CGE-LIF as a novel approach for the analysis of glycosphingolipid-derived glycans

Analysis of glycosphingolipid (GSL) glycosylation remains a major challenge in analytics. Current technologies, such as mass spectrometry (MS) or reversed-phase (RP)-HPLC are time-consuming and mostly low-throughput. Here we present a novel high-throughput-compatible approach for the analysis of GSL-derived glycans using capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF), which has so far been widely applied by us and others for Nglycan profiling. We show for the first time the adaption of CGE-LIF for the analysis of GSL glycans. GSL glycan head groups were efficiently released from the ceramide by digestion with ceramide glycanase, fluorescently labelled and could subsequently be separated by CGE-LIF. Using defined glycans, we built up a data base that currently comprises the migration times of 36 different glycan structures belonging to ganglioseries, globo- / isogloboseries and lacto- / neolactoseries. The separation power of CGE-LIF is capable to not only discriminate glycans differing in the number or type of monosaccharides but also clearly distinguishes peaks of structural isomers that differ either in the positioning of identical monosaccharides (e.g. GD1a-derived glycan vs. GD1b-derived glycan) or even in the type of linkage between identical monosaccharides (e.g. fucosyl lactotetra vs. fucosyl neolactotetra). We applied this novel approach for the analysis of GSL glycans of human induced pluripotent stem cells (hiPSCs). Based on our database, 14 different glycans could clearly be assigned and were confirmed by exoglycosidase digests. The majority of peaks could still be detected from as few as 105 cells. Subsequently we quantitatively compared GSLs of hiPSCs and cardiomyocytes (CMs) derived thereofby in vitro differentiation. This analysis revealed that levels of GSL-derived glycans with globo- (sialyl globopentaose / stage-specific embryonic antigen 4 (SSEA4), globopenta / SSEA3, globotetra) and lacto-series (fucosyl lactotetra / SSEA5, lactotetra) core structures were considerably reduced, whereas levels of ganglioseries glycans (GD3- derived glycan and GM3-derived glycan) were strongly augmented upon differentiation of hiPSCs. Taken together, once having established a comprehensive migration time database, CGE-LIF provides a cheap, easyto-use and fast access for the analysis of GSL glycans with superior sensitivity and specificity that can be simply scaled up to a high-throughput approach.