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Abstract

Carbohydrates and lipids are not only energy sources and structural components of cells but fulfill very diverse and important roles in a multitude of cellular processes. The combination of a carbohydrate and a lipid linked via a glycosidic bond yields a glycolipid. Glycolipids occur in all kinds of organisms where they constitute major components of cell membranes, mediate cell-cell interactions and modulate immune responses. Their biological activities are highly dependent on their exact three-dimensional structures; however, the elucidation of glycolipid structures is inherently challenging due to the non-template-driven regio- and stereoselective synthesis of the carbohydrate and additional variations of the lipid moiety, such as length, saturation and hydroxylation. The modular biosynthesis of glycolipids generates many different and often isomeric combinations of glycans and lipids, which are not always distinguishable by standard analytical workflows based on liquid chromatography-mass spectrometry. The structural analysis of isomeric glycolipids thus requires orthogonal techniques yielding complementary information. In some cases, ion mobility spectrometry can be used to distinguish isomers based on their different cross sections in the gas phase. Another promising structure-sensitive technique is cryogenic gas phase infrared spectroscopy, which falls under the term action spectroscopy and yields highly resolved, reproducible IR spectra of isolated ions in the clean gas phase environment.
In this work, sets of isomeric glycolipids were systematically investigated using ion mobility-mass spectrometry and cryogenic gas phase IR spectroscopy in helium nano- droplets. The obtained IR spectra of various isomeric glycosphingolipids bearing different monosaccharide headgroups are diagnostic for both the type of monosaccharide and the anomeric configuration because of very characteristic and reproducible absorption bands in the fingerprint region (1000–1150 cm^-1). A more complex trisaccharide headgroup still yields a well-resolved IR fingerprint. The influence of the lipid part was examined by comparing IR spectra of glycolipids bearing the same headgroup but a different sphingoid base or a diacylglycerol. The exchange of phytosphingosine for sphingosine does not alter the fingerprint region significantly but influences the intensity of the amine bending vibration, whereas the introduction of a diacylglycerol changes the whole spectrum. The data show that cryogenic gas phase IR spectroscopy is a powerful technique to distinguish glycolipids bearing isomeric glycans up to a certain size, anomeric configurations and also subtle variations in the lipid moiety. Furthermore, the applicability of IR spectroscopy to distinguish double bond isomers was tested on synthetic derivatives of 1-deoxysphingosine. Depending on the position and configuration of the C C double bond, distinct N H bending modes of the protonated amine are observed. Gas phase IR spectroscopy could thus fill a gap in lipidomics, where lipid double bond isomers still pose a major challenge.