Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QZ, UK.
J Am Soc Mass Spectrom. 2019 Jan;30(1):45-57. doi: 10.1007/s13361-018-2064-1. Epub 2018 Nov 20.
Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND-gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration. Graphical abstract ᅟ.
通过质谱（气相 HDX-MS）测量的气相氢/氘交换是探测蛋白质离子构象的快速方法。然而，将气相 HDX-MS 用于研究蛋白质复合物的结构和相互作用的应用尚未得到充分利用。在最大程度保留其天然结构的条件下使蛋白质离子化（天然 MS），可以在电喷雾电离（ESI）后毫秒内研究类似于溶液的构象，这使得可以在质谱仪内使用 ND 气快速氘代杂原子结合的非酰胺氢。在这里，我们探索了气相 HDX-MS 用于检查蛋白质-蛋白质复合物并提供有关其结合表面和气相解离结构后果的用途。通过气相 HDX-MS 分析了范围从 24 kDa 二聚体到 395 kDa 24 聚体的蛋白质复合物，随后进行碰撞诱导解离（CID）。因此，可以估计形成复合物涉及的可交换位点的数量。例如，细胞色素 c 或α-乳白蛋白的二聚体的每个亚基掺入的氘原子少于其未结合的单体对应物，这提供了参与复合物形成的杂原子结合侧链氢数量的度量。我们还研究了在蛋白质复合物解离时不对称电荷分配是否导致分子间 H/D 迁移。在较大的多聚体蛋白质复合物中，解离的单体显示出氘的显著增加。这表明在 CID 过程中，不对称电荷分配的一部分会发生分子间 H/D 迁移。我们讨论了几个可能解释这种增加的氘含量的模型，并发现仅涉及迁移电荷的氘原子可以解释在逐出的单体上观察到的大部分氘富集。总之，可以使用逐出亚基的氘含量来估计完整复合物的氘含量，并且对于大复合物，观察到的偏差可以通过电荷迁移来解释。摘要图。
