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Development of a Method to Quantitate Nematode Pheromone for Study of Small-Molecule Metabolism in Caenorhabditis elegans
journal contribution
posted on 2016-02-19, 19:54 authored by Kwang-Youl Kim, Hyoe-Jin Joo, Hye-Won Kwon, Heekyeong Kim, William S. Hancock, Young-Ki PaikPheromones produced by Caenorhabditis elegans are
considered key regulators of development, mating, and social behaviors
in this organism. Here, we present a rapid mass spectrometry-based
method (PheroQu) for absolute quantitation of nematode pheromones (e.g., daumone 1, 2, and 3) both in C.
elegans worm bodies (as few as 20 worms) and in liquid culture
medium. Pheromones were separated by ultra performance liquid chromatography
and monitored by a positive electrospray ionization detector in the
multiple-reaction monitoring mode. The daf-22 mutant
worms were used as surrogate matrix for calibration, and stable deuterated
isotope-containing pheromone was used as internal standard for measuring
changes in pheromones in N2 wild-type and other strains under different
growth conditions. The worm-body pheromones were extracted by acidified
acetonitrile solvent, and the secreted pheromones were extracted from
culture medium with solid-phase extraction cartridges. The run time
was achieved in less than 2 min. The method was validated for specificity,
linearity, accuracy, precision, recovery, and stability. The assay
was linear over an amount range of 2–250 fmol, and the limit
of quantitation was 2 fmol amounts for daumone 1, 2, and 3 in both
worm bodies and culture medium. With the PheroQu method, we were able
to identify the location of pheromone biosynthesis and determine the
changes in different pheromone types synthesized, according to developmental
stages and aging process. This method, which is simple, rapid, sensitive,
and specific, will be useful for the study of small-molecule metabolism
during developmental stages of C. elegans.