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）：研究是为了确定化学刺激对（R）-NMSAL（帕金森氏症神经毒素）的生物合成和代谢的影响，并在缺血条件下释放D-SER（最近鉴定出的神经递质 /调节剂）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法（MCE-MS/MS）的新手性分析方法，以进行单个细胞的高通量手性分析。我们计划将手性选择器分子连接到缩短的单壁碳纳米管上，然后将手性选择器键入的碳纳米管固定在通道中，从而产生高效且稳定的手性MECE分离通道。还提出了一种新的微芯片设计，该设计还可以与质谱仪的纳米 - ESI组件直接且简便的耦合，并将评估。手性MCE-MS/MS方法到位后，将进行拟议的代谢研究。尽管有充分的文献证明，（R）NMSAL诱导大鼠帕金森氏症，但研究其生物合成和代谢远非足够。我们计划将PC-12或SH-SY5Y细胞与氘标记的Salsolinol（即Sal-- ,,,,,,1）或（R）-NMSAL一起孵育。孵育后，使用开发的手性MCE-MS/MS方法来量化感兴趣化合物的细胞外和细胞内水平。我们希望从单细胞分析中检测到更多的代谢产物，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物谱，从而更好地了解该神经毒素的生物合成和代谢。我们还旨在研究在缺血条件下D-SER的细胞摄取和释放。一些实验室证据表明，D-SER参与引起缺血性脑损伤。但是，到目前为止，还没有对神经细胞对缺血的反应进行研究。在这项研究中，我们将部署暴露于氧气 - 葡萄糖剥夺（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，将将细胞与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将通过在不同时间点（1、5、10、30、60、120分钟）分析培养基或单细胞分析来量化。同时，OGD损伤引起的细胞损伤将通过caspase-3测定法进行评估。对于上述三个具体目的，我们的工作假设是：1）可以通过固定手性选择器键入的碳纳米管来制备高效且耐用的手性MCE分离通道，这将导致手性MCE-MS/MS方法的发展，以实现单个细胞的高透射手学分析； 2）暴露于酒精或氧化应激 - 诱导MN2+会影响（R） - NMSAL的生物合成和代谢； 3）由于细胞对缺血的反应，在缺血状态下，细胞摄取和D-SER的释放改变了。该项目开发的手性MCE-MS/MS分析方法将具有长期的生物医学研究价值，特别是用于探测涉及手性的细胞代谢。关于（R）-NMSAL和D-SER的代谢研究将有助于我们对分子生物学水平上某些神经系统疾病的理解，包括缺血性疾病下D-SER的神经学意义以及（R）-NMS-NMSAL诱导parkinsisism的机制。关键词：新型的生物分析方法，手性微芯片电泳质量光谱法，细胞水平的代谢研究，帕金森神经毒素，（R） - n-甲基甲硅烷醇，D-塞罗，缺血。 公共卫生相关性：该SC1应用程序中提出的研究旨在确定化学刺激对（R）-NMSAL（帕金森氏症神经毒素）的生物合成和代谢的影响，并表征了D-SER的细胞摄取和释放（最近鉴定出的神经递质 /Modulator）。为了实现研究目标，将开发基于微芯片电泳质谱法（MCE-MS/MS）的新手性分析方法，用于单个细胞的高通量手性分析。拟议的手性MCE-MS/MS方法的成功开发将对生物医学研究具有长期价值，特别是用于探测涉及手性的细胞代谢。关于（R）-NMSAL和D-SER的代谢研究将有助于我们对分子生物学水平上某些神经系统疾病的理解，包括缺血性疾病下D-SER的神经学意义以及（R）-NMS-NMSAL诱导parkinsisism的机制。