Chiral Microchip Electrophoresis - Mass Spectrometric Methods for Metabolic Studi

描述(申请人提供)：本研究旨在确定化学刺激对(R)-NMSal(一种帕金森病神经毒素)生物合成和代谢的影响，并表征细胞在缺血条件下对D-Ser(一种新近发现的神经递质/调节剂)的摄取和释放。为了实现这一研究目标，基于微芯片电泳法-串联质谱仪(MCE-MS/MS)的新型手性分析方法将被开发用于单细胞的高通量手性分析。我们计划将手性选择剂分子共价连接到缩短的单壁碳纳米管上，然后将手性选择剂键合的碳纳米管固定在通道中，生成高效稳定的手性MCE分离通道。还提出了一种新的微芯片设计，使MCE能够与质谱仪的纳米ESI组件直接和方便地耦合，并将对其进行评估。在手性MCE-MS/MS方法到位后，将进行拟议的代谢研究。虽然已有文献证明(R)-NMSal可诱导大鼠帕金森病，但对其生物合成和代谢的研究还远远不够。我们计划用氚标记的丹参素(即Sal-，，，1-d4)或(R)-NMSal孵育PC-12或SH-SY5Y细胞。孵育后，感兴趣的化合物的胞外和胞内水平将使用开发的手性MCE-MS/MS方法进行定量。我们预计将从单细胞分析中检测到更多的代谢物，因为代谢物在细胞内的浓度远远高于细胞外的浓度。因此，将获得更准确的代谢物分布，从而更好地了解这种神经毒素的生物合成和代谢。我们还旨在研究细胞在缺血条件下对D-丝氨酸的摄取和释放。一些实验室证据表明，D-丝氨酸参与了缺血性脑损伤。然而，目前还没有从D-丝氨酸的加工和利用方面对神经细胞对缺血的反应进行研究。在本研究中，我们将PC-12细胞和培养的皮层神经元置于缺氧-葡萄糖剥夺(OGD)环境中作为体外缺血模型。将细胞与D-丝氨酸-2，3，3-d3(D-丝氨酸-d3)或L-丝氨酸-2，3，3，-d3在正常或OGD条件下孵育。在不同的时间点(1、5、10、30、60、120min)，通过分析培养液或通过单细胞分析来定量细胞内和细胞外的D-Ser-d3。同时，用半胱氨酸天冬氨酸氨基转移酶-3(Caspase-3)法检测OGD损伤后的细胞损伤。针对上述三个具体目标，我们的工作假设是：1)通过将手性选择器结合的碳纳米管固定在通道中，可以制备高效和持久的手性MCE分离通道，这将导致高通量单细胞手性分析的手性MCE-MS/MS方法的发展；2)暴露于酒精或氧化应激诱导的Mn2+影响(R)-NMSal的生物合成和代谢；3)由于细胞对缺血的反应，细胞对D-丝氨酸的摄取和释放在缺血条件下发生改变。本项目开发的手性MCE-MS/MS分析方法将在生物医学研究中具有长期价值，特别是在探索涉及手性的细胞代谢方面。对(R)-NMSal和D-Ser的代谢研究将有助于我们从分子生物学水平上了解某些神经系统疾病，包括D-Ser在缺血条件下的神经学意义以及(R)-NMSal诱导帕金森症的机制。关键词：生物分析新方法、手性微芯片电质联用、细胞水平代谢研究、帕金森神经毒素、(R)-N-甲基沙龙醇、D-丝氨酸、缺血。 公共卫生相关性：本SC1申请中提出的研究旨在确定化学刺激对(R)-NMSal(一种帕金森神经毒素)生物合成和代谢的影响，并表征细胞在缺血条件下对D-Ser(一种新近发现的神经递质/调节剂)的摄取和释放。为了实现这一研究目标，基于微芯片电泳法-串联质谱仪(MCE-MS/MS)的新型手性分析方法将被开发用于单细胞的高通量手性分析。手性MCE-MS/MS方法的成功开发将对生物医学研究，特别是对涉及手性的细胞代谢的探测具有长期的价值。对(R)-NMSal和D-Ser的代谢研究将有助于我们从分子生物学水平上了解某些神经系统疾病，包括D-Ser在缺血条件下的神经学意义以及(R)-NMSal诱导帕金森症的机制。

DESCRIPTION (provided by applicant): The research is to determine the effects of chemical stimuli on the biosynthesis and metabolism of (R)-NMSal (a Parkinsonian neurotoxin) and to characterize the cellular uptake and release of D-Ser (a recently identified neurotransmitter /modulator) under ischemic conditions. To achieve the research goals, new chiral analytical methods based on microchip electrophoresis-tandem mass spectrometry (MCE-MS/MS) will be developed for high throughput chiral analysis of single cells. We plan to covalently attach chiral selector molecules onto shortened single walled carbon nanotubes and then to immobilize the chiral selector-bonded carbon nanotubes in the channel, producing highly effective and stable chiral MCE separation channels. A new microchip design that enables a direct and facile coupling of MCE with a nano-ESI assembly of a mass spectrometer is also proposed and will be assessed. After the chiral MCE-MS/MS method is in place, the proposed metabolic studies will be carried out. Although it is well documented that (R)-NMSal induces Parkinsonism in rats, study on its biosynthesis and metabolism is far from adequate. We plan to incubate PC-12 or SH-SY5Y cells with deuterium-labeled salsolinol (i.e. Sal-,,,1-d4) or (R)-NMSal. After incubation, both extracellular and intracellular levels of the compounds of interest will be quantified by using the developed chiral MCE-MS/MS method. We expect that more metabolites will be detected from single cell analysis because the intracellular concentrations of metabolites are much higher than their extracellular concentrations. Therefore, a more accurate metabolite profile will be obtained, leading to a better understanding of the biosynthesis and metabolism of this neurotoxin. We also aim to investigate the cellular uptake and release of D-Ser under ischemic conditions. Some lab evidences indicated that D-Ser was involved in causing ischemic brain damage. However, no studies on the responses of nerve cells to ischemia in terms of processing and utilizing D-Ser have been carried out so far. We will deploy PC-12 cells and cultured cortical neurons exposed to oxygen-glucose deprivation (OGD) as in vitro ischemia models in this research. The cells will be incubated with either D-Ser-2,3,3-d3 (D-Ser-d3) or L-Ser- 2,3,3,-d3 under normal or OGD conditions. Both intracellular and extracellular D-Ser-d3 will be quantified by analyzing the culture medium or through single cell analysis at different time points (1, 5, 10, 30, 60, 120 min). In parallel, OGD insult-induced cell injury will be assessed by Caspase-3 assay. For the above stated three specific aims, our working hypotheses are: 1) highly efficient and durable chiral MCE separation channels can be prepared by immobilizing chiral selector-bonded carbon nanotubes in the channel, which will lead to the development of chiral MCE-MS/MS methods for high throughput chiral analysis of single cells; 2) exposure to alcohol or oxidative stress- inducing Mn2+ affects the biosynthesis and metabolism of (R)-NMSal; 3) cellular uptake and release of D-Ser is altered under ischemic conditions as a result of the cells' responses to ischemia. The chiral MCE-MS/MS analytical methods developed in this project will have long-term value for biomedical research, particularly for probing cellular metabolism involving chirality. The metabolic studies on (R)-NMSal and D-Ser will contribute to our understanding of certain neurological diseases at the molecular biology level including the neurological significance of D-Ser under ischemic conditions and the mechanism by which (R)-NMSal induces Parkinsonism. Key words: Novel bioanalytical methods, chiral microchip electrophoresis-mass spectrometry, metabolic study at cellular levels, Parkinsonian neurotoxin, (R)-N- methylsalsolinol, D-serine, ischemia. PUBLIC HEALTH RELEVANCE: The research proposed in this SC1 application aims to determine the effects of chemical stimuli on the biosynthesis and metabolism of (R)-NMSal (a Parkinsonian neurotoxin) and to characterize the cellular uptake and release of D-Ser (a recently identified neurotransmitter /modulator) under ischemic conditions. To achieve the research goals, new chiral analytical methods based on microchip electrophoresis- tandem mass spectrometry (MCE-MS/MS) will be developed for high throughput chiral analysis of single cells. Successful development of the proposed chiral MCE-MS/MS methods will have long-term value for biomedical research, particularly for probing cellular metabolism involving chirality. The metabolic studies on (R)-NMSal and D-Ser will contribute to our understanding of certain neurological diseases at the molecular biology level including the neurological significance of D-Ser under ischemic conditions and the mechanism by which (R)-NMSal induces Parkinsonism.