Chiral Microchip Electrophoresis - Mass Spectrometric Methods for Metabolic Studi

手性微芯片电泳 - 代谢研究的质谱方法

• 批准号: 8029590
• 负责人: YIMING LIU
• 金额: $ 17.47万
• 依托单位: JACKSON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8029590
• 关键词: Affect; Anabolism; Area; Attention; Award; Beds; Biological; Biological Assay; Biomedical Research; Brain Ischemia; Carbon Nanotubes; Cell Culture Techniques; Cell Surface Receptors; Cells; Chemicals; Coupled; Coupling; Culture Media; Cultured Cells; Cyclodextrins; Data; Detection; Deuterium; Development; Evaluation; Financial Support; Funding Opportunities; Glucose; Glues; Glycopeptides; Goals; Grant; Health; High Pressure Liquid Chromatography; Immobilization; In Vitro; Incubated; Ions; Ischemia; Ischemic Brain Injury; Isotope Labeling; Kinetics; Label; Lead; Mainstreaming; Mass Spectrum Analysis; Metabolic; Metabolic Pathway; Metabolism; Methodology; Methods; Microchip Electrophoresis; Modeling; Molecular Biology; National Institute of Neurological Disorders and Stroke; Neuroglia; Neurologic; Neurons; Neuropharmacology; Neurotoxins; Neurotransmitters; Organism; Oxidative Stress; Oxygen; PC12 Cells; Parkinsonian Disorders; Phase; Procedures; Process; Proteins; Rattus; Research; Research Infrastructure; Research Personnel; Research Project Grants; Serine; Staging; Stimulus; Techniques; Time; Tubular formation; United States National Institutes of Health; Work; alcohol exposure; analytical method; base; career; caspase-3; cell injury; deprivation; design; enantiomer; experience; extracellular; high throughput analysis; improved; interest; killings; mass spectrometer; metabolic abnormality assessment; microchip; nano; nervous system disorder; neurochemistry; neurotoxicology; novel; response; salsolinol; serine containing aminolipid; single cell analysis; single walled carbon nanotube; tandem mass spectrometry; uptake

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.

描述（由申请人提供）：该研究旨在确定化学刺激对 (R)-NMSal（一种帕金森病神经毒素）生物合成和代谢的影响，并表征细胞摄取和释放 D-Ser（一种最近发现的神经递质） /调制器）在缺血条件下。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）的新手性分析方法，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，产生高效且稳定的手性MCE分离通道。还提出了一种新的微芯片设计，该设计可以将 MCE 与质谱仪的纳米 ESI 组件直接轻松地耦合，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。尽管(R)-NMSal可诱导大鼠帕金森病已有充分记录，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。我们还旨在研究缺血条件下细胞对 D-Ser 的摄取和释放。一些实验室证据表明 D-Ser 与引起缺血性脑损伤有关。然而，目前尚未开展神经细胞加工和利用D-Ser对缺血反应的研究。在本研究中，我们将使用暴露于氧糖剥夺（OGD）的 PC-12 细胞和培养的皮质神经元作为体外缺血模型。在正常或 OGD 条件下，细胞将与 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 分钟）的单细胞分析来定量。同时，OGD 损伤诱导的细胞损伤将通过 Caspase-3 测定进行评估。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剂键合的碳纳米管，可以制备高效、耐用的手性MCE分离通道，这将导致手性MCE-MS/的发展。单细胞高通量手性分析的 MS 方法； 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-Ser 的摄取和释放在缺血条件下发生改变。该项目开发的手性 MCE-MS/MS 分析方法将对生物医学研究具有长期价值，特别是对于探索涉及手性的细胞代谢。 (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理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诱发帕金森病的机制。关键词：新型生物分析方法，手性微芯片电泳-质谱法，细胞水平代谢研究，帕金森神经毒素，(R)-N-甲基野马酚，D-丝氨酸，缺血。 公共健康相关性：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 D-Ser（最近的一种在缺血条件下确定的神经递质/调节剂）。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）的新手性分析方法，用于单细胞的高通量手性分析。所提出的手性 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。 (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理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诱发帕金森病的机制。