Gas-Phase Cross-Linking with Ion/Ion Chemistry Coupled to Ion Mobility/Mass Spectrometry

离子/离子化学气相交联与离子淌度/质谱联用

• 批准号: 9807489
• 负责人: Ian Webb
• 金额: $ 20.7万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9807489
• 关键词: Address; Amino Acids; Amyloid; Binding Sites; Biological Process; Biomedical Research; Cell Mobility; Cells; Chemistry; Clinical Research; Clinical Treatment; Complex Mixtures; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Data Analyses; Dimensions; Disease; Dissociation; Drug Design; Electrons; Gases; Generations; Goals; Health; Heterogeneity; Human; Ions; Knowledge; Label; Lead; Location; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methods; Mission; Modification; Molecular Conformation; Molecular Weight; Nature; Nuclear Magnetic Resonance; Organism; Outcome; Parkinson Disease; Peptides; Pharmaceutical Preparations; Phase; Phosphorylation; Post-Translational Protein Processing; Production; Protein Analysis; Protein Dynamics; Proteins; Public Health; Quaternary Protein Structure; Reaction; Reaction Time; Reagent; Research; Research Design; Research Project Grants; Resolution; Roentgen Rays; Sampling; Shapes; Signal Transduction; Site; Speed; Structural Protein; Structure; System; Techniques; Technology; United States National Institutes of Health; alpha synuclein; crosslink; drug candidate; drug development; experimental study; field study; flexibility; innovation; ion mobility; link protein; mass spectrometer; millisecond; new technology; novel; preservation; protein complex; protein folding; protein protein interaction; protein structure; protein structure function; screening; small molecule; stoichiometry; tandem mass spectrometry; technology development; therapy design; tool; transcription factor; virtual

PROJECT SUMMARY Although many technologies exist for studying protein structures, none possess a combination of speed, selectivity, accuracy, resolution, and flexibility. The long-term goal is to use a suite of gas-phase chemistries for ion/ion reactions coupled to ion mobility (IM) and tandem mass spectrometry (MS) measurements for high- throughput, virtually sample-prep free, protein structure measurements. The overall objective for this exploratory project, which is the next step toward attaining our long-term goal, is to implement rapid ion/ion cross-linking reactions on an IM/MS platform, using tandem mass spectrometry to identify cross-linked sites. The rationale for the development of this technology is to combine the information from native IM/MS with information obtained from cross-linking in an experimental method conducted on the sub-second timescale. The increase in throughput, information, and lack of sample prep (compared to, e.g., X-ray diffractometry, nuclear magnetic resonance, or cryo-electron microscopy) is expected to advance biomedical research determining protein structure and function to where protein structural determinations can become rapid and routine. The overall objective of this application will be reached through the following Specific Aims: 1. Combine gas-phase ion/ion cross-linking of intact proteins with IM/MS measurements; and 2. Use IM combined with tandem MS to determine cross-linking locations for intact proteins. For the first aim, a variety of monomeric and multimeric proteins will be cross-linked in the gas-phase. Changes in overall structure between cross-linked and unmodified proteins, as well as between solution and gas-phase cross-linked proteins, will be measured by IM. Under the second aim, a combination of collision induced dissociation (CID) and electron capture dissociation (ECD) will be used to determine cross-linked sites. The proposed technology is innovative because it represents a substantive departure from the status quo by coupling cross-linking and native IM/MS analysis into one gas-phase mass spectrometry experiment, allowing rapid cross-linking analysis and providing multiple complementary measures of gas-phase protein structure. This new technology is significant because it is expected to become a rapid tool for high-throughput characterization of primary, secondary, tertiary, and quaternary protein structure. When fully developed, the technology has the potential to be used for complex mixtures of intact proteins and for rapid screening of interactions with small molecules/drugs, creating new opportunities in clinical research, treatment, and drug design.

项目概要 尽管存在许多用于研究蛋白质结构的技术，但没有一种技术能够同时具备速度、 选择性、准确性、分辨率和灵活性。长期目标是使用一套气相化学方法 离子/离子反应与离子淌度 (IM) 和串联质谱 (MS) 测量相结合，可实现高 通量、几乎无需样品准备的蛋白质结构测量。本次探索性的总体目标 该项目是实现我们长期目标的下一步，是实施快速离子/离子交联 IM/MS 平台上的反应，使用串联质谱法来识别交联位点。理由 这项技术的发展是将来自本地IM/MS的信息与所获得的信息结合起来 来自在亚秒时间尺度上进行的实验方法中的交联。增加的 通量、信息和样品制备的缺乏（与 X 射线衍射、核磁分析等相比） 共振或冷冻电子显微镜）有望推进确定蛋白质的生物医学研究 结构和功能使蛋白质结构测定变得快速和常规。整体 本应用的目标将通过以下具体目标来实现： 1. 结合气相离子/离子 通过 IM/MS 测量交联完整蛋白质； 2. 使用 IM 结合串联 MS 来确定 完整蛋白质的交联位置。对于第一个目标，各种单体和多聚体蛋白质将被 在气相中交联。交联和未修饰蛋白质之间整体结构的变化，如 以及溶液和气相交联蛋白之间的差异，将通过 IM 进行测量。在第二个目标下， 碰撞诱导解离（CID）和电子捕获解离（ECD）的组合将用于 确定交联位点。所提出的技术具有创新性，因为它代表了实质性的 通过将交联和本机 IM/MS 分析耦合到一个气相质量中来摆脱现状 光谱实验，允许快速交联分析并提供多种补充措施 气相蛋白质结构。这项新技术意义重大，因为它有望成为一种快速工具 用于一级、二级、三级和四级蛋白质结构的高通量表征。当完全 开发完成后，该技术有潜力用于完整蛋白质的复杂混合物和快速 筛选与小分子/药物的相互作用，为临床研究、治疗、 和药物设计。