Ion mobility spectrometry-mass spectrometry offers the potential to characterize structures of transient protein assemblies and protein isoforms by means of their orientationally-averaged momentum transfer cross-sections. A commonly observed phenomenon is the compaction of a protein in the ion mobility measurement, that is, the cross section measured for the protein by ion mobility spectrometry is smaller than the cross section expected for its native structure. Consequently, this compaction means that at least some structural changes of the protein must have occurred during the ion mobility measurement. A major challenge is then to identify which aspects of the solution structure are retained in the ion mobility measurement and which ones are not. Here, we apply our recently developed Structure Relaxation Approximation (SRA) method in conjunction with trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) to probe compaction of the human protein chemokine (C-C motif) ligand 5 (also CCL5). Ion mobility spectra are recorded for various charge states and solution conditions of CCL5 under both “soft” and collisionally-activated conditions. Our data show that the SRA reproduces the overall trends in the experimental spectra: (1) the compaction of the CCL5 structure as seen in the experiments; (2) the general increase in the cross section for the various charge states; and (3) the increase in cross section after collisional-activation. The SRA attributes the compaction of the CCL5 structure mainly to the folding of the unstructured N-terminus onto the central Greek key motif of CCL5. By contrast, the SRA indicates that native residue-residue contacts present in the NMR structure are largely retained. Additionally, our analysis indicates that accurate treatment of proton transfer processes during the structural relaxation process would significantly improve the structural interpretation of ion mobility data by the SRA.

离子迁移谱-质谱提供了通过其定向平均动量传递截面来表征瞬态蛋白质组件和蛋白质同种型结构的潜力。通常观察到的现象是在离子迁移率测量中蛋白质的压实，即通过离子迁移谱测量蛋白质的横截面小于其天然结构预期的横截面。因此，这种压实意味着在离子迁移率测量过程中至少必须发生蛋白质的某些结构变化。一个主要的挑战是确定溶液结构的哪些方面保留在离子迁移率测量中，哪些方面没有。这里，我们将我们最近开发的结构松弛近似 (SRA) 方法与俘获离子迁移谱-质谱 (TIMS-MS) 结合使用，以探测人类蛋白质趋化因子 (CC 基序) 配体 5（也是 CCL5）的压实。在“软”和碰撞激活条件下，针对 CCL5 的各种电荷状态和溶液条件记录离子迁移谱。我们的数据表明，SRA 再现了实验光谱中的总体趋势：（1）实验中看到的 CCL5 结构的压实；(2) 各种电荷态横截面的普遍增加；(3) 碰撞激活后横截面的增加。SRA 将 CCL5 结构的压缩主要归因于非结构化 N 端折叠到 CCL5 的中央希腊键基序上。相比之下，SRA 表明 NMR 结构中存在的天然残基-残基接触在很大程度上得以保留。此外，我们的分析表明，在结构弛豫过程中准确处理质子转移过程将显着改善 SRA 对离子迁移率数据的结构解释。