An integrated pipeline for next-generation protein interactomics

项目摘要 蛋白质是细胞过程的主要效应物。在活细胞中，蛋白质 相互作用的网络，通过稳定和短暂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。蛋白质的改变，包括蛋白质长度、氨基酸序列、表达水平的变化， 和亚细胞定位，可以导致形成改变的蛋白质相互作用网络， 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，创造了检测方法的需要， 并解剖相互作用体和任何此类改变。该项目将大大推进概念验证 - 产生旨在揭示相互作用组的组成的成熟平台技术的方法， PPI的行为在以前未实现的深度和准确性。所采取的方法类似于 结晶筛选，除了应用于内源性蛋白质复合物的亲和捕获，优化 样品制备与质谱法初步表征相结合。该项目将 展示了该平台的应用，除了检测蛋白质相互作用外，还包括生物化学和 大分子复合物的结构表征（以及其他可能性）。三个目标，包括 创新的方法和技术的综合，将使我们能够实现我们的目标：我们将（1）优化 获得相互作用组，（2）保存相互作用组用于生物分析测定，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捕获的成功率 实验设计，并在基础，生物医学和生物技术研究中找到应用，从蛋白质 复杂的发现和表征，样品储存，基础和临床检测开发，以及 工业工程

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.