Summary
The structural diversity of mucin-type O-glycans (O-GalNAc core-based) exceeds that of N-linked glycans by far. Structural analysis of this type of protein modification is hampered, however, by the unavailability of specific endo-acetylgalactosaminidases that would cleave the intact oligosaccharide from threonine or serine residues. Chemical cleavage is either performed by hydrazinolysis resulting in reducing glycans ready for terminal labeling reactions or by the classical reductive β-elimination, which yields the chemically inert alditols. Composition and sequence of the O-glycans can be analyzed by mass spectrometry using FAB-, MALDI-, or ESI-ionization technology. A significant increase in sensitivity and a facilitated fragmentation of the sugar chains can be achieved by methylation of free hydroxyl groups according to a modified procedure based on NaOH/DMSO and methyl iodide. Permethylated glycan alditols are readily separated on reversed-phase capillary columns under conditions similar to those used in peptide chromatography, and their sequences can be deduced from MS/MS spectra registered after collision-induced fragmentation of parental proton or sodium adduct ions using a Q-TOF mass spectrometer.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hanisch, F.-G. (2001) O-Glycosylation of the mucin-type. Biol. Chem. 382, 143–149
Iyer, R.N., Carlson, D.M. (1971) Alkaline borohydride degradation of blood group H substance. Arch. Biochem. Biophys. 142, 101–105
Lamblin, G., Boersma, A., Lhermitte, M., Roussel, P., Mutsaers, J.H., van Halbeek, H., Vliegenthardt, J.F. (1984) Further characterization, by combined high-performance liquid chromatography/1H-NMR approach, of the heterogeneity displayed by the neutral carbohydrate chains of human bronchial mucins. Eur. J. Biochem. 143, 227–236
Klein, A., Lamblin, G., Lhermitte, M., Roussel, P., Breg, J., van Halbeek, H., Vliegenthardt, J.F.G. (1988) Primary structure of neutral oligosaccharides derived from respiratory-mucus glycoproteins of a patient suffering from bronchiectasis, determined by combination of 500-MHz 1H-NMR spectroscopy and quantitative sugar analysis. Eur. J. Biochem. 171, 631–642
Hounsell, E.F., Lawson, A.M., Feeney, J., Gooi, H.C., Pickering, N.J., Stoll, M.S., Lui, S.C., Feizi, T. (1985) Structural analysis of the O-glycosidically linked coreregion oligosaccharides of human meconium glycoproteins which express oncofetal antigens. Eur. J. Biochem. 148, 367–377
Hanisch, F.-G., Uhlenbruck, G., Peter-Katalinic, J.,Egge, H., Dabrowski, J., Dabrowski, U. (1989) Structures of neutral O-linked polylactosaminoglycans on human skim milk mucins. J. Biol. Chem. 264, 872–883
Morelle, W., Faid, V., Michalski, J.-C. (2004) Structural analysis of permethylated oligosaccharides using electrospray ionization quadrupole time-of-flight tandem mass spectrometry and deutero-reduction. Rapid Commun. Mass Spectrom. 18, 2451–2464
Morelle, W., Slomianny, M.-C., Diemer, H., Schaeffer, C., Dorsselaer, A., Michalski, J.-C. (2004) Fragmentation characteristics of permethylated oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight (TOF/TOF) tandem mass spectrometer. Rapid Commun. Mass Spectrom. 18, 2637–2649
McNeil, M., Darvill, A.G., Aman, P., Franzen, L.-E., Albersheim, P. (1982) Structural analysis of complex carbohydrates using high-performance liquid chromatography, gas chromatography, and mass speectrometry. Meth. Enzymol. 83, 3–45
Dell, A. (1990) Preparation and desorption mass spectrometry of permethyl and peracetyl derivatives of oligosaccharides. Meth. Enzymol. 193, 647–660
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
Ciucanu, I., Kerek, F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res. 131, 209–217
Needs, P.W., Selvendran, R.R. (1993) Avoiding oxidative degradation during sodium hydroxide/methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide. Carbohydr. Res. 245, 1–10
Ciucanu, I., Costello, C.E. (2003) Elimination of oxidative degradation during the per-O-methylation of carbohydrazes. J. Am. Chem. Soc. 125, 16213–16219
Schulz, B.L., Packer, N.H., Karlsson, N.G. (2002) Small-scale analysis of O-linked oligosaccharides from glycoproteins and mucins separated by gel electrophoresis. Anal. Chem. 74, 6088–6097
Engelmann, K., Kinlough, C.L., Müller, S., Razawi, H., Baldus, S.E., Hughey, R.P., Hanisch, F.-G. (2005) Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles. Glycobiology 15, 1111–1124
Domon, B., Costello, C.E. (1988) A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J. 5, 397–409
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hanisch, FG., Müller, S. (2009). Analysis of Methylated O-Glycan Alditols by Reversed-Phase NanoLC Coupled CAD-ESI Mass Spectrometry. 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_8
Download citation
DOI: https://doi.org/10.1007/978-1-59745-022-5_8
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-774-7
Online ISBN: 978-1-59745-022-5
eBook Packages: Springer Protocols