Resource for Native Mass Spectrometry Guided Structural Biology

我们提出了一个本土质谱指导的结构生物学资源， 由包括MS仪器专家在内的科学家团队（Wysocki，OSU; Russell，Texas A&M）， 分离科学与电离耦合（Olesik，OSU; Badu，OSU; Holland; WVU）和计算 化学（Lindert，OSU）。本资源的目标是构建和验证集成工作流， 蛋白质：蛋白质、膜蛋白质：脂质和蛋白质：RNA复合物结构表征， 对一系列细胞和有机体过程至关重要。人们越来越欣赏 基于MS的方法在生物大分子结构表征中的关键作用和实用性， 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 创新的MS方法，以确定：（i）所有结合配偶体和完整复合物的m/z，（ii） 组分化学计量，（iii）异质性，如果存在的话，（iv）相对拓扑/结构/ 复合物中组分的构象多样性，以及（v）组装的层次结构，包括 单个亚基的结合亲和力。来自美国各地（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、白内障形成和神经系统疾病。我们 工作流程的目的是促进理解：（一）如何形成和动态变化的广泛， 大分子组装决定了它们的生物学作用，（ii） 这些复合物的组装/结构导致疾病，以及（iii）构建的基本原则 生物医学应用所需的合成模拟物。在此资源中，十个驱动生物医学项目 （DBPs）提供生物医学结构表征挑战，并作为驱动程序和测试床 五个技术研究与开发（TR&D）项目。TR& D提供：（一）有效的 纯化和递送非常适合MS的天然大分子复合物的分离方法，（ii） 有效的表面诱导解离和紫外光解离技术，（iii）测量 完整复合物及其非共价（子复合物）和共价解离产物具有高的 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。 计算工具将使用来自表面诱导解离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）。TR& D和仪器公司（沃茨，赛默， Bruker、Sciex、Phenomenex）以及一个国家实验室（PNNL）将协助技术开发 加快技术推广。我们的培训和传播活动（例如，工作坊，beta 设备安装）的目的是增加外联和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.