Fast Atom Bombardment Mass Spectrometric Analysis of Glycosphingolipids by Direct Desorption from Thin Layer Chromatography Plates: Application of the Method to a Receptor-Active 8-Sugar Glycolipid

Abstract

Glycosphingolipids separated on thin layer chromatograms were analyzed by direct negative-ion fast atom bombardment mass spectrometry (FAB-MS). The thin-layer plates were moved within the ion source with a motor-driven sample probe. The technique was optimized using glycosphingolipids containing 5- and 7-sugar residues; the Forssman glycolipid (GalNAcα3GalNAcβ3Galα4Galβ4GlcβCer) and A-7 (GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ3Galβ4GlcβCer). Spectra containing pseudo-molecular ions and fragment ions caused by cleavage at the glycosidic bonds were successfully obtained and due to the chromatographic separation it was also possible to record spectra of glycolipids with different ceramide compositions. This separation was maintained after blotting onto a hydrophobic membrane which also was analyzed by direct-desorption FAB-MS. The thin layer chromatography (TLC)/FAB-MS method was used to analyze a receptor-active glycosphingolipid from female C3H/HeN mice kidneys with eight sugar residues: Galβ4(Fucα3)GlcNAcβ6(Galβ3)GalNAcβ3Galα4Galβ4GlcβCer. Although the analysis was made on a complex mixture of glycosphingolipids without purification, the pseudo-molecular ion and fragment ions obtained were of high abundance and allowed sequence determination of this glycolipid that is recognized by uropathogenic P-fimbriated Escherichia coli.