Tajoddin Nastaran N, Konermann Lars
Department of Chemistry, The University of Western Ontario, London, Ontario N6A 5B7, Canada.
Anal Chem. 2022 Nov 8;94(44):15499-15509. doi: 10.1021/acs.analchem.2c03931. Epub 2022 Oct 28.
Differential scanning calorimetry (DSC) is a standard tool for probing the resilience of monoclonal antibodies (mAbs) and other protein therapeutics against thermal degradation. Unfortunately, DSC usually only provides insights into global unfolding, although sequential steps are sometimes discernible for multidomain proteins. Temperature-dependent hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has the potential to probe heat-induced events at a much greater level of detail. We recently proposed a strategy to deconvolute temperature-dependent HDX data into contributions from local dynamics, global unfolding/refolding, as well as chemical labeling. However, that strategy was validated only for a small protein (Tajoddin, N. N.; Konermann, L. 2020, 92, 10058). The current work explores the applicability of this HDX framework to the NIST reference mAb (NISTmAb), a large multidomain protein that is prone to aggregation and has three melting points. Using global fitting, we were able to model HDX profiles across the NISTmAb sequence between zero and 95 °C, and for time points between 15 s and 20 min. We uncovered the enthalpic and entropic contributions of local fluctuations that govern the conformational dynamics at low temperatures. The CH2 and CH3 domains were found to be increasingly affected by global unfolding/refolding in the vicinity of their melting points, although the transiently unfolded protein displayed significant residual protection. Global dynamics were not involved in the deuteration of the Fab domains (which have the highest melting point). Instead, global Fab unfolding was followed immediately by irreversible aggregation. Our results reveal that the thermodynamic HDX-MS strategy applied in this work is well suited for probing spatially resolved dynamics of thermally stressed large proteins such as mAbs, complementing data obtained by DSC.
差示扫描量热法（DSC）是一种用于探究单克隆抗体（mAb）和其他蛋白质治疗药物抗热降解能力的标准工具。不幸的是，DSC通常只能提供关于整体解折叠的见解，尽管对于多结构域蛋白质，有时可以辨别出连续的步骤。温度依赖性氢/氘交换（HDX）质谱（MS）有潜力在更高的细节水平上探究热诱导事件。我们最近提出了一种策略，将温度依赖性HDX数据解卷积为局部动力学、整体解折叠/重折叠以及化学标记的贡献。然而，该策略仅针对一种小蛋白质进行了验证（Tajoddin, N. N.; Konermann, L. 2020, 92, 10058）。当前的工作探索了这种HDX框架对NIST标准mAb（NISTmAb）的适用性，NISTmAb是一种大型多结构域蛋白质，易于聚集且有三个熔点。通过全局拟合，我们能够对NISTmAb序列在0至95°C之间以及15秒至20分钟之间的时间点的HDX谱进行建模。我们揭示了在低温下控制构象动力学的局部波动的焓和熵贡献。发现CH2和CH3结构域在其熔点附近越来越受到整体解折叠/重折叠的影响，尽管瞬时解折叠的蛋白质显示出显著的残余保护。全局动力学不参与Fab结构域（熔点最高）的氘代。相反，Fab结构域的整体解折叠紧接着是不可逆的聚集。我们的结果表明，本工作中应用的热力学HDX-MS策略非常适合探究热应激大蛋白质（如mAb）的空间分辨动力学，补充了DSC获得的数据。
