Chiral Microchip Electrophoresis - Mass Spectrometric Methods for Metabolic Studi

描述（由申请人提供）：本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，并表征缺血条件下细胞摄取和释放D-Ser（一种最近鉴定的神经递质/调节剂）的特征。为实现这一研究目标，将开发基于微芯片串联质谱（MCE-MS/MS）的单细胞手性分析新方法。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，产生高效稳定的手性MCE分离通道。一种新的微芯片设计，使直接和方便的耦合MCE与纳米ESI组装的质谱仪也提出了，并将进行评估。在手性MCE-MS/MS方法到位后，将进行拟定的代谢研究。虽然有充分的文献证明（R）-NMSal在大鼠中诱导帕金森综合征，但对其生物合成和代谢的研究还远远不够。我们计划将PC-12或SH-SY 5 Y细胞与氘标记的猪毛菜醇（即Sal-1，1-d4）或（R）-NMSal孵育。孵育后，将使用开发的手性MCE-MS/MS方法定量目标化合物的细胞外和细胞内水平。我们预计，更多的代谢物将从单细胞分析中检测到，因为代谢物的细胞内浓度远高于其细胞外浓度。因此，将获得更准确的代谢产物谱，从而更好地了解这种神经毒素的生物合成和代谢。我们还旨在研究缺血条件下细胞摄取和释放D-丝氨酸。实验室研究表明，D-丝氨酸参与了缺血性脑损伤的发生。然而，迄今为止还没有关于神经细胞在加工和利用D-丝氨酸方面对缺血的反应的研究。本研究将PC-12细胞和培养的皮层神经元暴露于氧-葡萄糖剥夺（OGD）作为体外缺血模型。在正常或OGD条件下，将细胞与D-Ser-2，3，3-d3（D-Ser-d3）或L-Ser-2，3，3-d3一起孵育。细胞内和细胞外D-Ser-d3将通过分析培养基或通过不同时间点（1、5、10、30、60、120 min）的单细胞分析进行定量。同时，将通过胱天蛋白酶-3测定评估OGD损伤诱导的细胞损伤。针对上述三个具体目标，我们的工作假设是：1）通过将手性选择剂键合的碳纳米管固定在通道中，可以制备出高效、耐用的手性MCE分离通道，这将导致手性MCE-MS/MS方法的发展，用于单细胞的高通量手性分析; 2）暴露于酒精或氧化应激诱导的Mn 2+影响（R）-NMSal的生物合成和代谢; 3）在缺血条件下，细胞对D-Ser的摄取和释放由于细胞对缺血的反应而改变。本计画所发展之手性MCE-MS/MS分析方法，对于生物医学研究，特别是对于探讨细胞代谢之手性，将具有长远的价值。对（R）-NMSal和D-Ser代谢的研究将有助于我们在分子生物学水平上理解某些神经系统疾病，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：新的生物分析方法，手性微芯片质谱，细胞水平的代谢研究，帕金森神经毒素，（R）-N-甲基猪毛菜醇，D-丝氨酸，缺血。 公共卫生相关性：本SC 1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，并表征缺血条件下细胞摄取和释放D-Ser（一种最近鉴定的神经递质/调节剂）的特征。为实现这一研究目标，将发展基于微芯片电泳-串联质谱（MCE-MS/MS）的单细胞手性分析新方法。手性MCE-MS/MS方法的成功开发将对生物医学研究具有长期价值，特别是对于探索涉及手性的细胞代谢。对（R）-NMSal和D-Ser代谢的研究将有助于我们在分子生物学水平上理解某些神经系统疾病，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。

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.