Systematic Exploration of the Human Interactome

描述(由申请人提供)：蛋白质组可以被视为相互作用的蛋白质模块的星座，这些模块被组织成细胞器、分子机器和信号转导网络。到目前为止，只有一小部分表达的蛋白质的相互作用伙伴是已知的，但蛋白质-蛋白质关联是理解蛋白质功能和活性的主要驱动因素。在这里，我们建议整合新兴的基于质谱学的技术，以创建一个高质量的图谱，描述广泛使用的人类细胞系统中哺乳动物蛋白质组的物理组织和连接。利用亲和层析-质谱仪(AP-MS)技术，提出了三个目标。1)独立表达、纯化和分类HEK293细胞中表达的大多数基因的蛋白质相互作用。这将包括作为单一、高通量和高质量传递的约10,000个基因，还将包括用于 精选的一组高度相互作用的蛋白质(~3,000)用于验证目的。2)在期间和 在目标1结束时，将对所有研究的蛋白质进行计算和信息学分析。这种分析将被用来确定假定的生物学功能，并根据已知的相互作用蛋白质的活性将这些蛋白质置于生物框架内。此外，在目标1结束时，所有形式的数据(从原始MS文件到FINA信息学分析)都将通过定制的基于网络的界面进行访问，并存放在NCBI以供进一步传播。3)在目标1和目标2(预计在项目的头两年内完成)完成后，将根据它们的网络连通性和可能的生物功能选择~2,000个蛋白质作为诱饵，用于使用6个不同的细胞系进行AP-MS实验。这一目标将有助于验证全球图谱，并为了解细胞和/或组织特异性蛋白质复合体提供一个独特的窗口。总之，这项工作为全球哺乳动物相互作用研究提供了起点，产生了包含全面和公正的物理相互作用集的重要资源。

DESCRIPTION (provided by applicant): The proteome can be viewed as constellations of interacting protein modules which are organized into organelles, molecular machines, and signal transduction networks. To date, the interaction partners for only a small fraction of expressed proteins are known, yet protein-protein associations are a major driving factor in understanding protein function and activity. Here we propose to integrate emerging mass spectrometry-based technologies to create a high-quality atlas describing the physical organization and connectivity of the mammalian proteome housed in widely used human cell systems. Using affinity-chromatography with mass spectrometry (AP-MS) techniques, three aims are proposed. 1) Independently express, purify, and catalog protein interactions for the majority of genes expressed in an HEK293 cell. This will include ~10,000 genes as a single, high-throughput and high-quality pass and will also include biological triplicate experiments for a selected set of highly interacting proteins (~3,000) for purposes of validation. 2) Both during and at the conclusion of aim 1, computational and informatics analysis will be performed for all proteins investigated. This analysis will be used to determine putative biological functions and place these proteins within a biological framework based on known activities for interacting proteins. Furthermore, at the conclusion of aim 1, all forms of the data (from raw MS files to fina informatics analysis) will be made accessible via a custom web-based interface and also deposited at NCBI for additional dissemination. 3) Upon the completion of aims 1 and 2 (expected to be done within the first 2 years of the project), ~2,000 proteins will be selected based on their network connectivity and putative biological function as baits for AP-MS experiments using six different cell lines. This aim will serve to both validate global maps and provide a unique window into cell- and/or tissue-specific protein complexes. In total, this work provides the launching point for global mammalian interactions studies, generating an important resource containing comprehensive and unbiased sets of physical interactions.