Characterizing complex protein states with next generation HX-MS concepts

该提案描述了氢/氘交换质谱（HX-MS）的新概念，用于表征与药物研究计划中药物开发活动相关的高度复杂的蛋白质系统。HX-MS提供连接蛋白质结构和蛋白质功能的构象数据。从概念上讲，HX过程提供了一个极好的机会来生成结构-活性关系图，并支持在药物研究的早期阶段选择先导物。然而，现有HX-MS技术的复杂性限制了它在这种情况下的使用。传统的解决方案具有低光谱容量和欠发达的数据分析程序，限制了HX-MS更简单的蛋白质系统和低通量。本文提出的研究将通过开发一种新的、可扩展的nanoHX-MS^2功能来克服这些障碍，该功能由评估潜在结构-活性关系的大量数据的信息学解决方案提供支持。我们将以我的实验室最近的HX创新为基础，结合分析仪器市场领先的技术提供商Sciex的新的基于qtof的质谱技术。该研究计划的主要挑战将是在不丢失序列覆盖的情况下分析超复杂蛋白质复合物或网络，并在数据收集和分析中消除人工干预的需要。通过有效利用MS^2维度，以及开发新的处理试剂和信息学策略，我们预计一个平台可以生成完整的蛋白质状态HX图，其复杂性至少是目前最佳功能的10倍。该平台将在制药合作伙伴的积极参与下开发，并有利于其药物开发工作。辉瑞将提供复杂蛋白质配体系统形式的测试方案，而基因泰克将提供治疗性蛋白质，以支持针对生物制剂市场的HX-MS^2操作衍生模式的开发。总之，我们预计我们的研究将解决在更广泛的药物研发市场实施HX-MS的主要障碍，并缩短商业化系统，软件和试剂的路径。

This proposal describes new concepts in hydrogen/deuterium exchange mass spectrometry (HX-MS) for characterizing highly-complex protein systems relevant to drug development activities in pharmaceutical research programs. HX-MS provides conformational data that link protein structure to protein function. Conceptually, HX processes offer an excellent opportunity to generate maps of structure-activity relationships and support the selection of leads in early stages of drug research. However, the complexity of available HX-MS technology has restricted its use in this context. Conventional solutions have low spectral capacity and under-developed data analysis routines, limiting HX-MS to simpler protein systems and low throughput. The research proposed here will overcome these barriers by developing a new, scalable nanoHX-MS^2 capability, supported by informatics solutions that evaluate the high volume of data for underlying structure-activity relationships. We will build on recent HX innovations from my laboratory, combined with new QTOF-based mass spectrometry technology from Sciex, a leading technology provider in the analytical instruments market. The major challenge for the research program will be to profile ultracomplex protein complexes or networks without loss of sequence coverage, and remove the need for manual intervention in both data collection and analysis. Through effective use of the MS^2 dimension, and the development of new processing reagents and informatics strategies, we anticipate a platform that can generate complete HX maps of protein states at least 10-fold more complex than current best capabilities. The platform will be developed with the active participation of pharmaceutical partners and benefit their drug development efforts. Pfizer will provide test scenarios in the form of complex protein-ligand systems, and Genentech will provide therapeutic proteins to support the development of a derivative mode of HX-MS^2 operation targeting the biologics market. Together, we anticipate that our research will address the key barriers to implementing HX-MS in a wider pharmaceutical R&D market, and shorten the path to commercializing systems, software and reagents.