Discrimination of neolacto-series gangliosides with alpha2-3- and alpha2-6-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 alpha2-3 and alpha2-6 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-Neu5Acalpha2-3Galbeta1-4GlcNAc-R and anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibodies. A clear-cut series of fragment ions for both types of isomeric gangliosides, carrying alpha2-3- and alpha2-6-linked neuraminic acid, respectively, was obtained by low-energy CID. Additionally, a characteristic ring cleavage was detected exclusively in all species with Neu5Acalpha2-6Galbeta1-4GlcNAc terminus, regardless of ceramide fatty acid and oligosaccharide chain lengths. The diagnostic (0,2)X(4/6) ions, generated by ring cleavage of an alpha2-6-linked neuraminic acid are accompanied by a simultaneous decrease of the corresponding Y(4)/Y(6) ions. These results suggest the unequivocal discrimination of individual alpha2-3- and alpha2-6-sialylated neolacto-series monosialogangliosides by distinct fragmentation patterns in low-energy CID tandem MS.
Institute for Medical Physics and Biophysics, University of Münster, Robert-Koch-Strasse 31, D-48149 Münster, Germany.
Iris
I
Meisen
Jasna
J
Peter-Katalinić
Johannes
J
Müthing
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004187:Disaccharides / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D005732:Gangliosides / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D006098:Granulocytes / Q000737:chemistry; D006801:Humans; D007124:Immunoenzyme Techniques; D007477:Ions / Q000737:chemistry; D007536:Isomerism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D004187:Disaccharides; D005732:Gangliosides; D007477:Ions; D009844:Oligosaccharides; C025447:sialogangliosides; C063866:N-acetylneuraminyl-(2-6)-galactose; C049367:N-acetylneuraminyl-(2-3)-galactose