Proteomics systems for integrative structural biology

综合结构生物学的蛋白质组学系统

• 批准号: RGPIN-2017-04879
• 负责人: Schriemer, David
• 金额: $ 9.03万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711276
• 关键词: Proteomics; systems; integrative; structural; biology

We present a program of research designed to provide new tools for characterizing and modeling the structure of complex protein systems. Our strategy for generating structure-rich biophysical data involves condensed-phase protein chemistries that code protein properties into quantities measurable by mass spectrometry, at all organizational scales (structural proteomics). There are four interrelated themes in the proposed program. First, we will investigate new protein bioconjugation strategies that involve fast-acting, light-triggered chemical reactions, and develop next-generation reagents for both covalent labeling mass spectrometry (CL-MS) and crosslinking mass spectrometry (XL-MS). We propose that separating the equilibrium protein solvation properties of these reagents from the photolytic process will provide unprecedented opportunities to increase the structural value of modeling restraints derived from both CL-MS (topographical mapping) and XL-MS (distance measurements). Second, we present concepts for implementing protein bioconjugation methods in complex mixtures and cells, to avoid reconstituting protein complexes. Reconstitution is a laborious and failure-prone process, thus overcoming this current requirement is critical step towards extending structural methods into a cellular context. The work in this theme will involve the production fast-isolation methods for removing complexes from in vitro translation media and cell lysates, as well as the development of in situ, proximity-based crosslinking methods. Third, the richness of structural data emerging from these activities will tax existing MS-based informatics, requiring new scoring algorithms, file management and reporting structures. We will augment our Mass Spec Studio software architecture with new plug-ins to accommodate the extraction of restraint data. All plug-ins will be made publicly available, and we will open-source the architecture for community participation. Fourth, new strategies will be developed, together with collaborators, that better utilize MS-generated structural data for accurately modeling large, complex protein systems. Current MS-based methods are rudimentary. We propose a strategy whereby structural proteomics data are used to transform free protein structures into their bound form, to enable a more accurate docking experiment further constrained by data. Routines will be built within our Studio framework to interface with key structure-modeling compute systems, in order to generate the first ever comprehensive data-to-structure resource in structural proteomics. Our research program will take an important step towards extending structural biology into the complex milieu in which proteins function. Progress will improve our understanding of biological mechanisms, and stimulate new strategies for the development of therapeutics.

我们提出了一个研究计划，旨在提供新的工具，复杂的蛋白质系统的结构表征和建模。我们生成结构丰富的生物物理数据的策略涉及凝聚相蛋白质化学，该化学将蛋白质特性编码为可通过质谱测量的数量，在所有组织尺度上（结构蛋白质组学）。在拟议的方案中有四个相互关联的主题。首先，我们将研究新的蛋白质生物缀合策略，涉及快速作用，光触发化学反应，并开发下一代共价标记质谱（CL-MS）和交联质谱（XL-MS）试剂。我们建议，从光解过程中分离这些试剂的平衡蛋白质溶剂化特性将提供前所未有的机会，以增加来自CL-MS（地形图）和XL-MS（距离测量）的建模约束的结构价值。其次，我们提出了在复杂的混合物和细胞中实施蛋白质生物缀合方法的概念，以避免重构蛋白质复合物。重组是一个费力且容易失败的过程，因此克服当前的要求是将结构方法扩展到细胞环境中的关键一步。这一主题的工作将涉及从体外翻译介质和细胞裂解物中去除复合物的生产快速分离方法，以及原位邻近交联方法的开发。第三，从这些活动中出现的丰富的结构数据将征税现有的MS为基础的信息学，需要新的评分算法，文件管理和报告结构。我们将增加我们的质谱工作室软件架构与新的插件，以适应约束数据的提取。所有插件都将公开提供，我们将开源社区参与的架构。第四，将与合作者一起开发新的策略，更好地利用MS生成的结构数据来准确建模大型复杂的蛋白质系统。目前基于MS的方法是基本的。我们提出了一种策略，即结构蛋白质组学数据被用来将自由蛋白质结构转化为它们的结合形式，使一个更准确的对接实验进一步约束的数据。将在我们的Studio框架内构建数据库，以与关键的结构建模计算系统对接，从而生成结构蛋白质组学中第一个全面的数据到结构资源。我们的研究计划将朝着将结构生物学扩展到蛋白质功能的复杂环境中迈出重要一步。进展将提高我们对生物学机制的理解，并刺激治疗方法开发的新策略。