title: Structural characterization of neutral glycosphingolipids by thin-layer chromatography coupled to matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight MS/MS.
abstract: 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.
authors.affiliation: Sphingolipid Expression Laboratory, Supra-Biomolecular System Research Group, Frontier Research System, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Nakamura
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Naoko
authors.initials: N
authors.lastname: Goto-Inoue
authors.forename: Chikako
authors.initials: C
authors.lastname: Yoshida-Noro
authors.forename: Akemi
authors.initials: A
authors.lastname: Suzuki
mesh_terms: D002855:Chromatography, Thin Layer / Q000379:methods*; D020383:Neutral Glycosphingolipids / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D020383:Neutral Glycosphingolipids