Advancing Native Mass Spectrometry for Probing Protein Equilibria and Dynamics

在化学系化学测量与成像项目的支持下，以及分子与细胞生物科学系分子生物物理项目的部分共同资助下，华盛顿大学的布什教授和他的团队正在开发新的方法来表征蛋白质及其复合物的平衡和动力学。在布什实验室开发的最先进的技术使溶液中生物分子的相互作用和结构的新测量成为可能。这些测量解决了生物分子和生物材料快速表征的未满足需求，包括它们的组装、异质性、质量和相似性。这些测量将有助于回答有关蛋白质和蛋白质复合物的结构和功能的重要问题，对于理解人类疾病的分子基础和开发治疗方法非常重要。在布什实验室工作的本科生和研究生接受测量科学、数据科学和技术交流方面的培训，为他们在各种各样的职业生涯中取得成功做好准备，从而提高美国的经济竞争力。对于溶液中蛋白质平衡和动力学的测量，特别是快速、敏感和容忍样品异质性的方法，还没有得到满足。原生离子迁移率（IM）质谱（MS）具有许多特性，使其能够很好地满足这些未满足的需求。然而，考虑到使用液相和气相测量确定的平衡性质之间的差异，降低了对本地IM-MS数据的信心，并阻碍了这些技术的广泛采用。本研究通过(1)使用、表征和控制伴随电喷雾电离的电化学诱导的样品变化来解决这些未满足的需求；(2)促进负离子电喷雾电离更广泛地应用于本地IM-MS；(3)使用可变温度电喷雾电离快速表征热分布和蛋白质折叠可逆的程度。这反过来将使本地IM-MS在生物物理学和结构生物学中发挥更大的作用，这将有助于了解生物过程和治疗人类疾病的新知识。本科生研究人员正在开发和制造低成本的仪器，以扩大化学测量的机会，并扩大研究的参与。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With support from the Chemical Measurement & Imaging Program in the Division of Chemistry and partial co-funding from the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences, Professor Bush and his group at the University of Washington are developing new methods to characterize the equilibria and dynamics of proteins and their complexes. State-of-the-art techniques developed in the Bush lab enable new measurements of the interactions and structures of biological molecules in solution. These measurements address unmet needs for the rapid characterization of biological molecules and biological materials, including their assembly, heterogeneity, quality, and similarity. These measurements will help answer important questions about the structure and function of proteins and protein complexes, important for understanding the molecular basis for human diseases and developing therapeutics. Undergraduate and graduate students working in the Bush lab are trained in measurement science, data science, and technical communication, which prepares them for success in a wide variety of careers that increase the economic competitiveness of the United States.There are unmet needs for measurements that probe protein equilibria and dynamics in solution, especially methods that are fast, sensitive, and tolerant of sample heterogeneity. Native ion mobility (IM) mass spectrometry (MS) has many attributes that make it well positioned to fulfill these unmet needs. However, concerns about differences between equilibrium properties determined using solution- and gas-phase measurements reduces the confidence in native IM-MS data and inhibits the broader adoption of these technologies. This research addresses these unmet needs by (1) using, characterizing, and controlling the electrochemically-induced changes to samples concomitant with electrospray ionization; (2) facilitating the broader adoption of negative ion electrospray ionization for native IM-MS; and (3) using variable-temperature electrospray ionization to rapidly characterize thermal profiles and the extent to which protein folding is reversible. This in turn will enable native IM-MS to play a greater role in biophysics and structural biology, which will contribute to new knowledge of biological processes and cures for human disease. Undergraduate student researchers are developing and fabricating low-cost instruments to expand access to chemical measurements and broaden participation in research.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.