Ascaroside Profiling of Caenorhabditis elegans Using Gas Chromatography-Electron Ionization Mass Spectrometry

Nematodes such as the model organism Caenorhabditis elegans produce various homologous series of l-ascarylose-derived glycolipids called ascarosides, which include several highly potent signals in intra and interspecies communication as well as cross-kingdom interactions. Given their low concentrations and large number of structurally similar components, mass spectrometric screens based on high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) are commonly employed for ascaroside detection and quantification. Here, we describe a complementary gas chromatography-electron ionization mass spectrometry (GC-EIMS) screen that utilizes an ascarylose-derived K1-fragment ion signal at m/z 130.1 [C₆H₁₄OSi]^+● to highlight known as well as yet unidentified ascaroside components in TMS-derivatized crude nematode exometabolome extracts. GC-EIMS-based ascaroside profiling of wild-type and mutant C. elegans facilitates the analysis of all basic ascarosides using the same ionization technique while providing excellent resolution for the complete homologous series with side chains ranging from 3 to 33 carbons. Combined screening for m/z 130.1 along with side chain-specific J1 [M - 173]⁺ and J2 [M - 291]⁺ fragment ions, as well as additional characteristic marker ions from α-cleavage, enables convenient structure assignment of ca. 200 components from wild-type and peroxisomal β-oxidation mutants including (ω - 1)-linked acyl, enoyl, β-hydroxyacyl, and 2-ketoalkyl ascarosides along with their (ω)-linked or α-methyl isomers and ethanolamide derivatives, as well as 2-hydroxyalkyl ascarosides. Given the widespread availability of GC-MS and its increasing popularity in metabolomics, this method will promote the identification of ascarosides in C. elegans and other nematodes.