Extending the applications of hydrogen exchange mass spectrometry

描述(申请人提供)：有大量的蛋白质和蛋白质复合体的功能依赖于结构灵活性和构象变化，这些仍然是未知和未知的，远远超出了当前分析方法的范围。其中许多蛋白质系统是基本生化途径的关键组成部分，是疾病过程的积极和消极因素，并与广泛的与人类健康有关的问题有关。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)质谱学(MS)可用于研究许多不适合用常规工具分析的蛋白质的构象和柔性。但对于那些当前最感兴趣的系统--包括蛋白质复合体、膜蛋白质和大(&gt；250 kDa)系统--现有的HX-MS技术是不够的。该项目的目标是开发、改进和简化HX MS方法，以克服该方法目前的局限性。我们将通过完成三个具体目标来实现这一目标：(1)将HX-MS和亲和捕获技术结合起来，以增加通过HX-MS研究蛋白质复合体时可以获得的信息量；(2)通过将纳米盘作为HX-MS研究膜蛋白质的适当载体，将构象研究扩展到膜蛋白质；(3)将离子迁移率光谱(IMS)整合到HX-MS工作流程中，以提高分辨率，从而大幅提高HX-MS可应用于蛋白质尺寸的上限。我们将使用创新的实验设计，在保持HX MS方法所需的限制性猝灭条件的同时，使特定蛋白质从复合体中定向分离成为可能。纳米盘将被用来为研究膜蛋白的构象和柔韧性提供相关的膜环境。预期的结果将是常规使用HX MS进行大型蛋白质复合体和膜蛋白质构象分析的路线图，通过对几个与疾病相关的测试蛋白质系统的详细构象分析展示了这一点。这些测试系统包括对HIV进展重要的复合体，白血病的细胞信号，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依赖的血液凝固重要的跨膜蛋白。这项研究具有重要意义，因为它有望推动蛋白质构象分析领域的发展，使以其他方式无法获得构象信息的蛋白质和蛋白质体系的构象和动力学分析成为可能。然后，这些工具将由我们自己和其他人协同使用，以研究各种各样的蛋白质、蛋白质复合体，以及蛋白质科学和医学领域的问题。 与公共健康相关：拟议的研究，即开发允许分析不适用于其他技术的蛋白质构象的方法，与公共健康相关，因为了解蛋白质结构对于了解异常的、与疾病相关的蛋白质功能至关重要。这些结果将为NIH开发新的生物分析方法的使命提供相关工具，以便为疾病诊断、治疗和预防方面的进展奠定基础。

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. PUBLIC HEALTH RELEVANCE: The proposed research, development of methods that allow for the analysis of the conformation of proteins not amenable to other techniques, is relevant for public health because understanding protein structure is essential to understanding abnormal, disease-related protein function. The results will provide tools relevant to NIH's mission to develop new bioanalytical methods in order to lay the foundation for advances in disease diagnosis, treatment and prevention.