Resource for Native Mass Spectrometry Guided Structural Biology

我们提出了一种自然质谱学指导的结构生物学资源，将被引导 由包括MS仪器专家在内的科学家团队(俄亥俄州立大学Wysocki；德克萨斯州A&M的Russell)， 与电离相结合的分离科学(Olesik，OSU；Badu，OSU；Holland；WVU)和计算 化学(俄亥俄州立大学林德特)。本资源的目标是构建和验证以下方面的集成工作流 蛋白质：蛋白质、膜蛋白质：脂质和蛋白质：RNA复合体的结构表征 对一系列细胞和生物体过程至关重要。人们越来越多地认识到 基于MS的方法在生物大分子结构表征中的关键作用和应用， 填补关键空白并补充其他结构生物学工具。因此，我们的工作计划利用 创新的MS方法来确定：(I)所有结合伙伴和完整络合物的m/z，(Ii) 组件化学计量，(Iii)异质性，如果存在，(Iv)相对拓扑/架构/ 复合体中各组分的构象多样性，以及(V)组装的层次，包括 单个亚基的结合亲和力。来自美国各地的调查人员(在华盛顿州、俄勒冈州、犹他州、 CA、AZ、TX、TN、OH、MD、MA)和国际站点将在以下方面贡献具有挑战性的生物医学项目 主题包括病毒性出血热、艾滋病毒、白内障形成和神经疾病。我们的 工作流旨在促进以下方面的理解：(I)如何在广泛的环境中形成和动态变化 范围广泛的大分子组合决定了它们的生物学作用，(Ii)在 这些复合体的组装/架构会导致疾病，以及(Iii)建筑的基本原则 生物医学应用所需的合成模拟物。在这个资源中，十个推动生物医学项目 (DBP)提供生物医学结构表征挑战，并充当驱动程序和试验台 五个技术研究与开发(T R&D)项目。研究与发展署提供：(I)有效 适合于MS的天然大分子络合物的分离纯化方法(II) 有效的表面诱导解离和紫外光解离技术，(Iii)测量 完整的络合物及其非共价(亚络合物)和共价高解离产物 分辨率离子迁移率(IM)和/或MS，以及(Iv)用于结构预测的计算工具。 计算工具将使用表面诱导的离解模式和碰撞交叉的约束 来自IM-MS实验和基于MS的溶液测量(H/D交换和 共价标记)。研发部门与仪器公司(Waters、Thermo、 以及一个国家实验室(PNNL)将帮助技术开发 并加快技术传播。我们的培训和传播活动(例如，讲习班、测试版 设备安装)旨在扩大覆盖面并最大限度地提高资源回报。

We propose a Resource for Native Mass Spectrometry Guided Structural Biology that will be led by a team of scientists including experts in MS instrumentation (Wysocki, OSU; Russell, Texas A&M), separation science coupled to ionization (Olesik, OSU; Badu, OSU; Holland; WVU), and computational chemistry (Lindert, OSU). The goal of this Resource is to build and validate an integrated workflow for structural characterization of protein:protein, membrane protein:lipid, and protein:RNA complexes that are critical for an array of cellular and organismal processes. There is a growing appreciation of the pivotal role and utility of MS-based approaches for structural characterization of biomacromolecules, filling critical gaps and complementing other structural biology tools. Thus, our workplan leverages innovative MS methods to determine: (i) m/z of all binding partners and the intact complex, (ii) component stoichiometry, (iii) heterogeneity, if present, (iv) the relative topology/architecture/ conformational diversity of components in the complex, and (v) the hierarchy of assembly, including binding affinities of individual subunits. Investigators from across the United States (in WA, OR, UT, CA, AZ, TX, TN, OH, MD, MA) and international sites will contribute challenging biomedical projects on topics including viral hemorrhagic fevers, HIV, cataract formation, and neurological disorders. Our workflow is designed to advance understanding of: (i) how formation of and dynamic changes in wide- ranging macro-molecular assemblies determine their biological roles, (ii) how alterations in assembly/architecture of these complexes lead to disease, and (iii) foundational principles for building synthetic mimics needed for biomedical applications. In this Resource, ten Driving Biomedical Projects (DBPs) provide biomedical structural characterization challenges and serve as drivers and test beds for five Technology Research and Development (TR&D) projects. The TR&Ds provide: (i) effective separation methods to purify and deliver native macromolecular complexes well suited for MS, (ii) effective surface-induced dissociation and UV-photodissociation technologies, (iii) measurement of the intact complexes and their non-covalent (sub-complex) and covalent dissociation products with high resolution ion mobility (IM) and/or MS, and (iv) computational tools for structure prediction. Computational tools will use restraints from surface-induced dissociation patterns and collision cross sections from IM-MS experiments, and from MS-based solution measurements (H/D exchange and covalent labeling). Existing ties between the TR&Ds and instrument companies (Waters, Thermo, Bruker, Sciex, Phenomenex) as well as a national laboratory (PNNL) will aid technology development and expedite technology dissemination. Our training and dissemination activities (e.g., workshops, beta device installations) are designed to increase outreach and maximize Resource payoffs.