Ligand-Receptor Dynamics and Cellular Responses Studied In Situ Using Venturi Easy Ambient Sonic-Spray Ionization Mass Spectrometry

使用文丘里易环境声喷雾电离质谱法原位研究配体受体动力学和细胞响应

• 批准号: 9245575
• 负责人: Richard N Zare
• 金额: $ 11.52万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9245575
• 关键词: Adrenergic Agents; Adsorption; Affinity; Agonist; Alpha Cell; Atmospheric Pressure; BRAIN initiative; Binding; Biological; Biological Models; Bipolar Disorder; Blood capillaries; Buffers; Cell Line; Cell physiology; Cell surface; Cells; Chemicals; China; Cholinergic Receptors; Collaborations; Competitive Binding; Complex; Coupled; Data; Detection; Development; Disease; Dopamine; Electrospray Ionization; Event; Exocytosis; Freezing; G-Protein-Coupled Receptors; Gases; Goals; Human Genome Project; In Situ; Investigation; Lasers; Ligands; Link; Liquid Chromatography; Liquid substance; Major Depressive Disorder; Mass Spectrum Analysis; Measurement; Measures; Mental Health; Mental disorders; Methods; Monitor; Muscarinic Acetylcholine Receptor; Nebulizer; Neurons; Neuropeptides; Neurotransmitters; Nitrogen; PC12 Cells; Pattern; Perfusion; Physiologic pulse; Preparation; Public Health; Reaction; Research; Resolution; Sampling; Schizophrenia; Signal Transduction; Silicon Dioxide; Sodium Chloride; Sonication; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stream; Surface; System; Systems Analysis; Techniques; Temperature; Time; Tissues; Tube; Universities; Vacuum; Work; base; capillary; ionization; neurotransmission; neurotransmitter release; novel; receptor; receptor binding; response; sample fixation; tool; ultraviolet; virtual; voltage

PROJECT SUMMARY Several studies link mental health problems such as major depression, bipolar disorder and schizophrenia with G-protein coupled receptors (GPCRs), including adrenergic, dopaminergic and cholinergic receptors. Simultaneously, there are more than 140 GPCRs identified through the human genome project where their endogenous ligand is still unknown. In search of these ligands, there is an emerging need for methods that can provide detailed information of ligand affinity and their putative cellular response. Investigations of ligand– receptor interactions should ideally take place as close as possible to the native state of a biological cell with minimal perturbation. Here, we propose to develop a novel probe of cell function, based on ambient sampling mass spectrometry (MS), where ligand–receptor interactions can be monitored in situ by forming a liquid junction between two fused silica capillaries and immersing them on top of a surface with cells acting as a biological model for neurotransmission. The first capillary acts as a perfusion system, the second capillary pulls on the liquid junction and delivers a spray of analytes at the MS-inlet for detection. The capability of measuring a ligand-receptor binding event, the subsequent release of neurotransmitters and any enzymatic conversion of the released molecules taking place on the cell surface is made possible by the multiplexing abilities of MS. The technique does not require derivatization to detect analytes and is non-invasive. Using the PC12 cell line, the affinity of several classic neurotransmitters will be screened against endogenous muscarinic receptors as a proof-of-concept for this novel method. The technique will also be used to perform measurements of cellular responses such as release of dopamine from PC12 cells. The establishment of the proposed technique is highly translational in the sense that it may be applied to virtually any type of tissue and ligand-receptor interaction. Understanding the complexity of cellular mechanisms in neurons is a challenging task as recognized by the BRAIN initiative and ligand-receptor interactions are a fundamental part of cell-to-cell signaling. The proposed groundbreaking research will develop a method for measuring ligand–receptor interactions on the surface of a cell using mass spectrometry. To detect and measure these events with ambient sampling coupled to MS has hitherto never been done. This is a challenging yet possible task that will be achieved through collaboration between the Zare group at Stanford University and the Zhong group at Central China Normal University. The project combines recent advances of ambient sonic spray ionization with classic microperfusion, nanoflow liquid chromatography and high-resolution MS, and is expected to interrogate ligand–receptor interactions on live cells.

项目摘要 几项研究将重度抑郁症，躁郁症和精神分裂症等心理健康问题与 G蛋白偶联受体（GPCR），包括肾上腺素，多巴胺能和胆碱能受体。 同时，通过人类基因组项目确定了140多个GPCR 内源配体仍然未知。为了寻找这些配体，对可以的方法有一个新兴的需求 提供配体亲和力及其推定的细胞反应的详细信息。配体调查 - 理想情况下应尽可能接近受体相互作用 最小扰动。在这里，我们建议基于环境采样来开发一种新颖的细胞功能探针 质谱法（MS），其中可以通过形成液体来原位监测配体 - 受体相互作用 两个熔融二氧化硅毛细血管之间的结，将它们浸入表面上，细胞充当 神经传递的生物学模型。第一个毛细管充当灌注系统，第二个毛细管拉动 在液体连接处，并在MS-INLET处提供一系列分析物进行检测。测量的能力 配体受体结合事件，随后的神经递质的释放和任何酶促转化 通过MS的多路复用能力使释放的分子发生在细胞表面。 该技术不需要衍生化来检测分析物，并且是无创的。使用PC12单元线， 几种经典神经递质的亲和力将针对内源性毒蕈碱受体进行筛选 这种新方法的概念概念。该技术还将用于对细胞进行测量 诸如从PC12细胞中释放多巴胺的反应。拟议技术的建立是 高度翻译的意义上可以将其应用于几乎任何类型的组织和配体受体 相互作用。 了解神经元中细胞机制的复杂性是一项挑战任务，如 大脑启动和配体受体相互作用是细胞对细胞信号传导的基本组成部分。提议 开创性的研究将开发一种测量在A表面上的配体 - 受体相互作用的方法 使用质谱法。用与MS的环境采样相结合的环境采样来检测和测量这些事件 迄今从未完成过。这是一个挑战，但可以通过协作来实现的任务 在斯坦福大学的Zare集团与中国师范大学的中央官方集团之间。这 项目结合了环境声音喷雾电离与经典微型灌注的最新进展 液相色谱和高分辨率MS，有望在对配体 - 受体相互作用上的相互作用 活细胞。