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

通过氢交换质谱研究蛋白质结构和动力学的集成系统

• 批准号: 422442-2012
• 负责人: Konermann, Lars
• 金额: $ 10.92万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2011
• 资助国家: 加拿大
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=495794
• 关键词: Integrated; System; Studying; Protein; Structure

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.

该提案是针对通过质谱法（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执行其生物学任务。这种结构/功能/动力学三元组仍然知之甚少。Konermann和其他人的开创性工作表明，氢/氘交换（HDX）MS是解决这一问题的有力工具。高度柔性的蛋白质区域经历快速HDX。同位素交换在刚性段中慢得多。HDX工作流程包括消化和随后在淬灭条件下通过液相色谱/MS进行的肽分析。单个蛋白质片段的氘代动力学可以从所得的质量分布推导出来。商业HDX-MS平台的最新发展极大地改变了竞争环境。几家美国实验室已经购买了这种nanoACQUITY/HDX技术，但这些系统都还没有在加拿大上市。与传统的自制系统相比，这种新平台大大提高了再现性、消化效率、鲁棒性和软件易用性。拟议中的设备将允许Konermann的研究计划显着扩展到跨膜蛋白领域，其中许多代表重要的药物靶点。具体而言，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购买了一台最先进的Synapt质谱仪，占HDX平台的一半。nanoACQUITY/HDX代表了仍然缺少的第二部分（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。如果不能为这些设备获得资金，申请人的研究计划将受到严重挫折。