Discrimination of neolacto-series gangliosides with [alpha]2-3-, and [alpha]2-6-linked N-acetylneuraminic acid by nanoelectrospray ionization low-energy collision-induced dissociation tandem quadrupole TOF MS

A combined strategy of thin-layer chromatography immunostaining and negative ion nanoelectrospray low-energy CID mass spectrometry was established for the differentiation of isomeric [alpha]2-3 and [alpha]2-6 sialytated neolactoseries monosialogangliosides from human granulocytes. The gangliosides investigated differed in the ceramide moiety by substitution with C16:0 or C24:1 fatty acid and in their oligosaccharide chains due to nLc4 and nLc6 core structures. With respect to the type of sialylation, the homogeneity of the HPLC-purified ganglioside fractions was verified by use of specific anti-Neu5Ac[alpha]2-3Gal[beta]1-4GlcNAc-R and anti-Neu5Ac[alpha]2-6Gal[beta]1-4GlcNAc-R antibodies. A clear-cut series of fragment ions for both types of isomeric gangliosides, carrying [alpha]2-3- and [alpha]2-6-linked neuraminic acid, respectively, was obtained by low-energy CID. Additionally, a characteristic ring cleavage was detected exclusively in all species with Neu5Ac[alpha]2-6Ga1[beta]1-4GlcNAc terminus, regardless of ceramide fatty acid and oligosaccharide chain lengths. The diagnostic [sup.0,2][X.sub.4/6] ions, generated by ring cleavage of an [alpha]2-6-linked neuraminic acid are accompanied by a simultaneous decrease of the corresponding [Y.sub.4]/[Y.sub.6] ions. These results suggest the unequivocal discrimination of individual [alpha]2-3- and [alpha]2-6-sialylated neolacto-series monosialogangliosides by distinct fragmentation patterns in low-energy CID tandem MS.