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。迄今为止，只有一小部分表达蛋白的相互作用伙伴是已知的，然而蛋白质-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的技术来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通性。使用亲和色谱-质谱（AP-MS）技术，提出了三个目标。1)独立表达、纯化和编目HEK293细胞中表达的大部分基因的蛋白相互作用。这将包括约10,000个基因作为一个单一的，高通量和高质量的传代，还将包括生物三重复实验

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.