Chiral Microchip Electrophoresis - Mass Spectrometric Methods for Metabolic Studi

描述（由申请人提供）：该研究旨在确定化学刺激对(R)-NMSal（一种帕金森神经毒素）的生物合成和代谢的影响，并表征D-Ser（一种最近发现的神经递质/调节剂）在缺血条件下的细胞摄取和释放。为了实现研究目标，将开发基于微芯片电泳-串联质谱（MCE-MS/MS）的手性分析新方法，用于单细胞的高通量手性分析。我们计划将手性选择器分子共价连接到缩短的单壁碳纳米管上，然后将手性选择器键合的碳纳米管固定在通道中，从而产生高效稳定的手性MCE分离通道。本文还提出了一种新的微芯片设计，可以直接和方便地将MCE与质谱仪的纳米esi组件耦合，并将对其进行评估。手性MCE-MS/MS方法到位后，将进行拟议的代谢研究。虽然(R)-NMSal诱导大鼠帕金森病的研究已得到充分证实，但对其生物合成和代谢的研究还远远不够。我们计划用氘标记的salsolinol（即Sal-…1-d4）或(R)- nmsal孵育PC-12或SH-SY5Y细胞。孵育后，将使用开发的手性MCE-MS/MS方法对感兴趣的化合物的细胞外和细胞内水平进行定量。我们期望从单细胞分析中检测到更多的代谢物，因为代谢物的细胞内浓度远高于细胞外浓度。因此，将获得更准确的代谢物谱，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D-Ser在缺血条件下的细胞摄取和释放。一些实验证据表明D-Ser参与了缺血性脑损伤的发生。然而，目前还没有关于神经细胞在缺血时对D-Ser的加工和利用的反应的研究。在本研究中，我们将使用缺氧葡萄糖剥夺（OGD）下的PC-12细胞和培养的皮质神经元作为体外缺血模型。将细胞与d - ser -2,3,3-d3 （D-Ser-d3）或L-Ser- 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、120分钟），通过分析培养基或单细胞分析来定量细胞内和细胞外的D-Ser-d3。同时，OGD胰岛素诱导的细胞损伤将通过Caspase-3检测进行评估。针对上述三个具体目标，我们的工作假设是：1)通过在通道中固定手性选择器键合的碳纳米管，可以制备出高效耐用的手性MCE分离通道，这将导致用于单细胞高通量手性分析的手性MCE-MS/MS方法的发展；2)暴露于酒精或氧化应激诱导的Mn2+会影响(R)- nmsal的生物合成和代谢；3)在缺血条件下，由于细胞对缺血的反应，细胞对D-Ser的摄取和释放发生改变。本项目开发的手性MCE-MS/MS分析方法将对生物医学研究具有长期价值，特别是对涉及手性的细胞代谢的探测。对(R)-NMSal和D-Ser的代谢研究将有助于我们在分子生物学水平上对某些神经系统疾病的认识，包括缺血条件下D-Ser的神经学意义以及(R)-NMSal诱导帕金森病的机制。关键词：新型生物分析方法，手性微芯片电泳-质谱，细胞水平代谢研究，帕金森神经毒素，(R)- n -甲基沙索林醇，d -丝氨酸，缺血

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.