Automated Specific Amino Acid Footprinting Mass Spectrometry: Repurposing an HDX Platform for Determining Reagent Feasibility.

Protein footprinting is a mass spectrometry (MS)-based approach to measure protein conformational changes. One approach, amino acid specific labeling, imparts an irreversible modification to protein side chains but requires careful selection of the reactive reagent and often time-consuming optimization of experimental parameters prior to submission to bottom-up analysis. In this work, we repurpose a hydrogen-deuterium exchange MS (HDX-MS) LEAP HDX system for automated specific amino acid footprinting MS, demonstrating its efficacy in reaction optimization and monitoring applicability to specific ligand binding systems. We screened reagent conditions for two model ligand-binding systems and demonstrate the method’s efficacy for measuring differences induced by ligand binding. Our proof-of-concept experiments provide a platform for rapidly screening specific amino acid reagents and reaction conditions for particular protein systems.

蛋白质足迹法是一种基于质谱（MS）的方法，用于测量蛋白质的构象变化。一种方法是氨基酸特异性标记，它对蛋白质侧链进行不可逆的修饰，但需要仔细选择反应试剂，并且在进行自下而上的分析之前，往往需要对实验参数进行耗时的优化。在这项工作中，我们将一个氢 - 氘交换质谱（HDX - MS）LEAP HDX系统重新用于自动化的特异性氨基酸足迹法质谱，证明了它在反应优化方面的功效以及对特定配体结合系统的监测适用性。我们针对两个模型配体结合系统筛选了试剂条件，并证明了该方法在测量配体结合所引起的差异方面的有效性。我们的概念验证实验为快速筛选特定蛋白质系统的特异性氨基酸试剂和反应条件提供了一个平台。