Quantitative Determination of High-Order Protein Structure with Native Ion Mobility-Mass Spectrometry and Computational Chemistry

利用天然离子淌度-质谱法和计算化学定量测定高级蛋白质结构

• 批准号: 10707524
• 负责人: JAMES STEPHEN PRELL
• 金额: $ 25万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707524
• 关键词: Adjuvant; Affinity; Architecture; Area; Behavior; Benchmarking; Buffers; Case Study; Cataract; Characteristics; Chemicals; Clinical; Collaborations; Collection; Complement; Complex; Computer software; Computing Methodologies; Cryoelectron Microscopy; Crystalline Lens; Crystallization; Data; Data Analyses; Dependence; Development; Disease; Dissociation; Entropy; Environment; Experimental Designs; Eye Lens Protein; Fingerprint; Gases; Goals; Health; Heating; Heterogeneity; Human; Ions; Kinetics; Label; Libraries; Lipids; Mass Spectrum Analysis; Measures; Membrane; Membrane Proteins; Methods; Modeling; Modernization; NMR Spectroscopy; Outcome; Pathway interactions; Pharmacologic Substance; Phase; Physiological; Physiology; Preparation; Process; Proteins; Reagent; Research; Research Personnel; Resistance; Resolution; Sampling; Shapes; Source; Specificity; Speed; Structure; Surface; Techniques; Temperature; Therapeutic; Thermodynamics; X-Ray Crystallography; aqueous; chemical property; computational chemistry; computerized tools; design; evidence base; experimental study; flexibility; heuristics; improved; instrument; instrumentation; ion mobility; ionization; ionization technique; mass spectrometer; molecular dynamics; open source; physical property; preservation; pressure; protein complex; protein structure; theories

PROJECT SUMMARY/ABSTRACT Characterizing the structures and interactions of biomolecules and their complexes is of fundamental importance in human physiology, disease, and therapeutics. Many of the advances of the last century in these areas are attributed to improvements in bioanalytical techniques and controlling the processes that underlie them. For example, x-ray crystallography, nuclear magnetic resonance spectroscopy, and cryoelectron microscopy have achieved atomic-level resolution of the structure of many thousands of proteins and protein complexes, and these methods are often complemented by Molecular Dynamics studies to further understand biomolecule structure and reactivity. However, these methods can be challenging to use for very small or highly heterogeneous samples or samples that require a membrane environment. Native Ion Mobility-Mass Spectrometry (IM-MS) is a complementary technique that ionizes and transfers intact biomolecules and complexes directly from buffered, aqueous solution into the gas-phase for mass and shape/size analysis, and modern sample preparation and data analysis methods make it highly suitable for membrane proteins in lipid environments as well as heterogeneous and polydisperse samples. In commonly available IM-MS instrumentation, Collision Induced Dissociation and Unfolding are used to activate native biomolecular ions by colliding them repeatedly with neutral buffer gas until they dissociate or unfold, and recently-introduced Surface Induced Dissociation and Unfolding activate ions via a single, controlled collision with a hard surface inside the mass spectrometer. These native IM-MS methods can be extremely useful for profiling the composition, size, and shape of biomolecules and their complexes with exquisite chemical specificity, sensitivity, and speed. However, two major hurdles to the use of these methods for accurate, quantitative interpretation of biomolecule domain, surface, and interface structure are the lack of a flexible, robust method for computing and interpreting the energy required to induce the observed structural changes and a dearth of reliable benchmark values. Here, we tackle these challenges with a combined computational and experimental approach aimed at producing a “universal,” validated ion activation model that can be readily used for across many commonly used native MS and IM-MS platforms and by producing a benchmark library for prototypical local and large-scale interactions that govern protein unfolding, dissociation, and surface labeling. Expected outcomes include open-source, publicly available software for researchers world-wide to model unfolding/dissociation energetics for their own samples, heuristics for the design of effective gas-phase surface-labeling reagents, and a quantitative understanding of cataract-associated human eye lens protein heterooligomerization as a case study. The long- term goal of the project is to facilitate the acquisition and interpretation of decisive structural and dynamical information for a wide range of biomolecules and complexes relevant to human health.

项目摘要/摘要 表征生物分子及其复合物的结构和相互作用是基本的 在人类生理，疾病和治疗中的重要性。上个世纪的许多进步 区域归因于进步的生物分析技术和控制基础的过程 他们。例如，X射线晶体学，核磁共振光谱和冷冻电子 显微镜已经实现了数千种蛋白质和蛋白质结构的原子水平分辨率 复合物，这些方法通常通过分子动力学研究完成，以进一步理解 生物分子结构和反应性。但是，这些方法可能会挑战非常小的或高度的 需要膜环境的异质样品或样品。本地离子迁移率质量 光谱法（IM-MS）是一种完整的技术，它使完整的生物分子和转移转移 直接从缓冲，水溶液中的复合物进入气相，以进行质量和形状/尺寸分析，并 现代样品制备和数据分析方法使其非常适合脂质中的膜蛋白 环境以及异质和多分散样品。在常见的IM-MS中 仪器，碰撞诱导的解离和展开用于激活天然生物分子离子 用中性缓冲气体反复碰撞它们，直到它们解离或展开，并且最近引入的表面 通过单个，受控的碰撞诱导解离并展开激活离子，并在内部有硬表面 质谱仪。这些天然IM-MS方法对于分析组成，大小， 生物分子及其复合物的形状具有独特的化学特异性，灵敏度和速度。 但是，使用这些方法的两个主要障碍，用于准确，定量解释生物分子 域，表面和界面结构缺乏用于计算和解释的灵活，可靠的方法 诱导观察到的结构变化和可靠基准值死亡所需的能量。这里， 我们通过一种旨在产生的计算和实验方法来应对这些挑战 “通用”，经过验证的离子激活模型，可以在许多常用的本机MS上很容易使用 和IM-MS平台，并通过生产用于原型本地和大规模互动的基准库 控制蛋白质的展开，解离和表面标记。预期的结果包括开源 全球研究人员的公开软件，以建模为自己的展开/解离能量 样品，用于设计有效气相表面标签试剂的启发式方法，定量 理解与白内障相关的人眼镜蛋白杂产蛋白作为案例研究。长期 该项目的术语目标是促进对决定性结构和动态的获取和解释 与人类健康相关的各种生物分子和复合物的信息。