engleski

Characterization of Ganglioside Composition of Selected Fetal Human Brain Regions Combining Chip-Based Mass Spectrometry and Thin-Layer Chromatography

Gangliosides (GGs), a class of sialic acid-containing glycosphingolipids, are constituents of external leaflet of plasma membranes in mammalian tissues ; their highest content and pattern complexity are present in brain cells. Their compositions vary in different brain regions and characteristically change during ontogenesis ; specific changes of the normal pattern have been observed in various neuropathological conditions. Certain GG species are involved in key brain developmental processes: neuritogenesis, cell motility, axon guidance, apoptosis, cell-to-cell recognition. Here, we represent data of compositional and structural characterization of native GG mixtures and fractions from 24 weeks of gestation fetal human brain neocortical regions that correspond to prospective precentral and postcentral gyrus (i.e. prospective primary motor and primary somatosensory cortex, respectively). The native GG mixtures were extracted and purified from the tissue homogenates. For analysis, our recently introduced mass spectrometry (MS) approaches were combined with high-performance thin-layer chromatography (HPTLC). GG fractions separated by HPTLC using suitable chloroform/methanol/0.2% aq. CaCl2 developing system(s) were visualized with resorcinol-HCl reagent. Also, the GG fractions were isolated by preparative TLC. MS screening and sequencing analyses were performed on a High Capacity Ion Trap Ultra (PTM discovery) mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled with a NanoMate robot incorporating ESI 400 Chip technology (Advion BioSciences, Ithaca, USA). Tandem MS was carried out by collision-induced dissociation. HPTLC GG patterns of the analyzed fetal brain regions were similar, containing fractions migrating as GM4, GM3, GM2, nLM1, GM1, GD3, GD1a, GD2/GD1-α /GT3, GD1b, GX, GT1b and GQ1b ; the GD1a-migrating fraction was quantitatively dominant. By MS analysis it was distinguished that all HPTLC-separated bands contained several distinct species. More than 140 species were detected in each GG mixture by MS1 screening in negative ion mode. Most of GG forms with defined oligosaccharide structure were represented by several species differing by the composition of ceramide portion. The relative abundance of individual species was moderately different in two analyzed regions. Higher number and higher total abundance of mono- and disialylated structures was found in the prospective postcentral comparing to the precentral gyrus. In contrast, the prospective precentral gyrus contained larger number of polysialylated (tri- to even hexasialylated) structural variations. Species such as tetra- and pentasialo-forms, O-Ac-GM3, di-O-Ac-GD3 and O-Ac-GT3, and to a certain extent GM4, GT3, Fuc-GT1 and GalNAc-GT1 were considerably more abundant in both fetal brain regions than previously observed in adult human brain ; this further confirms their categorization as fetal brain markers and developing brain-associated species.

fetal human brain; gangliosides; mass spectrometry

nije evidentirano