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Abstract

Nematodes such as the model organism Caenorhabditis elegans produce homologous series of L-ascarylose-derived glyco-lipids 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 HPLC-ESI-MS/MS are commonly employed for ascaroside detection and quantification. Here, we describe a complementary 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 exo-metabolome 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 sidechains ranging from 3 to 33 carbons. Combined screening for m/z 130.1 along with sidechain-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.