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Anal Chem. 2006 Aug 15;78(16):5736-43.

Structural characterization of neutral glycosphingolipids by thin-layer chromatography coupled to matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight MS/MS.

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Sphingolipid Expression Laboratory, Supra-Biomolecular System Research Group, Frontier Research System, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.


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.

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