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

项目摘要 几项研究将精神健康问题，如重度抑郁症，双相情感障碍和精神分裂症， G蛋白偶联受体（GPCR），包括肾上腺素能、多巴胺能和胆碱能受体。 同时，通过人类基因组计划确定了140多个GPCR， 内源性配体仍然未知。为了寻找这些配体，出现了对能够 提供了配体亲和力及其推定细胞反应的详细信息。配体的研究- 受体相互作用理想地应尽可能接近生物细胞的天然状态发生， 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），其中可以通过形成液体来原位监测配体-受体相互作用 两个熔融石英毛细管之间的连接处，并将它们浸没在表面顶部，细胞充当 神经传递的生物模型。第一个毛细管充当灌注系统，第二个毛细管拉动 并在MS入口处提供分析物喷雾用于检测。测量能力 配体-受体结合事件，随后神经递质的释放和神经递质的任何酶促转化， 通过MS的多路复用能力使得在细胞表面上发生的释放分子成为可能。 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PC 12细胞系， 将筛选几种经典神经递质对内源性毒蕈碱受体的亲和力， 对这种新方法的概念验证。该技术还将用于测量蜂窝 反应，如从PC 12细胞释放多巴胺。该技术的建立是 在某种意义上它可以应用于几乎任何类型的组织和配体-受体 互动 理解神经元中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 突破性的研究将开发出一种方法来测量表面上的配体受体相互作用， 用质谱仪分析细胞。为了检测和测量这些事件与环境采样耦合到MS 迄今为止从未做过。这是一项具有挑战性但又有可能完成的任务，将通过合作来实现 斯坦福大学的Zare小组和华中师范大学的Zhong小组之间。的 该项目结合了环境声波喷雾电离与经典的微灌注，纳米流的最新进展 液相色谱和高分辨率MS，并预计将询问配体-受体相互作用， 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.