Extending the applications of hydrogen exchange mass spectrometry

扩展氢交换质谱的应用

• 批准号: 8707489
• 负责人: JOHN R ENGEN
• 金额: $ 29.55万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8707489
• 关键词: Address; Affinity; Back; Behavior; Biochemical Pathway; Biological; Blood coagulation; Complex; Complex Mixtures; Detergents; Deuterium; Development; Digestion; Disease; Environment; Equilibrium; Experimental Designs; Foundations; HIV; Health; Human; Human Pathology; Hydrogen; Immobilization; Inflammation; Integral Membrane Protein; Label; Mass Spectrum Analysis; Mediating; Medicine; Membrane; Membrane Proteins; Methodology; Methods; Micelles; Mission; Molecular Conformation; Neurodegenerative Disorders; Outcome; Pain; Peptides; Peripheral; Phospholipids; Positioning Attribute; Prevention; Process; Protein Analysis; Protein Conformation; Protein Dynamics; Proteins; Protocols documentation; Public Health; Relative (related person); Research; Science; Signal Transduction; Spectrometry; System; Techniques; Temperature; Testing; Therapeutic; Translating; Vitamin K; Work; analytical method; base; biophysical techniques; combat; design; disease diagnosis; flexibility; human disease; improved; innovation; interest; ion mobility; leukemia; member; mimetics; nanodisk; protein complex; protein function; protein structure; rapid technique; research and development; research study; success; tool

DESCRIPTION (provided by applicant): There are a great number of proteins and protein complexes whose function depends on structural flexibility and conformational changes that remain uncharted and unknown and well beyond the reach of current analytical methodologies. Many of these protein systems are critical components of essential biochemical pathways, contributors - both positive and negative - to disease processes, and relevant to a broad range of human health-related problems. Progress in understanding these systems will depend on development of new tools capable of studying the structural basis of function in these proteins. Hydrogen exchange (HX) mass spectrometry (MS) can be used for studies of conformation and flexibility of many proteins not suitable for analysis with conventional tools. But for those systems of greatest current interest - including protein complexes, membrane proteins, and large (>250 kDa) systems - existing HX MS techniques are inadequate. The objective of this project is to develop, refine and reduce to common practice HX MS methods that overcome the current limitations of the approach. We will do this by completing three specific aims: (1): Combine HX MS and affinity capture techniques to enhance the amount of information that can be obtained in the study of protein complexes through HX MS; (2): Extend conformational studies to membrane proteins by adapting the use of nanodiscs as appropriate vehicles for membrane proteins studied by HX MS; (3): Integrate ion mobility spectrometry (IMS) into the HX MS workflow to enhance the resolving power and thereby substantially increase the upper limit of protein size to which HX MS can be applied. We will use innovative experimental design to make possible directed isolation of specific proteins from complexes while maintaining the restrictive quench conditions required by HX MS methodology. Nanodiscs will be used to provide relevant membrane environments for the study of conformation and flexibility of membrane proteins. The expected outcome will be a roadmap for the routine use of HX MS for conformational analysis of large protein complexes and membrane proteins, demonstrated through the detailed conformational analysis of several disease- relevant test protein systems. These test systems include complexes important for HIV progression, cellular signaling in leukemia, membrane-associated proteins important for pain, inflammation and neurodegenerative disorders, and a transmembrane protein important in vitamin-K dependent blood coagulation. The proposed research is significant because it is expected to advance the field of protein conformational analysis with mass spectrometry, making possible analysis of the conformation and dynamics of proteins and protein systems for which conformational information cannot otherwise be obtained. These tools would then be applied by ourselves and in coordination with others to study a huge variety of proteins, protein complexes, and problems in the fields of protein science and medicine.

描述（由申请人提供）：有大量的蛋白质和蛋白质复合物，其功能取决于结构灵活性和构象变化，这些变化仍然是未知的和未知的，远远超出了当前分析方法的范围。许多这些蛋白质系统是重要生化途径的关键组成部分，对疾病过程有积极和消极的贡献，并且与广泛的人类健康相关问题相关。理解这些系统的进展将取决于能够研究这些蛋白质功能的结构基础的新工具的开发。氢交换 (HX) 质谱 (MS) 可用于研究许多不适合使用常规工具进行分析的蛋白质的构象和灵活性。但对于当前最受关注的系统 - 包括蛋白质复合物、膜蛋白和大型 (>250 kDa) 系统 - 现有的 HX MS 技术还不够。该项目的目标是开发、完善和简化 HX MS 方法的通用实践，以克服该方法当前的局限性。我们将通过完成三个具体目标来实现这一目标：(1)：结合 HX MS 和亲和捕获技术，以增加通过 HX MS 研究蛋白质复合物时可以获得的信息量； (2)：通过使用纳米圆盘作为 HX MS 研究膜蛋白的适当载体，将构象研究扩展到膜蛋白； (3)：将离子迁移谱 (IMS) 集成到 HX MS 工作流程中，以增强分辨率，从而大幅提高 HX MS 可应用的蛋白质大小上限。我们将使用创新的实验设计，使从复合物中定向分离特定蛋白质成为可能，同时保持 HX MS 方法所需的限制性淬灭条件。纳米圆盘将用于为膜蛋白的构象和柔性研究提供相关的膜环境。预期结果将是常规使用 HX MS 对大型蛋白质复合物和膜蛋白进行构象分析的路线图，通过对几种疾病相关测试蛋白质系统的详细构象分析进行证明。这些测试系统包括对 HIV 进展重要的复合物、白血病中的细胞信号传导、对疼痛、炎症和神经退行性疾病重要的膜相关蛋白，以及对维生素 K 依赖性凝血重要的跨膜蛋白。这项研究意义重大，因为它有望推动质谱法蛋白质构象分析领域的发展，使对无法通过其他方式获得构象信息的蛋白质和蛋白质系统的构象和动力学分析成为可能。然后我们自己并与他人协调应用这些工具来研究各种各样的蛋白质、蛋白质复合物以及蛋白质科学和医学领域的问题。