College of Life Sciences, Beijing Normal University , Beijing 100875, China.
Anal Chem. 2013 Oct 1;85(19):9281-7. doi: 10.1021/ac402025c. Epub 2013 Sep 19.
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.
在有利条件下，秀丽隐杆线虫幼虫经过一系列蜕皮周期后成长为有生殖能力的成虫。当环境条件恶劣时，它们会停滞为 dauer 幼虫。dafachronic acid (DA)，一种秀丽隐杆线虫的类固醇激素，是生殖发育所必需的。在这里，我们报告了一种用于秀丽隐杆线虫中 DA 绝对定量的质谱 (MS) 方法。优化了从秀丽隐杆线虫中提取 DA 的方法，以实现回收率大于 83%。DA 的 MS 灵敏度在与 2-吡啶甲胺进行羧基衍生化后提高了 100 倍。高分辨率选择离子监测 (HR-SIM) 在 Q-Exactive 轨道阱质谱仪上的性能优于同一仪器上的靶向-MS2 和三重四极杆质谱仪 TSQ Quantum Discovery 上的选择反应监测 (SRM)。该 HR-SIM 方法的定量下限低至 1 pg DA，可在秀丽隐杆线虫生殖发育过程中实现内源性 DA 的绝对定量。我们发现，在野生型 (WT) 线虫中，DA 从 L1 幼虫阶段的 0.04 ± 0.02 ng/mg 蛋白增加到 L2 幼虫阶段的 1.21 ± 0.67 ng/mg 蛋白，在 L3 阶段后再次下降。相比之下，由于胰岛素、TGF-β 或 cGMP 信号通路中断而具有持续 dauer 形成表型的四个遗传突变体在 L2 阶段的 DA 水平非常低（低于 WT 的 15%）。这些突变体能够逃脱 dauer 命运，并且当供应外源性 DA 时，它们中的大多数都能生长为有生殖能力的成虫。因此，L2 阶段的 DA 激增对秀丽隐杆线虫的生殖发育至关重要。
