There is international interest among glycobiologists to find a method for the isolation of intact unreduced O-linked oligosaccharides from glycoprotein. They can be used as biomarkers for diseases and with the reactive reducing end can be further modified for analysis or attachment to other proteins for use as immunostimulants. We have found such a method. We are able to obtain mass spectra for the non-reductive � eliminated molecule from bovine thyroglobulin and mass spectra for the reductively � eliminated O-linked oligosaccharide alditol from bovine thyroglobulin. We can support a structure for each that is the complete molecule. The latter of the two produces a molecule with hydride insertion reductions of two sulfate substitutions. In the spectra of the non-reduced O-linked oligosaccharides of bovine thyroglobulin we can support structures that have these substitutions. All other mass spectral comparisons of the � eliminated compounds offer support for the integrity of the structure of the non-reductively �-eliminated O-linked oligosaccharide. We also do graded hydrolysis and HPAEC-PAD for both the O-linked oligosaccharide and the O-linked oligosaccharide alditol. We are able to identify the reducing sugar in this way. With these methods we are able to partially identify the structure of the O-linked oligosaccharide of bovine thyroglobulin. The sugar composition is fucose, N-acetyl glucosamine and glucose. Glucose is the reducing sugar. We propose that the sulfate esters are substituted on the two primary alcohol positions of glucose and N-acetyl glucosamine. We found the most effective conditions for � elimination by allowing glucose to stand at ambient temperature for from 40 to 70 minutes at pH 11.4, sodium hydroxide. We monitored whether there were any mannose from isomerization formed in the reaction versus time. We found at the conditions, 50 minutes at ambient temperature and at pH 11.4, sodium hydroxide, that there was no mannose formed

Simple method for the non-reductive β elimination of o-linked oligosaccharide from glycoprotein

245th National Meeting (2013)

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