Glycolipids are involved in essential biological pathways related to cell-cell communication, cellular growth, host-pathogen interaction, and signal transduction. They significantly impact the lipidome beyond the bulk lipid classes usually observed in state-of-the-art lipidomics assays. Glycolipidomics is still in its infancy as there is a lack of commercially available standards and measurement and identification strategies to resolve native glycolipids. Traditional analytical methods offer class-specific glycolipid detection only. Mass spectrometry (MS) enables monitoring hundreds of glycolipid species with different lipid compositions in parallel that can be identified based on their mass-to-charge ratio and fragmentation pattern. High-resolution mass spectrometry is particularly useful in providing high mass accuracy and reducing hybrid spectra and false annotations of glycolipids. In order to obtain the highest structural resolution, we developed new HRMS-based strategies for complex glycolipid analysis, including field asymmetric ion mobility mass spectrometry (FAIMS) or alternative fragmentation strategies using (1) multistage fragmentation (MSn), (2) ultraviolet photodissociation mass spectrometry (UVPD) and (3) electron-induced dissociation. Using these strategies, it was possible to resolve glycolipids up to the molecular lipid species level, including information about the glycan head group and the glycolipid class as well as the ceramide and fatty acid composition of the lipid part [1]. Additionally, we provide an automated annotation workflow for glycolipids based on the freely available Lipid Data Analyzer (LDA) [1], [2]. In native and differentiated human mesenchymal stem cells, we successfully annotated 254 ganglioside species, including 137 unique gangliosides, enabling us to report the highest ganglioside numbers so far[1]. Our analytical workflow paves the way for probing glycolipid-based biochemical processes, shedding light on the enigmatic processes of glycolipids.
References:
[1] Hohenwallner, K. et al., Decoding Distinct Ganglioside Patterns of Native and Differentiated Mesenchymal Stem Cells by a Novel Glycolipidomics Profiling Strategy. JACS Au 2022, 2 (11), 2466-2480
[2] Panzenboeck, L. et al., Chasing the major sphingolipids on earth: Automated annotation of plant glycosyl inositol phospho ceramides by glycolipidomics. Metabolites 2020, 10, 375
