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

• 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
• 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

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.

项目摘要 蛋白质是细胞过程的主要效应者。在活细胞的背景下，蛋白质形成 通过稳定的和瞬时的大分子络合物发挥其功能的相互作用网络。 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体 一间牢房。蛋白质的变化，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，可以导致改变的蛋白质相互作用网络的形成，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，创造对检测方法的需求 并解剖交叉处和任何这样的变化。该项目将大大推进概念验证 一种产生成熟的平台技术的方法，旨在揭示交互的组成和 PPI在以前未实现的深度和准确性上的行为。所采取的方法类似于 晶体筛选，除应用于内源蛋白复合体的亲和捕获外，优化 样品制备与质谱学初步表征相结合。这个项目将 演示该平台在检测蛋白质相互作用之外的应用，包括生化和 大分子络合物的结构表征(以及其他可能性)。三个目标，包括 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 获取互动文件，(2)保存互动文件以进行生物分析，以及(3)解释互动文件 在计算工具的帮助下。生成的知识将提高亲和力捕获的成功率 基于蛋白质的实验设计及其在基础、生物医学和生物技术研究中的应用 复杂的发现和表征，到样品存储、基础和临床分析开发，以及 工业工程学。