Microflow Liquid Chromatograph for Ultrasensitive Steroid Profiling by Mass Spectrometry

压力对所有脊椎动物都有重要的生理影响。尤其值得一提的是，早期生活压力会对生理学产生“编程”作用，其后果会持续到成年。糖皮质激素是调节应激反应的类固醇激素。这些激素由肾上腺分泌，并通过血液到达其众多的靶细胞。此外，令人兴奋的新研究表明，糖皮质激素在大脑、淋巴器官和生殖组织中局部合成和代谢。我的NSERC发现拨款和发现加速器补充专注于早期发育过程中大脑和淋巴器官中糖皮质激素的局部合成。特别是，我将研究环境应激源的调节，作用机制，以及局部糖皮质激素的长期影响。科利尔教授的发现基金专注于UDP-葡萄糖醛酸基转移酶。这些酶通过新陈代谢降低局部糖皮质激素水平，并在发育和繁殖中发挥关键作用。我们的两个项目都集成了多种技术，包括分子生物学、生物信息学和LC-MS/MS(LC-MS/MS)。*对于这两个项目，测量显微解剖组织：脑和淋巴器官(SOMA)或生殖组织(Collier)中糖皮质激素的局部水平是至关重要的。这项关键任务面临着技术挑战，特别是对特定和超灵敏分析的需要。在特异性方面，质谱学是金标准，我们的实验室是开发类固醇质谱学分析的先驱。在敏感性方面，我们实验室的类固醇检测是世界上最敏感的之一。然而，类固醇不容易电离，这限制了测量低量类固醇(.MicroLC可以与我们现有的最先进的质谱仪无缝集成。无法获得所需的设备将极大地推迟我们对局部糖皮质激素的研究。我们迫切需要这种设备来了解压力是如何“深入皮肤”影响生理的。此外，质谱学的进步将加速全球的内分泌研究，并为我们的HQP配备学术界和工业界高度需要的尖端技术技能。从这个项目中获得的基础知识将影响到各种学科，包括内分泌学、酶学、生理学、神经科学、免疫学和分析化学。

Stress has major physiological effects in all vertebrates. In particular, early-life stress can “program” physiology, with enduring consequences that last into adulthood. Glucocorticoids are steroid hormones that mediate the stress response. These hormones are secreted from the adrenal glands and reach their numerous target cells via the blood. In addition, exciting new research shows that glucocorticoids are locally synthesized and metabolized within the brain, lymphoid organs, and reproductive tissues.******My NSERC Discovery Grant and Discovery Accelerator Supplement focus on the local synthesis of glucocorticoids in the brain and lymphoid organs during early development. In particular, I will examine the regulation by environmental stressors, mechanisms of action, and long-lasting effects of local glucocorticoids. Prof. Collier's Discovery Grant focuses on UDP-glucuronosyltransferase enzymes. These enzymes reduce local levels of glucocorticoids via metabolism and play critical roles in development and reproduction. Both of our projects integrate multiple techniques, including molecular biology, bioinformatics, and liquid chromatography tandem mass spectrometry (LC-MS/MS).******For both projects, it is critical to measure local levels of glucocorticoids in microdissected tissue: brain and lymphoid organs (Soma) or reproductive tissues (Collier). This key task faces technical challenges, in particular the need for specific and ultrasensitive assays. With regard to specificity, mass spectrometry is the gold standard, and our labs are pioneers in developing mass spectrometry assays for steroids. With regard to sensitivity, the steroid assays in our labs are among the most sensitive in the world. However, steroids do not ionize readily, which limits the ability to measure low amounts of steroids (. The microLC can be integrated seamlessly with our existing state-of-the-art mass spectrometers. Failure to obtain the requested equipment will greatly delay our studies on local glucocorticoids.******This equipment is urgently needed to understand how stress “gets under the skin” to affect physiology. In addition, the advances in mass spectrometry will accelerate endocrine research worldwide and equip our HQP with cutting-edge technical skills that are in high demand in academia and industry. The fundamental knowledge gained from this project will impact a variety of disciplines, including endocrinology, enzymology, physiology, neuroscience, immunology, and analytical chemistry.