Profiling of sphingolipids in Caenorhabditis elegans by two-dimensional multiple heart-cut liquid chromatography -...

Sphingolipids exert important functions in cells, ranging from stabilising the cell membrane to bioactive signalling in signal transduction pathways. Changed concentrations of sphingolipids are associated with, among others, neurodegenerative and cardiovascular diseases. In this work, we present a novel two-dimensional liquid chromatography method (2D-LC) coupled to tandem mass spectrometry (MS/MS) for the identification of ceramides, hexosylceramides and sphingomyelins in the model organism Caenorhabditis elegans (C. elegans). The method utilises a multiple heart-cut approach with a hydrophilic interaction liquid chromatography (HILIC) separation in the first dimension. The fractions of the sphingolipid classes were cut out and thereby separated from the abundant glycerolipids, which offers a simplified sample preparation and a high degree of automation as it compensates the alkaline depletion step usually conducted prior to the chromatographic analysis. The fractions were stored in a sample loop and transferred onto the second column with the combination of two six port valves. A reversed phase liquid chromatography was performed as the second dimension and allowed for a separation of the species within a sphingolipid class and according to the fatty acid moiety of the sphingolipid. The segregation of the abundant glycerolipids and the reduced matrix effects allowed for better identification of low abundant species, especially dihydro-sphingolipids with a saturated sphingoid base. In addition, the separation of the three fractions was carried out parallel to the separation and equilibration in the first dimension, which leads to no extension of the analysis time for the 2D-LC compared to the one-dimensional HILIC method. In total 45 sphingolipids were detected in the C. elegans lipid extract and identified via accurate mass and MS/MS fragments.