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伴侣是已知的，但蛋白质-蛋白质缔合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，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的哺乳动物蛋白质组的物理组织和连接。采用亲和色谱-质谱联用技术（AP-MS），提出三个目标。1)独立表达、纯化和编目HEK293细胞中表达的大多数基因的蛋白质相互作用。这将包括约10，000个基因作为一个单一的，高通量和高质量的通行证，还将包括生物学的一式三份实验， 选择一组高度相互作用的蛋白质（约3，000）用于验证目的。2)期间和 在目标1结束时，将对所研究的所有蛋白质进行计算和信息学分析。该分析将用于确定推定的生物学功能，并基于相互作用蛋白质的已知活性将这些蛋白质置于生物学框架内。此外，在目标1结束时，所有形式的数据（从原始MS文件到最终信息学分析）都将通过一个基于网络的自定义界面提供，并存放在NCBI以供进一步传播。3)在完成目标1和2（预计将在项目的前2年内完成）后，将根据其网络连接性和推定的生物学功能选择约2，000种蛋白质作为使用6种不同细胞系的AP-MS实验的诱饵。这一目标将有助于验证全球地图，并提供一个独特的窗口到细胞和/或组织特异性蛋白质复合物。总之，这项工作提供了全球哺乳动物相互作用研究的出发点，产生了一个重要的资源，包含全面和公正的物理相互作用。 公共卫生相关性：发现蛋白质的功能对于了解其在异常生物学（包括人类疾病）中的作用非常重要。阐明蛋白质的功能和调节的关键步骤是确定其相互作用伙伴。因此，一个全面的人类相互作用组图谱将有望对人类健康的所有学科的研究产生重大影响。

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. PUBLIC HEALTH RELEVANCE: Discovering a protein's function is important for understanding its role in abnormal biology including human disease. A critical step in elucidating a protein's function and regulation is determining its interaction partners. Thus, a comprehensive human interactome map would be expected to significantly impact on research across potentially all disciplines of human health.