CAREER: Next-Generation Ion Mobility-Mass-Spectrometry of Protein-ligand and Multiprotein Complexes

在化学系化学测量和成像计划的支持和分子和细胞生物科学系分子生物物理计划的共同资助下，密歇根大学的Brandon Ruotolo教授和他的团队将开发一种结合离子迁移率-质谱仪(IM-MS)技术的纳米电喷雾电离(NESI)，作为确定药物分子在多蛋白复合体中结合的全球结构后果的关键分析工具。这种非常规方法将首先去除天然状态多蛋白质/药物复合体周围的水，然后将离子电荷注入这些解溶的蛋白质，然后根据它们的大小/形状和它们在气相中的质量对这些蛋白质复合体离子进行分类。开发NESI-IM-MS以获得药物/蛋白质相互作用的详细信息需要1)对去溶蛋白质在真空中受到过量热能时如何改变其形状和结构(例如通过蛋白质展开)有更深入的物理理解，以及2)发明可靠的方案，使用这种内部温度诱导的蛋白质/药物复合体展开作为快速筛选新的多蛋白质抑制剂的手段。拟议研究的智力价值主要包括开发上述新的分析工具，并结合对蛋白质展开过程的更深层次的化学-物理类型描述，在没有大量溶剂的情况下进行操作。鉴于多蛋白质复合体的普遍重要性，我们预计我们的工作将产生普遍影响，但我们预计在IM-MS和蛋白质结构社区中的影响特别大。拟议研究的更广泛影响包括：(A)对参与研究的研究生和本科生进行关于最先进的IM-MS仪器、蛋白质纯化、蛋白质配基筛选、单分子反应动力学、气相动力学和蛋白质折叠/结构的高度跨学科培训。(B)吸纳来自历史上代表性不足的群体的学生，部分是通过国家科学基金会资助的REU方案。(C)在密歇根大学开发增强的质谱学课程，包括为本科教育设计的新颖实验室练习和仪器，以及为研究生设计的单元讲座课程。(D)通过密歇根大学的密歇根数学和科学学者方案向高中生推广。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry and co-fund from the Molecular Biophysics program in the Division of Molecular and Cellular Biosciences, Professor Brandon Ruotolo and his group at the University of Michigan will develop a nano-electrospray ionization (nESI) combined with ion mobility-mass spectrometry (IM-MS) technique as a key analytical tool for ascertaining the global structural consequences of drug molecule binding within multiprotein complexes. This unconventional approach will start by removing the water surrounding native-state multiprotein/drug complexes, it will then imbue these desolvated proteins with ionic charge, and then subsequently sort these protein complex ions according to both their size/shape and their mass in the gas phase. Developing nESI-IM-MS so that detailed information on drug/protein interactions can be obtained requires 1) a deeper physical understanding of how desolvated proteins can change their shape and structure (e.g. through protein unfolding) when given excess thermal energy in a vacuum and 2) the invention of robust protocols that use such internal temperature-induced protein/drug complex unfolding as a means of rapidly screening for new multiprotein inhibitors. The intellectual merits of the proposed research primarily include the development of the new analytical tools described above, combined with a deeper chemical-physics type description of the protein unfolding processes operative in the absence of bulk solvent. Given the general importance of multiprotein complexes we expect that our work will be generally impactful, but we expect especially high impact within both the IM-MS and protein structure communities. The broader impacts of the proposed research include (a) the highly interdisciplinary training of participating graduate and undergraduate students in state-of-the-art IM-MS instrumentation, protein purification, protein-ligand screening, unimolecular reaction dynamics, gas-phase kinetics, and protein folding/structure. (b) The inclusion of students from historically underrepresented groups, in part through an NSF-funded REU program. (c) Developing an enhanced mass spectrometry curriculum at the University of Michigan, including novel laboratory exercises and instrumentation designed for undergraduate education, as well as a modular lecture course designed for graduate students. (d) Outreach to high school students through the Michigan Math and Science Scholars program at the University of Michigan.