Protein Protocols Protein Protocols

830 Hounsell, Davies, and Smith
6. The resulting oligosaccharide containing fractions are then derivatized for LSIMS and
GC-MS or analyzed by NMR.
4. Notes
1. Reverse-phase or normal-phase HPLC may also be required for the complete separation
of some oligosaccharide isomers.
2. To prevent anomerization, the oligosaccharides can be reduced to their alditols (either
after PNGase F digestion or hydrazinolysis). The inclusion of a 3 H-label on reduction will
give increased sensitivity over UV. Alternatively, the oligosaccharides can be fluorescently
labeled at their reducing terminus (with 2-amino-benzamide or 2-amino-pyridine)
by reductive amination to give a fluorescent chromophore and increased sensitivity. Sensitivity
of detection may also be increased by the postcolumn addition of 300 mM NaOH
and pulsed amperometric detection as described for HPAEC-PAD.
References
1. Hase, S. (1993) Analysis of sugar chains by pyridylamination, in Methods in Molecular
Biology, vol. 14: Glycoprotein Analysis in Biomedicine (Hounsell, E. F., ed.), Humana,
Totowa, NJ, pp. 69–80.
2. Kakehi, K. and Honda, S. (1993) Analysis of carbohydrates in glycoproteins by high performance
liquid chromatography and high performance capillary electrophoresis, in Methods
in Molecular Biology, vol. 14: Glycoprotein Analysis in Biomedicine (Hounsell, E. F.,
ed.), Humana, Totowa, NJ, pp. 81–98.
3. Smith, D. D., Davies, M. J., Hounsell, E. F. (1994) Structural profing of oligosaccharides
of glycoproteins, in Methods in Molecular Biology, vol. 32: Basic Protein and Peptide
Protocols (Walker, J. M., ed.), Humana, Totowa, NJ, pp. 143–155.
4. Davies, M. J. and Hounsell, E. F. (1995) Comparison of separation modes for high performance
liquid chromatography of glycoprotein- and proteoglycan-derived oligosaccharides.
J. Chromatogr. 720, 227–234.