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.

项目概要 多项研究将重度抑郁症、双相情感障碍和精神分裂症等心理健康问题与 G 蛋白偶联受体 (GPCR)，包括肾上腺素能受体、多巴胺能受体和胆碱能受体。 同时，通过人类基因组计划鉴定了超过 140 个 GPCR，它们的 内源配体仍然未知。在寻找这些配体的过程中，迫切需要能够 提供配体亲和力及其假定细胞反应的详细信息。配体的研究—— 理想情况下，受体相互作用应尽可能接近生物细胞的天然状态 最小扰动。在这里，我们建议开发一种基于环境采样的新型细胞功能探针 质谱 (MS)，可以通过形成液体来原位监测配体-受体相互作用 两个熔融石英毛细管之间的连接处，并将它们浸入表面顶部，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 位于液接部上，并在 MS 入口处喷射分析物以进行检测。测量能力 配体-受体结合事件，随后释放的神经递质以及任何酶促转化 MS 的多重功能使得释放的分子发生在细胞表面成为可能。 该技术不需要衍生化来检测分析物，并且是非侵入性的。使用PC12细胞系， 将针对内源性毒蕈碱受体筛选几种经典神经递质的亲和力，作为 这种新颖方法的概念验证。该技术还将用于执行细胞的测量 PC12 细胞释放多巴胺等反应。所提出技术的建立是 高度转化是指它几乎可以应用于任何类型的组织和配体受体 相互作用。 正如科学家们所认识到的，理解神经元细胞机制的复杂性是一项具有挑战性的任务。 大脑主动性和配体-受体相互作用是细胞间信号传导的基本组成部分。拟议的 突破性的研究将开发一种测量配体-受体相互作用的方法 细胞使用质谱分析。为了通过与 MS 耦合的环境采样来检测和测量这些事件， 迄今为止从未做过。这是一项具有挑战性但可能的任务，将通过协作来实现 斯坦福大学的扎尔小组和华中师范大学的钟小组之间。这 该项目将环境声波喷雾电离的最新进展与经典的微灌注、纳流相结合 液相色谱和高分辨率 MS，并有望探究配体-受体相互作用 活细胞。