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Introduction Temporal lobe epilepsy (TLE) accompanied by hippocampal sclerosis (HS) is one of the most common forms of epilepsy in humans. The molecular events underlying seizures involve changes in membrane ion homeostasis leading to altered neuronal excitability. Gangliosides are membrane glycoconjugates, expressed with large structural diversity and abundance in human brain. Numerous (patho)physiological effects of gangliosides have been confirmed, including regulation of membrane ion transport. However, potential role of gangliosides in molecular pathogenesis of TLE-HS has not been explored in details. In this study we have developed a high resolution (HR) MSn approach using nanoESI Orbitrap MS to assess the changes in the ganglioside pattern in adult hippocampus affected by TLE vs. normal hippocampus aiming to detect specific TLE-associated structures. Methods The native ganglioside mixtures analyzed in this study were purified from human hippocampus affected by TLE (sample HipE) and age-matched normal hippocampus tissue (sample Hip), used as the control. Gangliosides were extracted in chloroform: methanol: water (1:2:0.75 by vol.), separated using phase partition and repartition, collected and purified. The MS experiments were conducted under identical conditions, in the negative ion mode on a LTQ Orbitrap Velos from Thermo Fisher Scientific (Bremen, Germany) equipped with nanoESI source. The MS scans were acquired with the resolution set to 100000 (for m/z 400). Multistage mass spectrometry (MSn) was carried out by higher energy collision dissociation (HCD) at variable collision energies within 35-80 eV range to enhance a high coverage of fragment ions. Preliminary data For nanoESI HR MS screening and HCD MSn fragmentation experiments, 10 µL of the 5 pmol·μL−1 HipE and 10 µL of the 5 pmol·μL−1 Hip solutions in MeOH, were infused by (-) nanoESI into the Orbitrap MS under identical instrumental conditions. Both screening mass spectra featured a rich molecular ion pattern, confirming not only the presence of a high number of glycoforms, but also a diversity of the ceramide composition for certain species. The optimized nanoESI HR MS conditions allowed the multicharging of ganglioside molecules, enhanced the ionization of long-chain polysialylated GT1 and GQ1 structures and provided a fair ionization/detection of minor components in the analyzed samples. In the comparative assay, the accurate mass measurement allowed the discrimination of 72 different ganglioside species in HipE vs. 51 in Hip sample. Unlike Hip control, HipE mixture was found enriched in polysialylated gangliosides. No less than 26 species belonging to GD class i.e. GD1, GD2 and GD3, 23 to GT class i.e. GT1 and GT3 glycoforms and 20 of GQ1 type were discovered for the first time associated to TLE. Another characteristic of HipE mixture was the occurrence of tetrasialo fucogangliosides and compounds having the oligosaccharide chain modified by either O-acetylation or CH3COO-. Since HR MS screening has indicated that the structures in the GQ1 class dominate numerically the HipE ganglioside extract and may represent markers of TLE, in the final stage of research we have conducted a detailed structural analysis by HCD MSn using as the precursor ion the [M-4H+]4- detected at m/z 603.7844, which, according to mass calculation, corresponds to GQ1(d18:1/18:0) species. MS2-MS4, generated by accumulating scans at variable collision energies, gave rise to fragmentation patterns supporting the presence of GQ1b(d18:1/18:0) structural isomer in HipE. Detected specific alterations of ganglioside structures and composition might contribute to molecular pathogenesis of TLE. Novel aspect The gangliosidome of human hippocampus affected by temporal lobe epilepsy was determined by high resolution multistage mass spectrometry. |
