Discrimination of Neolacto-Series Gangliosides with α2−3- and α2−6-Linked N-Acetylneuraminic Acid by Nanoelectrospray Ionization Low-Energy Collision-Induced Dissociation Tandem Quadrupole TOF MS

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Abstract
A combined strategy of thin-layer chromatography immunostaining and negative ion nanoelectrospray low-energy CID mass spectrometry was established for the differentiation of isomeric α2−3 and α2−6 sialylated neolacto-series 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-Neu5Acα2−3Galβ1−4GlcNAc-R and anti-Neu5Acα2−6Galβ1−4GlcNAc-R antibodies. A clear-cut series of fragment ions for both types of isomeric gangliosides, carrying α2−3- and α2−6-linked neuraminic acid, respectively, was obtained by low-energy CID. Additionally, a characteristic ring cleavage was detected exclusively in all species with Neu5Acα2−6Galβ1−4GlcNAc terminus, regardless of ceramide fatty acid and oligosaccharide chain lengths. The diagnostic 0,2X4/6 ions, generated by ring cleavage of an α2−6-linked neuraminic acid are accompanied by a simultaneous decrease of the corresponding Y4/Y6 ions. These results suggest the unequivocal discrimination of individual α2−3- and α2−6-sialylated neolacto-series monosialogangliosides by distinct fragmentation patterns in low-energy CID tandem MS.