Core-branching pattern and sequence analysis of mannitol-terminating oligosaccharides by neoglycolipid technology

The occurrence of mannitol-terminating oligosaccharides (2-substituted or 2 ,6-disubstituted) among the O-glycans released by alkaline borohydride trea tment from glycoproteins of the nervous system has prompted the development of a microscale method to analyze the core-branching pattern and sequence by the neoglycolipid (NGL) technology, analogous to a method previously des cribed for Gal-NAcol-terminating oligosaccharides (M. S. Stoll, E. F. Houns ell, A. M. Lawson, W. Chai, and T. Feizi, Eur. J. Biochem. 189, 499-507, 19 90). The approach involves the selective cleavage at the core mannitol by m ild periodate treatment and analysis of the reaction products as NGLs by in situ TLC/liquid secondary ion mass spectrometry. Oxidation conditions have been optimized using as reference compounds 2-, 3-, 4-, or 6-monosubstitut ed mannobi-itols, 3,6-disubstituted mannitol-terminating pentasaccharides, and 2-mono- and 2,6-disubstituted mannitol-terminating neutral and sialylat ed oligosaccharides isolated from brain glycopeptides. When a 2:1 molar rat io of periodate to alditol is used, the core mannitol is cleaved at the C3- C4 threo-diol bond and in the absence of a threo-diol cleavage occurs to a lesser extent at erythro-diols. Saccharide ring diols are not cleaved under these conditions, and it is also shown that the side chain of sialic acid on the oligosaccharide is largely unaffected. Substituents at 2- and 6-posi tions of the core mannitol can be identified, and the method is applicable to neutral and sialylated oligosaccharide alditols. Typically, the starting material is 5 nmol of oligosaccharide and 0.5-1 nmol of derivatives is app lied for analysis. By this strategy, the core-branching pattern and positio n of sialic acid of two branched monosialylated mannitol-terminating oligos accharide isomers have been determined. (C) 1999 Academic Press.