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Comparative Analysis of Ganglioside Composition from Two Fetal Human Brains at Early Stage of Development

Gangliosides (GG) are the most complex group of glycosphingolipids, characterized by the sialylated carbohydrate chain and differing in both the hydrophilic and hydrophobic moieties. Their highest content and structural diversity are found in mammalian brain tissues. They are located in the external leaflet of neural plasma membranes, obligatory present in microdomains and acting as cell surface recognition sites. Certain GG species have been implicated in key processes of brain development such as neuritogenesis, directional cell motility, axon guidance and cell adhesion. The amount and expression patterns of GGs drastically change during the embryonic to postnatal stages, and these changes continue during brain maturation and aging. In this study, we have performed compositional and structural analysis of native GG mixtures from two fetal human brains at different gestational age: 13 and 23 weeks of gestation. The comparative study was performed employing advanced chip-mass spectrometry (MS) and high-performance thin-layer chromatography (HPTLC) methods. Total gangliosides were isolated and purified as native mixtures from tissue homogenates and spectrophotometrically quantified. HPTLC was used for compositional analysis following resorcinol-HCl staining of separated ganglioside patterns. The 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. The HPTLC ganglioside patterns of analyzed fetal brain samples were similar, with quantitatively dominant GD1a- and more expressed GQ1b-migrating fraction, as characteristic for fetal human brain. Although showing resemblance in number of detected GG species, comparative MS screening of two different fetal brain samples showed high variability of ceramide portions for the majority of ganglioside forms defined by oligosaccharide moieties. All major GG forms were also represented by their fucosylated and/or O-acetylated counterparts. The abundance of so-called fetal brain markers was higher than previously detected in adult human brain, as expected. Here we represent, to our knowledge, the first MS data regarding GG composition at the earliest fetal brain developmental stage so far analysed.

gangliosides; human fetal brain; early development; mass spectrometry

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