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

职业：蛋白质-配体和多蛋白复合物的下一代离子淌度质谱分析

• 批准号: 1253384
• 负责人: Brandon Ruotolo
• 金额: $ 45万
• 依托单位: Regents of the University of Michigan - Ann Arbor
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2019-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1253384&HistoricalAwards=false
• 关键词: CAREER; Next; Generation; Ion; Mobility

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.

在化学系化学测量和成像项目的支持下，在分子和细胞生物科学系分子生物物理项目的共同资助下，密歇根大学的布兰登·鲁托洛教授和他的小组将开发一种纳米电喷雾电离（nESI）结合离子迁移率-质谱（IM-MS）技术作为一个关键的分析工具，用于确定药物分子结合多蛋白复合物内的全球结构后果。这种非常规的方法将通过去除天然状态多蛋白质/药物复合物周围的水开始，然后将这些去溶剂化的蛋白质注入离子电荷，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物离子进行分类。 开发nESI-IM-MS，以便获得药物/蛋白质相互作用的详细信息，需要1）对去溶剂化蛋白质如何改变其形状和结构有更深入的物理理解（例如，通过蛋白质解折叠），以及2）发明了使用这种内部温度诱导的蛋白质解折叠的稳健方案。药物复合物展开作为快速筛选新的多蛋白抑制剂的手段。 拟议研究的智力优势主要包括开发上述新的分析工具，结合更深入的化学物理类型描述的蛋白质展开过程中的散装溶剂的情况下运作。考虑到多蛋白复合物的普遍重要性，我们预计我们的工作将具有普遍的影响力，但我们预计在IM-MS和蛋白质结构社区中的影响力特别高。 拟议研究的更广泛影响包括：（a）参与研究生和本科生在最先进的IM-MS仪器，蛋白质纯化，蛋白质配体筛选，单分子反应动力学，气相动力学和蛋白质折叠/结构方面的高度跨学科培训。 (b)包括来自历史上代表性不足的群体的学生，部分通过NSF资助的REU计划。(c)在密歇根大学开发增强的质谱课程，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模块化讲座课程。 (d)通过密歇根大学的密歇根数学和科学学者计划与高中生进行外联。