An integrated pipeline for next-generation protein interactomics

下一代蛋白质相互作用组学的集成管道

基本信息

批准号：
10061613
负责人：
John Paul LaCava
金额：
$ 50.29万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10061613
关键词：
Address Affect Affinity Amino Acid Sequence Behavior Biochemical Biochemical Reaction Biochemistry Biological Biological Assay Biotechnology Cataloging Cell Maintenance Cell Separation Cell physiology Cells Chemicals Collaborations Complex Computer software Coupled Crude Extracts Cryoelectron Microscopy Data Development Devices Disease Electron Microscopy Engineering Environment Experimental Designs Filtration Functional disorder Future Goals Graph Health Human In Vitro Industrialization Knowledge Lead Length Macromolecular Complexes Maintenance Maps Mass Spectrum Analysis Methods Molecular Monitor Outcome Particle Size Patients Pharmacologic Substance Physiological Preparation Procedures Protein Structure Initiative Proteins Proteomics Quality Control Reaction Reporting Research Research Personnel Resected Sampling Structure Techniques Technology Temperature Testing Tissue Extracts Tissues Tube Work X-Ray Crystallography clinical assay development computerized tools crosslink experimental study human disease human interactome improved in vivo innovation macromolecule next generation parallelization preservation protein complex protein protein interaction protein structure screening solid state structural biology success tissue culture tool tumor

项目摘要

Project Summary Proteins are the chief effectors of cellular processes. Within the context of the living cell, proteins form networks of interactions to exert their functions through both stable and transient macromolecular complexes. These protein-protein interactions (PPIs), and interactions with other biomolecules, constitute the interactome of a cell. Alterations in proteins, including changes to protein length, amino acid sequence, expression level, and subcellular localization, can lead to the formation of altered protein interaction networks that cause cellular dysfunction and disease. Therefore, to understand molecular and cellular function and dysfunction, to understand health and disease, we must understand interactomes, creating a need for methods that detect and dissect interactomes and any such alterations. This project will significantly advance a proof-of-concept method to produce a mature platform technology aimed at revealing the compositions of interactomes and behaviors of PPIs at previously unrealized depth and accuracy. The approach taken is akin to a crystallographic screen, except applied to the affinity capture of endogenous protein complexes, optimizing sample preparation in conjunction with initial characterization by mass spectrometry. This project will demonstrate the application of the platform beyond detecting protein interactions, to include biochemical and structural characterization of macromolecular complexes (among other possibilities). Three Aims, composed of an innovative synthesis of methods and technologies, will permit us to achieve our goals: we will (1) optimize access to interactomes, (2) preserve interactomes for bioanalytical assays, and (3) interpret interactomes aided by computational tools. The knowledge generated will improve success rates in affinity capture experimental design and find applications in basic, biomedical, and biotechnology research, from protein complex discovery and characterization, to sample storage, basic and clinical assay development, and industrial engineering.

项目概要蛋白质是细胞过程的主要效应物。在活细胞的背景下，蛋白质形成相互作用网络通过稳定和瞬时的大分子复合物发挥其功能。这些蛋白质-蛋白质相互作用（PPI）以及与其他生物分子的相互作用构成了相互作用组一个细胞的。蛋白质的改变，包括蛋白质长度、氨基酸序列、表达水平的改变，和亚细胞定位，可以导致蛋白质相互作用网络改变的形成，从而导致细胞功能障碍和疾病。因此，要了解分子和细胞的功能和功能障碍，了解健康和疾病，我们必须了解相互作用组，从而需要检测方法并剖析相互作用组和任何此类改变。该项目将显着推进概念验证方法产生成熟的平台技术，旨在揭示相互作用组的组成和 PPI 的行为具有以前未实现的深度和准确性。所采取的方法类似于晶体筛选，除了应用于内源蛋白复合物的亲和捕获外，优化样品制备与质谱初步表征相结合。该项目将展示该平台的应用超出检测蛋白质相互作用的范围，包括生化和大分子复合物的结构表征（以及其他可能性）。三个目标，组成方法和技术的创新综合将使我们能够实现我们的目标：我们将 (1) 优化获取相互作用组，(2) 保存相互作用组用于生物分析，以及 (3) 解释相互作用组借助计算工具。生成的知识将提高亲和力捕获的成功率蛋白质的实验设计并在基础、生物医学和生物技术研究中找到应用复杂的发现和表征，样品存储，基础和临床测定开发，以及工业工程。