Structural characterization of neutral glycosphingolipids by thin-layer chromatography coupled to matrix-assisted...

Rapid and convenient structural analysis of neutral glycosphingolipids (GSLs) was achieved by direct coupling of thin-layer chromatography (TLC) to matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF) MS/MS. Positions of unstained GSL spots on developed TLC plates were determined by comparison to orcinol-stained references. A matrix solution of 2,5-dihydroxybenzoic acid (DHB) in acetonitrile/water (1:1 v/v) was then added directly to the unstained GSL spots, and the GSLs were directly analyzed by MALDI-QIT-TOF MS. The acetonitrile/water DHB solution proved to be suitable for MS/MS structural analysis with high sensitivity. MS/MS and MS/MS/MS of GSLs yielded simple and informative spectra that revealed the ceramide and long-chain base structures, as well as the sugar sequences. Hydroxy fatty acids in ceramide provided characteristic MS/MS fragment ions. GSLs were stained with primuline, a nondestructive dye, after TLC development, and successfully analyzed by MALDI-QIT-TOF MS/MS with high sensitivity. Immunostaining of GSLs after TLC development is a powerful method for characterizing antibody-specific sugars, but not ceramides. By coupling TLC-immunostaining of GSLs to MALDI-QIT-TOF MS/MS, we were able to identify both the sugar and the ceramide structures. The detection limits of asialo GM1 (Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1-1'Cer) were 25 and 50 pmol in primuline staining and immunostaining, respectively.