Environmental Molecular Sciences Laboratory , Pacific Northwest National Laboratory , 3335 Innovation Boulevard , Richland , Washington 99354 , United States.
Anal Chem. 2020 Jan 21;92(2):1788-1795. doi: 10.1021/acs.analchem.9b03469. Epub 2020 Jan 7.
The extent to which noncovalent protein complexes retain native structure in the gas phase is highly dependent on experimental conditions. Energetic collisions with background gas can cause structural changes ranging from unfolding to subunit dissociation. Additionally, recent studies have highlighted the role of charge in such structural changes, but the mechanism is not completely understood. In this study, native top down (native TD) mass spectrometry was used to probe gas-phase structural changes of alcohol dehydrogenase (ADH, 4mer) under varying degrees of in-source activation. Changes in covalent backbone fragments produced by electron capture dissociation (ECD) or 193 nm ultraviolet photodissociation (UVPD) were attributed to structural changes of the ADH 4mer. ECD fragments indicated unfolding started at the N-terminus, and the charge states of UVPD fragments enabled monitoring of charge migration to the unfolded regions. Interestingly, UVPD fragments also indicated that the charge at the "unfolding" N-terminus of ADH decreased at high in-source activation energies after the initial increase. We proposed a possible "refolding-after-unfolding" mechanism, as further supported by monitoring hydrogen elimination from radical a-ions produced by UVPD at the N-terminus of ADH. However, "refolding-after-unfolding" with increasing in-source activation was not observed for charge-reduced ADH, which likely adopted compact structures that are resistant to both charge migration and unfolding. When combined, these results support a charge-directed unfolding mechanism for protein complexes. Overall, an experimental framework was outlined for utilizing native TD to generate structure-informative mass spectral signatures for protein complexes that complement other structure characterization techniques, such as ion mobility and computational modeling.
非共价蛋白质复合物在气相中保留天然结构的程度在很大程度上取决于实验条件。与背景气体的能量碰撞会导致结构变化，范围从展开到亚基解离。此外，最近的研究强调了电荷在这种结构变化中的作用，但机制尚不完全清楚。在这项研究中，使用天然自上而下（native TD）质谱法来探测在不同程度的源内激活下醇脱氢酶（ADH，4mer）的气相结构变化。电子俘获解离（ECD）或 193nm 紫外光解离（UVPD）产生的共价骨架片段的变化归因于 ADH 4mer 的结构变化。ECD 片段表明展开始于 N 端，并且 UVPD 片段的电荷状态能够监测电荷迁移到展开区域。有趣的是，UVPD 片段还表明，ADH 的“展开”N 端的电荷在源内激活能增加后最初增加后会降低。我们提出了一种可能的“展开后折叠”机制，这进一步得到了通过监测 UVPD 在 ADH 的 N 端产生的自由基 a-离子从氢键中消除的支持。然而，对于电荷减少的 ADH，并没有观察到随着源内激活而增加的“展开后折叠”，这可能采用了对电荷迁移和展开都具有抗性的紧凑结构。总之，这些结果支持了蛋白质复合物的电荷导向展开机制。总体而言，概述了一个利用天然 TD 生成蛋白质复合物结构信息丰富的质谱特征的实验框架，该框架补充了其他结构特征技术，如离子淌度和计算建模。
