Multiplex single-cell analysis of intracellular protein signaling dynamics

摘要 单细胞分析方法的最新进展为细胞异质性提供了一个清晰的观点 及其相关的机制和治疗方面的影响。然而，细胞内的单细胞研究 蛋白质信号活动可能会被信号事件的动态性质所混淆。此外，还有一个 迫切需要开发临床样本的非遗传分析方法。这些必备条件要求 单细胞方法，可以询问细胞内蛋白质信号动力学，同时兼容 其他下游分析方法。最近开发的一种原型方法使动态探测成为可能 细胞内蛋白质信号在单细胞分辨率下的活性，不需要遗传修饰。技术 是基于细胞内传递的表位靶向多肽探针，一种基于微孔的单细胞芯片和 高速成像技术。然而，存在许多障碍，挑战了这一点的普遍性 探讨并限制其在生物医学上的应用。在这项提案中，目的是显著推进这一证明- 概念技术。包括五个具体目标：开发一个自动筛查方案，以改善 筛选效率，实施独特的双环肽库以获得更高的结合亲和力，使用细胞 渗透剂作为一种更具普适性的传递方法，实现了多种信号的同时分析 蛋白质，并将这项技术与其他单细胞检测方法相结合。这一集成的多路传输流水线 将提供一种使能技术，承诺更深入地了解蛋白质之间的相互作用 生物过程中的信号活动、蛋白质表达水平和代谢。

ABSTRACT Recent developments on single-cell analytical methods have provided a clarifying view on cellular heterogeneity and its associated mechanistic and therapeutic implications. Nevertheless, single-cell studies on intracellular protein signaling activities can be confounded by the dynamic nature of signaling events. In addition, there is a pressing need of developing non-genetic analytical methods for clinical samples. These requisites call for a single-cell approach that can interrogate intracellular protein signaling dynamics while being compatible with other downstream analytical methods. A recently developed prototype method has enabled dynamic probing of intracellular protein signaling activities at single-cell resolution, without genetic modifications. The technology was based on intracellularly delivered epitope-targeting peptide probes, a microwell-based single-cell chip and high-speed imaging techniques. However, many obstacles exist that challenge the generalizability of this approach and limit its biomedical application. In this proposal, the aim is to significantly advance this proof-of- concept technology. Five specific goals are included: develop an automated screening protocol for improved screening efficiencies, implement a unique bicyclic peptide library for higher biding affinities, employ cell permeabilizers as a more generalizable delivery method, achieve simultaneous analysis of multiple signaling proteins, and integrate this technology with other single-cell detection methods. This integrated multiplex pipeline will provide an enabling technology that promises a more in-depth understanding of the interplay among protein signaling activities, protein expression levels and metabolism in biological processes.