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.

项目概要/摘要 表征生物分子及其复合物的结构和相互作用至关重要 在人类生理学、疾病和治疗学中具有重要意义。上个世纪在这些领域取得的许多进步 这些领域归因于生物分析技术的改进和控制基础过程 他们。例如，X射线晶体学、核磁共振光谱学和冷冻电子学 显微镜已经实现了数千种蛋白质和蛋白质结构的原子级分辨率 复合物，这些方法通常辅以分子动力学研究，以进一步了解 生物分子结构和反应性。然而，这些方法对于非常小的或高度大的应用可能具有挑战性。 异质样品或需要膜环境的样品。自然离子淌度-质量 光谱测定 (IM-MS) 是一种补充技术，可电离并转移完整的生物分子并 复合物直接从缓冲水溶液进入气相进行质量和形状/尺寸分析，以及 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。在常用的 IM-MS 中 仪器、碰撞诱导解离和解折叠用于通过以下方式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，以及最近推出的 Surface 诱导解离和展开通过与内部硬表面的一次受控碰撞来激活离子 质谱仪。这些原生 IM-MS 方法对于分析成分、尺寸、 具有精确的化学特异性、灵敏度和速度的生物分子及其复合物的形状和形状。 然而，使用这些方法对生物分子进行准确、定量的解释存在两个主要障碍 域、表面和界面结构缺乏灵活、鲁棒的计算和解释方法 引起观察到的结构变化所需的能量以及缺乏可靠的基准值。这里， 我们通过计算和实验相结合的方法来应对这些挑战，旨在产生 “通用”、经过验证的离子激活模型，可轻松用于许多常用的天然 MS 和 IM-MS 平台，并为典型的本地和大规模交互生成基准库 控制蛋白质的解折叠、解离和表面标记。预期成果包括开源、 供世界各地的研究人员为自己的展开/解离能量学建模的公开软件 样品、有效气相表面标记试剂设计的启发式方法以及定量方法 了解白内障相关的人眼晶状体蛋白异源寡聚化作为案例研究。长- 该项目的长期目标是促进决定性的结构和动力的获取和解释 与人类健康相关的各种生物分子和复合物的信息。