†University of Edinburgh, School of Chemistry, West Mains Road, Edinburgh EH9 3JJ, United Kingdom.
‡University of Manchester, School of Chemistry, Manchester Institute of Biotechnology, Michael Barber Centre for Collaborative Mass Spectrometry, 131 Princess Street, Manchester M1 7DN, United Kingdom.
Anal Chem. 2015 Jun 16;87(12):6271-9. doi: 10.1021/acs.analchem.5b01063. Epub 2015 Jun 3.
The thermal stability and strength of interactions in proteins are commonly measured using isothermal calorimetry and differential scanning calorimetry providing a measurement that averages over structural transitions that occur as the proteins melt and dissociate. Here, we apply variable temperature ion mobility mass spectrometry (VT-IM-MS) to study the effect of temperature on the stability and structure of four multimeric protein complexes. VT-IM-MS is used here to investigate the change in the conformation of model proteins, namely, transthyretin (TTR), avidin, concanavalin A (conA), and human serum amyloid P component (SAP) at elevated temperatures prior, during, and after dissociation up to 550 K. As the temperature of the buffer gas is increased from 300 to 350 K, a small decrease in the collision cross sections ((DT)CCS(He)) of protein complexes from the values at room temperature is observed, and is associated with complex compaction occurring close to the reported solution T(m). At significantly higher temperatures, each protein complex undergoes an increase in (DT)CCS(He) and in the width of arrival time distributions (ATD), which is attributed to extensive protein unfolding, prior to ejection of a highly charged monomer species. This approach allows us to decouple the distinct gas phase melting temperature (T(m)) from the temperature at which we see subunit dissociation. The thermally induced dissociation (TID) mechanism is observed to initially proceed via the so-called "typical" (CID) dissociation route. Interestingly, data collected at higher analysis temperature suggests that the TID process might be adapting more "atypical" dissociation route.
蛋白质中相互作用的热稳定性和强度通常使用等温热力学和差示扫描量热法来测量，这提供了一种平均测量值，涵盖了蛋白质熔融和离解过程中发生的结构转变。在这里，我们应用变温离子淌度质谱（VT-IM-MS）来研究温度对四个多聚体蛋白质复合物稳定性和结构的影响。VT-IM-MS 用于研究模型蛋白构象的变化，即转甲状腺素蛋白（TTR）、亲和素、伴刀豆球蛋白 A（conA）和人血清淀粉样蛋白 P 成分（SAP）在离解前、离解中和离解后在升高温度下（高达 550 K）的变化。随着缓冲气体温度从 300 升高到 350 K，从室温下的值观察到蛋白质复合物的碰撞截面（(DT)CCS(He)）略有减小，这与接近报道的溶液 T(m)时发生的复合物紧缩有关。在温度显著升高的情况下，每个蛋白质复合物的 (DT)CCS(He) 和到达时间分布（ATD）的宽度增加，这归因于在高度带电的单体物种被喷射之前广泛的蛋白质展开。这种方法使我们能够将明显的气相熔融温度（T(m)）与我们观察到的亚基离解温度区分开来。观察到热诱导解离（TID）机制最初通过所谓的“典型”（CID）解离途径进行。有趣的是，在较高分析温度下收集的数据表明，TID 过程可能正在适应更多的“非典型”解离途径。
