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Multiplex single-cell analysis of intracellular protein signaling dynamics

细胞内蛋白质信号传导动力学的多重单细胞分析

基本信息

批准号：
10152653
负责人：
Min Xue
金额：
$ 18.43万
依托单位：
UNIVERSITY OF CALIFORNIA RIVERSIDE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-07 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10152653
关键词：
Adopted Affect Affinity BRAF gene Bar Codes Biological Biological Assay Biological Process Biomedical Research Cell Line Cells Characteristics Clinical Complex Data Set Development Disease Progression Disease Resistance Eligibility Determination Epitopes Event Exercise Exhibits Fluorescent Probes Genetic Goals Heterogeneity Image Imaging Techniques Immobilization Immune response Institution Intracellular Signaling Proteins Libraries MAPK8 gene MCF7 cell MEKs Mass Spectrum Analysis Measurement Metabolic Metabolism Methods Microscope Modification Monitor Nature Oncology Peptide Library Peptides Pharmaceutical Preparations Phosphorylation Process Protein Analysis Protein Dynamics Proteins Proteomics Proto-Oncogene Proteins c-akt Protocols documentation Research Personnel Resistance development Resolution Sampling Scanning Signal Transduction Signaling Protein Speed Statistical Data Interpretation Surface System Technology Therapeutic Time Treatment Efficacy Uncertainty Work analytical method base biological systems cell type design detection method experience fluorophore improved inhibitor/antagonist insight non-genetic protein expression prototype screening single cell analysis single cell proteins single cell technology success therapeutic evaluation tumor progression

项目摘要

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.

摘要单细胞分析方法的最新发展为细胞异质性提供了一个清晰的视角及其相关的机制和治疗意义。然而，单细胞研究细胞内蛋白质信号传导活性可能被信号传导事件的动态性质所混淆。此外还有迫切需要开发用于临床样品的非遗传分析方法。这些问题需要一个单细胞方法，可以询问细胞内蛋白质信号传导动力学，同时与其他下游分析方法。最近开发的原型方法使动态探测在单细胞分辨率下的细胞内蛋白质信号传导活性，而没有遗传修饰。技术基于细胞内递送的表位靶向肽探针，基于微孔的单细胞芯片，高速成像技术。然而，存在着许多障碍，对这一点的普遍性提出了挑战。限制了它的生物医学应用。在这项提案中，目的是大大推进这一证明- 概念技术五个具体目标包括：开发一个自动筛选协议，筛选效率，实现独特的双环肽文库以获得更高的结合亲和力，使用细胞透化剂作为一种更普遍的传递方法，实现了多种信号的同时分析，蛋白质，并将该技术与其他单细胞检测方法相结合。这种集成的多路复用管道将提供一种使能技术，有望更深入地了解蛋白质之间的相互作用，信号活性、蛋白质表达水平和生物过程中的代谢。