Identification and characterization by electrospray mass spectrometry of endogenous Drosophila sphingadienes

Abstract

Sphingolipids comprise a complex group of lipids concentrated in membrane rafts and whose metabolites function as signaling molecules. Sphingolipids are conserved in Drosophila, in which their tight regulation is required for proper development and tissue integrity. In this study, we identified a new family of Drosophila sphingolipids containing two double bonds in the long chain base (LCB). The lipids were found at low levels in wild-type flies and accumulated markedly in Drosophila Sply mutants, which do not express sphingosine-1-phosphate lyase and are defective in sphingolipid catabolism. To determine the identity of the unknown lipids, purified whole fly lipid extracts were separated on a C18-HPLC column and analyzed using electrospray mass spectrometry. The lipids contain a LCB of either 14 or 16 carbons with conjugated double bonds at C4,6. The Δ4,6-sphingadienes were found as free LCBs, as phosphorylated LCBs, and as the sphingoid base in ceramides. The temporal and spatial accumulation of Δ4,6-sphingadienes in Sply mutants suggests that these lipids may contribute to the muscle degeneration observed in these flies.

Keywords

• high-performance liquid chromatography
• sphingolipids
• HPLC
• sphingadiene
• sphingosine-1-phosphate lyase
• muscle