Extending the applications of hydrogen exchange mass spectrometry

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基本生化途径的关键组成部分，是疾病过程的积极和消极贡献者，并与广泛的人类健康相关问题有关。了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发展。氢交换（HX）质谱（MS）可用于研究许多不适合用传统工具分析的蛋白质的构象和柔韧性。但是对于那些目前最感兴趣的系统——包括蛋白质复合物、膜蛋白和大型（bb0 250 kDa）系统——现有的HX - MS技术是不够的。该项目的目标是开发、完善和减少HX质谱方法，以克服当前方法的局限性。我们将通过完成三个具体目标来实现这一目标：(1)：结合HX质谱和亲和捕获技术，以增强通过HX质谱在蛋白质复合物研究中可以获得的信息量；(2)将构象研究扩展到膜蛋白，采用纳米片作为HX质谱研究膜蛋白的合适载体；(3)：将离子迁移谱法（IMS）集成到HX MS工作流程中，提高分辨率，从而大幅提高HX MS可应用的蛋白质大小上限。我们将采用创新的实验设计，使从复合物中定向分离特定蛋白质成为可能，同时保持HX质谱方法所需的限制性猝灭条件。纳米片将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HX质谱在大型蛋白质复合物和膜蛋白构象分析中的常规应用路线图，通过对几种疾病相关测试蛋白系统的详细构象分析来证明。这些检测系统包括对HIV进展具有重要意义的复合物、白血病中的细胞信号、对疼痛、炎症和神经退行性疾病具有重要意义的膜相关蛋白，以及对维生素k依赖性凝血具有重要意义的跨膜蛋白。这项研究具有重要意义，因为它有望推动质谱分析蛋白质构象领域的发展，使分析蛋白质和蛋白质系统的构象和动力学成为可能，否则无法获得构象信息。然后，这些工具将被我们自己应用，并与他人协同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。

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.