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High-throughput mass spectrometric determination of ganglioside expression and structure in human fetal cerebellum

Cerebellum, the so-called "small brain", is located in the posterior fossa of the skull, above the stem and toward the back of the brain. It is a region that processes information from many diverse sources, including the spinal cord, the brain stem and the cerebral cortex, and projects them to different centers in the brain involved in postural adaptations and generation of movements. Gangliosides, sialylated glycosphingolipids, are mostly building components of the outer leaflet of plasma membranes of all mammalian cell types ; however, their highest concentration is present in the central nervous system. In the framework of our research targeting the exhaustive brain ganglioside mapping, we have shown previously, that ganglioside composition exhibits developmental, ageing and topographic specificity. Moreover, we have reported that adult cerebellum shows characteristic differences in the composition of its major species, when compared to those regions of the cerebrum that we studied [1]. Here, we have extended our research towards the investigation of native ganglioside expression and structure in human fetal cerebellum. In this regard, we have applied our recently developed fully automated chip-based nanoelectrospray ionization (nanoESI) high capacity trap mass spectrometry (HCT MS) approach for the analysis in high-throughput regime of two ganglioside mixtures purified from fetal cerebellum (15 and 40 gestational weeks). Screening mass spectra obtained in the negative ion mode under restrictive acquisition time conditions enabled detection of over 50 different glycoforms of GM4, GM3, GM2, GD3, GM1/nLM1/LM1, GD1/nLD1, GT1, GD2 and GT3-type as well as polysialylated GQ1 and GP1 structures considered as fetal cerebellum markers. Besides the rich ganglioside pattern, a clear gestational stage-dependent ganglioside expression was observed. Multistage mass spectrometry (MS2-MS4) carried out by collision-induced dissociation (CID) yielded structural-informative sequencing patterns for several polysialylated species. Obtained results also demonstrated that the combination of fully automated chip-ESI with HCT MSn is able to provide reliable ganglioside high-throughput analysis at subpicomolar level of sensitivity within experiments lasting less than five minutes.

gangliosides determination; fetal human cerebellum; chip-based HCT MS

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