Next-Generation Collision Induced Unfolding Analysis of Proteins

在化学系化学测量和成像项目的支持下，以及分子和细胞生物科学系的部分共同资助下，密歇根大学的Brandon Ruotolo教授和他的团队正在开发强大的新工具，用于表征蛋白质的三维结构——生命过程中的关键分子。具体来说，Ruotolo团队正在扩大“碰撞诱导展开”（CIU）的能力，这是一种通过快速去除正常环境中蛋白质分子周围的水来研究蛋白质结构的方法，然后诱导它们展开，同时使用一种称为离子迁移-质谱（IM-MS）的最先进工具来监测它们的大小和形状。该方法有可能彻底改变蛋白质稳定性评估的细节和获取速度。潜在的应用包括生物治疗蛋白及其操作模式的快速分析。这项工作为对化学测量和制药科学之间的界面研究感兴趣的学生提供了极好的培训。该研究有三个主要目标：(1)对影响行波型离子迁移率分离器峰宽的因素进行完整的物理描述，并根据标准验证所得预测；(2)开发下一代软件，以二维和三维高参数化多模态高斯拟合的形式将IM峰宽的解释纳入CIU分析工作流程；(3)创建并行的多维CIU方法，能够同时快速跟踪蛋白质混合物在许多电荷状态下的展开模式，即使存在显著的化学噪声。新的CIU工具正在使用对生物技术部门具有重要意义的治疗性蛋白质进行测试。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 and Imaging Program in the Division of Chemistry, and with partial co-funding from the Division of Molecular and Cellular Biosciences, Professor Brandon Ruotolo and his group at the University of Michigan are developing powerful new tools for characterizing the 3-dimensional structure of proteins - key molecules in life processes. Specifically, the Ruotolo group is expanding the capabilities of "collision induced unfolding" (CIU), an approach which studies protein structures by quickly removing the water which surrounds protein molecules in their normal environment, then induces their unfolding while monitoring their size and shape using a state-of-the-art tool known as ion mobility-mass spectrometry (IM-MS). The approach has the potential to revolutionize the detail and acquisition speed with which protein stability can be evaluated. Potential applications include the rapid analysis of biotherapeutic proteins and their mode of operation. The work provides excellent training for students interested in research at the interface between chemical measurements and the pharmaceutical sciences.The research addresses three main goals: (1) creating a complete physical description of factors impacting the peak width for traveling-wave-type ion mobility separators, and validating the resulting predictions against standards; (2) developing next-generation software that incorporates the interpretation of IM peak width into CIU analysis workflows in the form of highly-parameterized multi-modal Gaussian fitting in both two and three dimensions; and (3) creating parallelized, multi-dimensional CIU methods capable of rapidly tracking the unfolding patterns of protein mixtures across many charge states simultaneously, even in the presence of significant chemical noise. The new CIU tools are being tested using therapeutic proteins of significance to the biotechnology sector.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.