Garcia Natalie K, Deperalta Galahad, Wecksler Aaron T
Department of Protein Analytical Chemistry, Genentech Inc., South San Francisco, CA 94080, United States.
Protein Pept Lett. 2019;26(1):35-43. doi: 10.2174/0929866526666181128141953.
Biotherapeutics, particularly monoclonal antibodies (mAbs), are a maturing class of drugs capable of treating a wide range of diseases. Therapeutic function and solutionstability are linked to the proper three-dimensional organization of the primary sequence into Higher Order Structure (HOS) as well as the timescales of protein motions (dynamics). Methods that directly monitor protein HOS and dynamics are important for mapping therapeutically relevant protein-protein interactions and assessing properly folded structures. Irreversible covalent protein footprinting Mass Spectrometry (MS) tools, such as site-specific amino acid labeling and hydroxyl radical footprinting are analytical techniques capable of monitoring the side chain solvent accessibility influenced by tertiary and quaternary structure. Here we discuss the methodology, examples of biotherapeutic applications, and the future directions of irreversible covalent protein footprinting MS in biotherapeutic research and development.
Bottom-up mass spectrometry using irreversible labeling techniques provide valuable information for characterizing solution-phase protein structure. Examples range from epitope mapping and protein-ligand interactions, to probing challenging structures of membrane proteins. By paring these techniques with hydrogen-deuterium exchange, spectroscopic analysis, or static-phase structural data such as crystallography or electron microscopy, a comprehensive understanding of protein structure can be obtained.
生物治疗药物，尤其是单克隆抗体（mAb），是一类不断成熟的药物，能够治疗多种疾病。治疗功能和溶液稳定性与一级序列正确折叠成高阶结构（HOS）以及蛋白质运动（动力学）的时间尺度相关。直接监测蛋白质HOS和动力学的方法对于绘制治疗相关的蛋白质-蛋白质相互作用以及评估正确折叠的结构非常重要。不可逆共价蛋白质足迹质谱（MS）工具，如位点特异性氨基酸标记和羟基自由基足迹，是能够监测受三级和四级结构影响的侧链溶剂可及性的分析技术。在此，我们讨论不可逆共价蛋白质足迹质谱在生物治疗研发中的方法、生物治疗应用实例以及未来方向。
使用不可逆标记技术的自下而上质谱为表征溶液相蛋白质结构提供了有价值的信息。实例包括表位作图和蛋白质-配体相互作用，以及探测膜蛋白具有挑战性的结构。通过将这些技术与氢-氘交换、光谱分析或晶体学或电子显微镜等静态相结构数据相结合，可以全面了解蛋白质结构。
