CAREER: Developing hybrid IMS/OMS-MS/MS techniques for protein complex structure characterization

在化学部化学测量和成像计划以及生物基础设施部生物研究仪器开发（IDBR）计划的支持下，西弗吉尼亚大学的Valentine教授及其团队正在开发用于研究复杂生物分子三维形状的科学仪器和技术。这项开发工作旨在研究蛋白质复合物。蛋白质在生命过程中起着至关重要的作用。当这些分子不能形成正确的三维形状时，疾病往往会发生，因此能够确定这些生物分子的结构以及它们的组分如何形成关键形状变得重要。然而，由于它们相对较大的尺寸和其他因素，很难确定许多分子的精确结构。Valentine教授开发的新仪器和技术是确定这些蛋白质结构的重要方法。瓦伦丁教授还从事教育推广工作，以提高在科学，技术，工程和数学领域的中学和高中教师的培训，从而使许多学生受益。蛋白质复合物结构的表征以及共存蛋白质物种的结构对现代分析技术提出了挑战。瓦伦丁教授正在开发新的气相分离仪器和技术来研究这些问题。这项工作有三大任务。首先，混合漂移管，结合传统的离子迁移谱（IMS）分离泛音迁移谱（OMS）分析的构建和耦合与现有的线性离子阱质谱仪。该方法可以允许探测在非常短的时间尺度（微秒和更低）上发生的微小构象变化（~4%），增强当前的结构评估方法。第二，新技术用于表征与亨廷顿病相关的蛋白质系统。通过串联质谱法（MS）对由亨廷顿（Htt）蛋白的部分形成的多聚体肽离子进行OMS-氢氘交换（HDX）分析。将结果与从IMS-OMS-MS分析获得的结果进行比较，以提供用于在真实世界样品上测试新仪器和技术的手段，并提供在测量过程中正在经历结构转变的物种形式的新信息。最后的任务是使用溶液相HDX结合OMS-MS/MS研究来表征这些相同的蛋白质系统，以解开溶液和气相结构的连接，并提供有关气相中构象建立的信息。 这项研究可能为亨廷顿病的进展提供重要线索。 教育部分建立战略，提高初中和高中科学教学的功效，开发本科课程，介绍本科生的研究，并为研究生提供独特的研究和教学机会。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry and the Instrument Development for Biological Research (IDBR) Program in the Division of Biological Infrastructure, Professor Valentine at West Virginia University and his group are developing scientific instrumentation and techniques for studying the three-dimensional shapes of complex, biological molecules. This developmental work is aimed at the study of protein complexes. Proteins play a crucial role in life processes. When such molecules do not form the correct three dimensional shapes, disease often ensues and therefore it becomes important to be able to determine the structures of these biological molecules and how their components come to form critical shapes. However, because of their relatively large sizes and other factors, it is difficult to determine accurate structures for many molecules. The new instrumentation and techniques developed by Professor Valentine are important ways for determining the structures of such proteins. Professor Valentine is also engaged in educational outreach efforts to improve the training of Middle- and High-School teachers in the science, technology, engineering and mathematical fields thereby benefitting many students. The characterization of protein complex structures as well as the structures of coexisting protein species presents one challenge to modern analytical techniques. Professor Valentine is developing novel gas-phase separation instrumentation and techniques to study these problems. This work has three major tasks. First, a hybrid drift tube that combines traditional ion mobility spectrometry (IMS) separations with overtone mobility spectrometry (OMS) analysis is constructed and coupled with an existing linear ion trap mass spectrometer. The approach may allow the probing of minor conformational changes (~4%) occurring on very short timescales (microsecond and lower), enhancing current methods for structural assessments. Second, the novel technique is used to characterize a protein system associated with Huntington's Disease. Multimeric peptide ions formed by portions of the huntingtin (Htt) protein are subjected to OMS-hydrogen deuterium exchange (HDX) analysis with tandem mass spectrometry (MS). The results are compared with those obtained from IMS-OMS-MS analysis to provide a means for testing the new instrumentation and techniques on a real-world sample as well as provide new information in the form of species that are undergoing structural transformations during the course of the measurement. The final task is characterizing these same protein systems using solution-phase HDX combined with OMS-MS/MS studies to unravel linking of solution- and gas-phase structures as well as to provide information regarding conformation establishment in the gas-phase. This research may provide important clues about Huntington's Disease progression. The educational component establishes strategies for improving the efficacy of middle- and high-school science instruction, develops undergraduate curricula, introduces undergraduate students to research, and provides unique research and instructional opportunities for graduate students.