Analysis of Ascarosides from Caenorhabditis elegans Using Mass Spectrometry and NMR Spectroscopy
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The nematode Caenorhabditis elegans secretes a family of water-soluble small molecules, known as the ascarosides, into its environment and uses these ascarosides in chemical communication. The ascarosides are derivatives of the 3,6-dideoxysugar ascarylose, modified with different fatty acid-derived side chains. C. elegans uses specific ascarosides, which are together known as the dauer pheromone, to trigger entry into the stress-resistant dauer larval stage. In addition, C. elegans uses specific ascarosides to control certain behaviors, including mating attraction, aggregation, and avoidance. Although in general the concentration of the ascarosides in the environment increases with population density, C. elegans can vary the types and amounts of ascarosides that it secretes depending on the culture conditions under which it has been grown and its developmental history. Here, we describe how to grow high-density worm cultures and the bacterial food for those cultures, as well as how to extract the culture medium to generate a crude pheromone extract. Then, we discuss how to analyze the types and amounts of ascarosides in that extract using mass spectrometry and NMR spectroscopy.
Key wordsDauer Ascarosides Pheromone C. elegans Mass spectrometry NMR spectroscopy dqf-COSY
Funding for this work was provided by the National Institutes of Health (GM87533 to R.A.B.) and the Human Frontiers Science Program (RGY0042/2010 subcontract to R.A.B.). Our work on the triple quadrupole mass spectrometer was supported in part by the National Institutes of Health and National Center for Research Resources CTSA grant 1UL 1RR029890. We would like to thank Dr. Tim Garrett (University of Florida) for helpful suggestions on the LC-MS/MS parameters and Dr. Ion Ghiviriga (University of Florida) for helpful suggestions on the NMR parameters. We would like to thank Prof. Justin R. Ragains (Louisiana State University) for providing the synthetic ascaroside standards that are used in our work.
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