Abstract
Glycosphingolipids and phospholipids from white matter of human hippocampus, were analyzed by a sequential procedure of two-dimensional TLC (2d-TLC), transference of separated lipids to a PVDF membrane by a TLC-Blot equipment and Mass spectrometry (MS) analysis with an ion-trap type MALDI-TOF equipment. The method is simple and quick; very small amount of sample (0.1 mg of brain tissue) is enough to analyze all lipid components. The 2d-TLC provided excellent separation and the MS analyses allowed identifying the characteristic profile of molecular species for individual glycolipids and phospholipids. The results of MS analyses on gangliosides showed that di- and tri-sialogangliosides are richer in d20:1 sphingosine-containing ceramide than monosialogangliosides, suggesting the presence of sialylation selection after GM1 gangliosides. Then we analyzed ganglioside molecular species obtained from different brain regions by using MS imaging technology. The MS images of individual gangliosides provided clear visual profiles in terms of molecular species distribution. The imaging profiles were region dependent and also indicated that the sialyltransferase toward GM1 ganglioside prefers to select d20:1 sphingosine containing molecule. This technology provides visual characterization of individual phospholipid and glycosphingolipid molecular species and informs us about the metabolic characterization of target tissue, opening a new gate for colorful lipidomics research.
Highlights
The gangliosides were named according to the nomenclature of Svennerholm [1]
The combination of thin layer chromatography (TLC) separation properties and the spectra obtained from mass spectrometry (MS) analysis has been used as complementary structural information [12,13,14,15,16]. We combined these two technologies, and for new lipidomics research, we introduced two more technologies, TLC-Blot which is a transfer of lipids separated on High performance thin layer chromatography (HPTLC)-plate to PVDF membrane and MS imaging to the blotted lipid bands on the membrane
We presented the usefulness of this TLC-Blot/MALDI-TOF MS system for detail structural analysis of complex GSLs such as gangliosides with high sensitivity [21]
Summary
The gangliosides were named according to the nomenclature of Svennerholm [1]. The glycosphingolipids (GSLs) are amphiphatic molecules consisting of one hydrophilic sugar chain and one hydrophobic ceramide moiety. GSLs are involved in a wide range of events such as cell-surface recognition, microbial pathogenesis [2,3], cell differentiation, and immunological recognition [4,5,6] They have been identified as components of raft microdomain [7], where they play important roles in the early events of signal transduction by arranging the environment of receptor or ligand molecules [8]. In these events, their carbohydrate moieties and ceramide moieties are very important to facilitate the formation of microdomain
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