title: Absolute quantification of a steroid hormone that regulates development in Caenorhabditis elegans.
abstract: Under favorable conditions, Caenorhabditis elegans larvae grow into reproductive adults after a series of molting cycles. When environmental conditions are harsh, they arrest as dauer larvae. Dafachronic acid (DA), a C. elegans steroid hormone, is required for reproductive development. Here, we report a mass spectrometry (MS) method for absolute quantitation of DA in C. elegans. The extraction of DA from C. elegans was optimized to achieve a recovery rate of greater than 83%. The MS sensitivity to DA increased 100-fold after carboxyl group derivatization with 2-picolylamine. High-resolution selected ion monitoring (HR-SIM) on a Q-Orbitrap mass spectrometer Q Exactive outperformed targeted-MS2 on the same instrument and selected reaction monitoring (SRM) on a triple-quadrupole mass spectrometer TSQ Quantum Discovery. With a limit of quantification as low as 1 pg of DA, the HR-SIM method enables absolute quantification of endogenous DA during the reproductive development of C. elegans. We found that in wild-type (WT) worms, DA increases from 0.04 ± 0.02 ng/mg protein in the L1 larval stage to 1.21 ± 0.67 ng/mg protein in the L2 larval stage and decreases again after the L3 stage. In comparison, four genetic mutants that have a constitutive dauer-formation phenotype due to disrupted insulin, TGF-β, or cGMP signaling all have a very low DA level in the L2 stage (below 15% of the WT). These mutants are able to escape the dauer fate and most of them grow into fertile adults when supplied with exogenous DA. Therefore, a DA spike in the L2 stage is critical for the reproductive development of C. elegans.
authors.affiliation: College of Life Sciences, Beijing Normal University , Beijing 100875, China.
authors.forename: Tie-Mei
authors.initials: TM
authors.lastname: Li
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Xiangke
authors.initials: X
authors.lastname: Li
authors.forename: Xiao-Jun
authors.initials: XJ
authors.lastname: Ding
authors.forename: Yao
authors.initials: Y
authors.lastname: Wu
authors.forename: Li-Feng
authors.initials: LF
authors.lastname: Zhao
authors.forename: She
authors.initials: S
authors.lastname: Chen
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Lei
authors.forename: Meng-Qiu
authors.initials: MQ
authors.lastname: Dong
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000254:growth & development* / Q000378:metabolism*; D002782:Cholestenes / Q000032:analysis* / Q000378:metabolism; D013058:Mass Spectrometry; D015394:Molecular Structure
chemical_list: D002782:Cholestenes; C533800:dafachronic acid