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

项目总结 几项研究将严重抑郁、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(GPCRs)，包括肾上腺素能、多巴胺和胆碱能受体。 同时，通过人类基因组计划确定了140多个GPCR，它们的 内源性配体尚不清楚。在寻找这些配体的过程中，出现了对能够 提供配基亲和力及其可能的细胞反应的详细信息。配基的研究- 理想情况下，受体的相互作用应尽可能接近生物细胞的天然状态 微扰最小。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 质谱学(MS)，其中配体-受体的相互作用可以通过形成液体来原位监测 两个熔融二氧化硅毛细管之间的连接，并将它们浸泡在表面的顶部，细胞充当 神经传递的生物学模型。第一根毛细血管起到灌流系统的作用，第二根毛细血管 在液体连接处，并在MS入口处喷射分析物进行检测。测量能力 配体-受体结合事件，随后神经递质的释放和任何酶转化 发生在细胞表面的释放分子是通过MS的多路传输能力来实现的。 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， 几种经典神经递质的亲和力将作为一种内源性M受体进行筛选 这一新方法的概念验证。这项技术还将用于对细胞进行测量 PC12细胞释放多巴胺等反应。建议的技术的建立是 高度翻译的意义是，它可以应用于几乎任何类型的组织和配体受体 互动。 了解神经元细胞机制的复杂性是一项具有挑战性的任务， 大脑的主动性和配体-受体的相互作用是细胞间信号传递的基本部分。建议数 开创性的研究将开发一种测量配体-受体表面相互作用的方法 细胞使用质谱学。为了利用耦合到MS的环境采样来检测和测量这些事件 到目前为止，还没有人这么做过。这是一项具有挑战性但有可能通过协作实现的任务 在斯坦福大学的扎尔小组和中部中国师范大学的中小组之间。这个 该项目将环境声波喷雾电离的最新进展与经典的微灌注、纳米流相结合 高效液相色谱和高分辨MS，有望研究配体-受体之间的相互作用 活细胞。

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.