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 treatment 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 described for GalNAcol-terminating oligosaccharides (M. S. Stoll, E. F. Hounsell, A. M. Lawson, W. Chai, and T. Feizi, Eur. J. Biochem. 189, 499-507, 1990). The approach involves the selective cleavage at the core mannitol by mild 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-monosubstituted mannobi-itols, 3,6-disubstituted mannitol-terminating pentasaccharides, and 2-mono- and 2,6-disubstituted mannitol-terminating neutral and sialylated oligosaccharides isolated from brain glycopeptides. When a 2:1 molar ratio 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-positions 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 applied for analysis. By this strategy, the core-branching pattern and position of sialic acid of two branched monosialylated mannitol-terminating oligosaccharide isomers have been determined.
Chai
W
W
Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Hospital, Watford Road, Harrow, Middlesex, HA1 3UJ, United Kingdom. w.chai@ic.ac.uk
Yuen
C T
CT
Feizi
T
T
Lawson
A M
AM
D000818:Animals; D001923:Brain Chemistry; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D007536:Isomerism; D008353:Mannitol / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D010084:Oxidation-Reduction; D010504:Periodic Acid; D017421:Sequence Analysis / Q000379:methods
D009844:Oligosaccharides; D010504:Periodic Acid; D008353:Mannitol