An Integrated System for Studying Protein Structure and Dynamics by Hydrogen Exchange Mass Spectrometry

这项建议是为了建立一个纳米ACQUITY/HDX系统，该系统是用质谱仪(MS)研究蛋白质结构和动力学所必需的。蛋白质的细胞功能与其结构密切相关。然而，蛋白质也必须经历大量的构象运动才能完成它们的生物学任务。这种结构/功能/动力学三位一体仍然知之甚少。Konermann和其他人的开创性工作揭示了氢/氢交换(HDX)MS是解决这一问题的强大工具。高度灵活的蛋白质区域会经历快速的HDX。在刚性段中，同位素交换要慢得多。HDX的工作流程包括消化和随后在淬火条件下用液相色谱/质谱仪进行的多肽分析。从得到的质量分布可以推断出单个蛋白质片段的氢化动力学。最近商业HDX-MS平台的发展极大地改变了竞争环境。几个美国实验室已经购买了这种纳米ACQUITY/HDX技术，但这些系统还没有在加拿大上市。与传统的自制系统相比，这一新平台提供了极大改进的重复性、消化效率、健壮性和软件易用性。拟议中的设备将使科纳曼的研究计划显著扩展到跨膜蛋白质领域，其中许多蛋白质代表着重要的药物靶标。具体地说，纳米ACQUITY/HDX将允许研究跨膜渗透、分子马达的机制(与Dunn)以及细菌膜酶的结构特征(与Valvano)。利用从其他地方获得的资金，科纳曼能够购买一台最先进的Synapt质谱仪，它代表了HDX平台的一半。纳米ACQUITY/HDX代表仍然缺失的另一半(用于液体处理/消化/层析)。目前的建议为HQP培训提供了极好的机会。如果不能为这种设备获得资金，将对申请者的研究计划造成严重挫折。

This proposal is for a nanoACQUITY/HDX system that is required for studying protein structure and dynamics by mass spectrometry (MS). The cellular functions of proteins are intimately linked to their structures. However, proteins also have to undergo a multitude of conformational motions to perform their biological tasks. This structure/function/dynamics triad remains poorly understood. Pioneering work by Konermann and others has revealed that hydrogen/deuterium exchange (HDX) MS is a powerful tool for addressing this problem. Protein regions that are highly flexible undergo rapid HDX. Isotope exchange is much slower in rigid segments. The HDX workflow involves digestion and subsequent peptide analysis by liquid chromatography/MS under quench conditions. The deuteration kinetics of individual protein segments can be deduced from the resulting mass profiles. The recent development of a commercial HDX-MS platform has dramatically changed the playing field. Several US labs already purchased this nanoACQUITY/HDX technology, but none of these systems are available in Canada yet. This new platform provides greatly improved reproducibility, digestion efficiency, robustness, and software ease-of-use compared to conventional home-built systems. The proposed equipment will allow a significant expansion of Konermann's research program into the area of transmembrane proteins, many of which represent important drug targets. Specifically, the nanoACQUITY/HDX will allow studies on transmembrane permeation, the mechanism of molecular motors (with Dunn), as well as the structural characterization of bacterial membrane enzymes (with Valvano). Using funds obtained elsewhere, Konermann was able to purchase a state-of-the-art Synapt mass spectrometer that represents one half of the HDX platform. The nanoACQUITY/HDX represents the second half that is still missing (for liquid handling/digestion/chromatography). The current proposal offers superb opportunities for HQP training. Failure to secure funding for this equipment would be a severe setback for the applicants' research programs.