Early stage fetal neocortex exhibits a complex ganglioside profile as revealed by high resolution tandem mass spectrometry

Abstract

In this study we report on the first mass spectrometric (MS) investigation of gangliosides and preliminary assessment of the expression and structure in normal fetal neocortex in early developmental stages: 14th (Neo14) and 16th (Neo16) gestational weeks. Ganglioside analysis was carried out using a hybrid quadrupole time-of-flight (QTOF) MS with direct sample infusion by nanoelectrospray ionization (nanoESI) in the negative ion mode. Under optimized conditions a large number of glycoforms i.e. 75 in Neo14 and 71 in Neo16 mixtures were identified. The ganglioside species were found characterized by a high diversity of the ceramide constitution, an elevated sialylation degree (up to pentasialylated gangliosides-GP1) and sugar cores modified by fucosylation (Fuc) and acetylation (O-Ac). Direct comparison between Neo14 and Neo16 revealed a prominent expression of monosialylated structures in the Neo16 as well as the presence of a larger number of polysialylated species in Neo14 which constitutes a clear marker of rapid development-dependant changes in the sialylation. Also the MS screening results highlighted that presumably O-acetylation process occurs faster than fucosylation. CID MS/MS under variable collision energy applied for the first time for structural analysis of a fucosylated pentasialylated species induced an efficient fragmentation with generation of ions supporting Fuc-GP1d isomer in early stage fetal brain neocortex.

Structural Analysis of Unsaturated Glycosphingolipids Using Shotgun Ozone-Induced Dissociation Mass Spectrometry

“…Specifically, different double bond positions could result in distinct structural conformations thus influencing antigenicity and ultimately, recognition by immune cells [15–19]. Studies also show that some of the potential glycosphingolipid biomarkers of brain development and the underlying diseases are in fact, unsaturated, yet rarely are they identified down to the finest structural detail [12, 20–23]. As such, comprehensive structural determination of intact glycosphingolipids is warranted.…”

Modern separation techniques coupled to high performance mass spectrometry for glycolipid analysis

“…While the qualitative densitometric analysis indicated some differences between the proportions of GG fractions within the two samples (Fig. ), the subsequent MS investigation of the separated fractions not only substantiated the HPTLC results, but also discovered brain development dependent changes in the degree of sialylation and O ‐acetylation of GGs.…”

“…In 2014 Ghiulai et al . optimized HPTLC combined with nanoESI QTOF MS and MS/MS for a comparative GG profiling in healthy fetal neocortex in two early developmental stages, 14th and 16th gestational weeks. The glass backed HPTLC‐plates developed in a solvent system containing CHCl 3 , MeOH and 12 mM MgCl 2 , sprayed with resorcinol reagent and heated for 30–45 min were subjected to laser densitometric scanning at 580 nm for relative quantification of individual gangliosides.…”

“…However, given the minute amount of samples extracted from the biological matrices and the high heterogeneity of GLs, a series of difficulties may arise in their mapping and structural determination. Among the biophysical analytical methods , MS, especially when hyphenated with various separation techniques, based on its high sensitivity, detection limit, speed of analysis and variety of its applications, was demonstrated as a key technology for a reliable mapping and structural sequencing of GLs, which could further serve for biomarker identification. Besides, the possibility to perform structural analysis by employing MS/MS or multistage MS (MS n ) constitutes a supreme advantage, allowing a complete and reliable pattern determination in complex mixtures.…”

Targeting of tumor-associated gangliosides with antibodies affects signaling pathways and leads to cell death including apoptosis

“…Gangliosides are enriched in nerve tissues. Mass spectroscopy, along with other techniques, can give important insight into diversity and changes of their contents related to localization within brain tissues, stages of nerve structures development, and when affected by diseases [3,4]. Cellular roles of gangliosides are already well described, including their involvement in pathological states, e.g., cancer, neurodegenerative, autoimmune diseases or genetically inherited gangliosidoses [5][6][7][8].…”