A MASS SPECTROMETER FOR PROTEIN FOOTPRINTING

描述(申请人提供)：质谱学是鉴定蛋白质和确定其结构和性质的有力工具。科学的 社区经常使用基于MS的蛋白质组学。一种新的机会，蛋白质足迹，正在出现，它利用了这些结合了高效液相色谱和质谱学的精致蛋白质组学能力。这些技术的发展是由越来越多的生物医学和其他领域的合作项目推动的。十多年来，华盛顿大学质谱学资源(WUMSR)在蛋白质足迹的发展中发挥了关键作用，是支持其扩展的理想场所。虽然它还没有X射线结晶学和核磁共振的分辨率，但基于MS的足迹明显比CD、荧光、拉曼、吸光度、SPR和量热等低分辨率工具更具特异性。此外，当蛋白质及其复合体处于天然状态时，就会产生足迹，从而提供许多重要的热力学和动力学参数的相关测量。足迹方法包括氢-氚交换(H/DX)、蛋白质的快速光化学氧化(FPOP)和其他氨基酸特定的化学修饰。为了实现和传播蛋白质足迹的承诺，我们建议增加一个高性能的LC/MS系统，并全职支持这项研究。拟议的仪器将用于满足在蛋白质生物化学和医学方面有问题的17名合作者的需求。这些蛋白质对癌症、病毒感染、疼痛管理和大脑疾病，主要是阿尔茨海默病(AD)有影响。这不仅对能源和光合作用有影响，而且也有影响。这些方法将使蛋白质折叠和展开的研究成为可能，允许确定重要多肽之间的界面(例如，A？涉及AD)，确定包括潜在药物在内的小配体的结合界面，并测量结合的亲和力。我们将把这些测量扩展到蛋白质组件，包括那些与膜结合的组件。考虑到足迹实验产生的数据“约束”蛋白质和蛋白质-复杂结构，就像在核磁共振中一样，我们预测，即使在没有高分辨率结构数据的情况下，我们最终也可以使用这些数据来获得“粗粒”结构。这一领域的进展将对广泛的蛋白质科学产生影响，从而对改善人类健康产生影响。

DESCRIPTION (provided by applicant): Mass spectrometry is a powerful tool for the identification of proteins and the determination of their structures and properties. The scientific community routinely employs MS-based proteomics. A new opportunity, protein footprinting, is emerging that takes advantage of these refined proteomics capabilities that combine high performance liquid chromatography and mass spectrometry. The development of these techniques is driven by a growing number of collaborative projects in biomedical science and other fields. The Washington University Mass Spectrometry Resource (WUMSR) has played a key role in the development of protein footprinting for more than a decade and is an ideal place to support its expansion. While it does not yet have the resolution of X-ray crystallography and NMR, MS-based footprinting is significantly more specific than the low resolution tools of CD, fluorescence, Raman, absorbance, SPR, and calorimetry. In addition footprinting occurs while the proteins and their complexes are in their native states, affording relevant measurements of many important thermodynamic and kinetic parameters. Footprinting approaches include hydrogen deuterium exchange (H/DX), fast photochemical oxidation of proteins (FPOP), and other amino acid specific chemical modifications. To fulfill and disseminate the promise of protein footprinting, we propose to add a high performance, LC/MS system and commit it full time to support this research. The proposed instrument will be used to address the needs of 17 collaborators who have problems in protein biochemistry and medicine. The proteins have implications in cancer, viral infections, pain management, and brain disorders, principally Alzheimer's disease (AD). There are implications as well as in energy and photosynthesis. The approaches will enable studies of protein folding and unfolding, permit the determination of interfaces between important peptides (e.g., A? with implications in AD), determine binding interfaces of small ligands including potential drugs, and measure the affinities of binding. We will extend these measurements to protein assemblies, including those that are membrane-bound. Given that the footprinting experiments produce data that "constrains" protein and protein-complex structure, just as in NMR, we predict that we can ultimately use these data to obtain "coarse-grained" structures, even in the absence of high resolution structural data. Progress in this area will impact a broad range of protein science with consequent effects on improving human health.