Proteomics systems for integrative structural biology

我们提出了一个研究计划，旨在为描述和模拟复杂蛋白质系统的结构提供新的工具。我们生成结构丰富的生物物理数据的策略涉及浓缩相蛋白质化学，它将蛋白质属性编码成所有组织规模的可通过质谱学测量的数量(结构蛋白质组学)。拟议的计划有四个相互关联的主题。首先，我们将研究涉及快速反应、光触发化学反应的蛋白质生物偶联新策略，并开发用于共价标记质谱学(CL-MS)和交联质谱学(XL-MS)的新一代试剂。我们认为，将这些试剂的平衡蛋白质溶剂化性质从光解过程中分离出来，将提供前所未有的机会来增加来自CL-MS(地形测绘)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现蛋白质生物结合方法的概念，以避免重组蛋白质复合体。重建是一个费力且容易失败的过程，因此，克服目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生产用于从体外翻译介质和细胞裂解物中去除复合体的快速分离方法，以及开发原位、基于邻近的交联法。第三，这些活动产生的丰富的结构性数据将给现有的基于MS的信息学带来负担，需要新的评分算法、文件管理和报告结构。我们将使用新的插件来增强我们的MassSpec Studio软件架构，以适应约束数据的提取。所有插件都将公开可用，我们将开源该架构以供社区参与。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的方法还很初级。我们提出了一种策略，利用结构蛋白质组学数据将自由蛋白质结构转换为它们的结合形式，以便能够更准确地对接实验，进一步受到数据的限制。我们将在我们的Studio框架内建立例程，以与关键的结构建模计算机系统进行接口，以便在结构蛋白质组学中生成第一个全面的数据到结构资源。我们的研究计划将迈出重要的一步，将结构生物学扩展到蛋白质发挥功能的复杂环境中。进展将提高我们对生物学机制的理解，并为治疗学的发展激发新的战略。

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.