Department of Biochemistry, College of Life Sciences and Biotechnology, Yonsei Proteome Research Center, Yonsei University, Seoul, Korea.
Anal Chem. 2013 Mar 5;85(5):2681-8. doi: 10.1021/ac4001964. Epub 2013 Feb 14.
Pheromones 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.
秀丽隐杆线虫产生的信息素被认为是该生物发育、交配和社会行为的关键调节剂。在这里，我们提出了一种基于快速质谱的方法（PheroQu），用于绝对定量线虫信息素（例如，daumone 1、2 和 3），无论是在秀丽隐杆线虫虫体（低至 20 条虫）还是在液体培养基中。信息素通过超高效液相色谱分离，并在多反应监测模式下通过正电喷雾电离检测器监测。daf-22 突变体蠕虫被用作校准的替代基质，稳定氘代同位素含量的信息素被用作内部标准，用于测量不同生长条件下 N2 野生型和其他菌株中信息素的变化。虫体信息素通过酸化乙腈溶剂提取，分泌的信息素用固相萃取小柱从培养基中提取。运行时间不到 2 分钟。该方法经过特异性、线性、准确性、精密度、回收率和稳定性验证。该测定在 2-250 fmol 的量范围内呈线性，daumone 1、2 和 3 的定量限在虫体和培养基中均为 2 fmol。使用 PheroQu 方法，我们能够根据发育阶段和衰老过程确定信息素生物合成的位置以及不同类型信息素的合成变化。这种方法简单、快速、灵敏、特异性强，将有助于研究秀丽隐杆线虫发育阶段中小分子代谢。
