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

该提案是用于通过质谱（MS）研究蛋白质结构和动力学所需的nanoACQUITY/HDX系统。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来完成它们的生物任务。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/氘交换（HDX）质谱是解决这一问题的有力工具。高度灵活的蛋白质区域经历快速HDX。刚性段的同位素交换要慢得多。HDX工作流程包括在淬火条件下的消化和随后的液相色谱/质谱分析。单个蛋白质片段的氘化动力学可以从所得的质量谱中推断出来。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nanoACQUITY/HDX技术，但这些系统在加拿大还没有可用。与传统的自制系统相比，这个新平台大大提高了再现性、消化效率、健壮性和软件易用性。该设备将使Konermann的研究项目显著扩展到跨膜蛋白领域，其中许多是重要的药物靶点。具体来说，nanoACQUITY/HDX将允许研究跨膜渗透、分子马达机制（与Dunn合作）以及细菌膜酶的结构表征（与Valvano合作）。利用从别处获得的资金，Konermann能够购买最先进的Synapt质谱仪，该质谱仪代表了HDX平台的一半。nanoACQUITY/HDX代表了仍然缺失的另一半（用于液体处理/消化/色谱）。目前的提案为HQP培训提供了极好的机会。如果不能保证为这些设备提供资金，将对申请人的研究项目造成严重的挫折。

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.