oligosaccharide using individual or mixtures of specific exoglycosidases. N-glycans have to be fractionated from mixtures prior to sequence analysis to assign this type of structural information to a specific glycan. Enzymatic sequencing can as well be applied to oligosaccharide mixtures to evaluate the occurrence of distinct oligosaccharide motives of functional and/or structural interest.
Here we describe the application of enzymatic sequence analysis to a mixture of N-glycans released from α1-acid glycoprotein. The experimental conditions are optimized for detection of possible Lewis X structures after stepwise exoglycosidase digestion by MALDI-TOF mass spectrometry. However, the described method is generally applicable to analyze other structural properties of N-glycans using (respective) specific exoglycosidases.
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References
Edge CJ, Rademacher TW, Wormald MR, Parekh RB, Butters TD, Wing DR, Dwek RA (1992) Fast sequencing of oligosaccharides: the reagent-array analysis method. Proc Natl Acad Sci U S A 89:6338–6342
Frado LY, Strickler JE (2000) Structural characterization of oligosaccharides in recombinant soluble interferon receptor 2 using fluorophore-assisted carbohydrate electrophoresis. Electrophoresis 21:2296–2308
Gohlke M, Mach U, Nuck R, Zimmermann-Kordmann M, Grunow D, Flieger C, Volz B, Tauber R, Petri T, Debus N, Reutter W (2000) Carbohydrate structures of soluble human L-selectin recombinantly expressed in baby-hamster kidney cells. Biotechnol Appl Biochem 32:41–51
Kornfeld R, Kornfeld S (1985) Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 54:631–664
Snider MD (1984) Biosynthesis of glycoproteins: formation of N-linked oligosaccharides. In: Ginsburg V, Robbins PW (eds) Biology of carbohydrates, vol 2. John Wiley and Sons, New York, NY, pp 163–198
Lucka L, Fernando M, Grunow D, Kannicht C, Horst AK, Nollau P, Wagener C (2005) Identification of Lewis x structures of the cell adhesion molecule CEACAM1 from human granulocytes. Glycobiology 15:87–100
Bogoevska V, Nollau P, Lucka L, Grunow D, Klampe B, Uotila LM, Samsen A, Gahmberg CG, Wagener C (2007) DC-SIGN binds ICAM-3 isolated from peripheral human leukocytes through Lewis x residues. Glycobiology 17(3):324–333
Uchida Y, Tsukada Y, Sugimori T (1979) Enzymatic properties of neuraminidases from Arthrobacter ureafaciens. J Biochem 86:1573–1585
Kannicht C, Lucka L, Nuck R, Reutter W, Gohlke M (1999) N-Glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures. Glycobiology 9:897–906
Appel RD, Bairoch A, Hochstrasser DF (1994) A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server. Trends Biochem Sci 19:258–260
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Kannicht, C., Grunow, D., Lucka, L. (2019). Enzymatic Sequence Analysis of N-Glycans by Exoglycosidase Cleavage and Mass Spectrometry: Detection of Lewis X Structures. In: Kannicht, C. (eds) Post-Translational Modification of Proteins. Methods in Molecular Biology, vol 1934. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9055-9_5
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DOI: https://doi.org/10.1007/978-1-4939-9055-9_5
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