Propagating Error through Traveling-Wave Ion Mobility Calibration
Propagating Error through Traveling-Wave Ion Mobility Calibration
复制标题
通过行波离子淌度校准传播误差
DOI：
10.1021/jasms.1c00144
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发表时间：
2021
影响因子：
3.2
通讯作者：
Gallagher, Elyssia S.
中科院分区：
文献类型：
--
作者：
Edwards, Alexis N.;Tran, Hien M.;Gallagher, Elyssia S.
天然质量谱(MS)用于阐明蛋白质复合体的化学计量，并通过在气相中保持天然的、非共价的相互作用来定量结合相互作用。然而，电离迫使蛋白质形成特定的构象，失去与溶剂化蛋白质结构相关的溶液相动力学。气相结构与溶液中的结构或与其他气相离子群体的比较具有许多生物学意义。首先，分析气相中保持的各种构象可以提供对蛋白质溶液相能量格局的洞察。蛋白质及其复合体的气相构象可以用离子迁移率(IM)光谱进行研究。具体地说，漂移管(DT)-IM利用均匀的电场来推动一群气相离子穿过包含中性气体的区域。通过测量气相离子的迁移率(K)，用户能够计算平均动量转移截面(DTCCS)，它提供了关于离子的结构信息。相反，在行波离子迁移率谱(TWIMS)中，TWCCS值不能直接从离子的迁移率得出，必须在校准后确定。尽管所需的校准增加了不确定度，但通常只报告计算的TWCCS的平均和标准偏差，考虑到与重复测量相关的不确定度，这只是总不确定度的一小部分。在这里，我们校准了一台TWIMS仪器，并获得了四种蛋白质的TWCCSN2和TWCCSN2→H值：细胞色素、泛素、脱脂肌红蛋白和全息肌红蛋白。我们表明，与只报告TWCCS的标准偏差相比，通过校准传播误差导致计算的TWCCS值的数量显著增加，这些值与文献值(DTCCS)在实验误差范围内一致。加入这种额外的不确定性可以更全面地评估蛋白质离子的气相构象，使天然IM-MS采样的结构能够与其他已报道的结构进行比较，无论是实验结构还是计算结构。
Native mass spectrometry (MS) is used to elucidate the stoichiometry of protein complexes and quantify binding interactions by maintaining native-like, noncovalent interactions in the gas phase. However, ionization forces proteins into specific conformations, losing the solution-phase dynamics associated with solvated protein structures. Comparison of gas-phase structures to those in solution, or to other gas-phase ion populations, has many biological implications. For one, analyzing the variety of conformations that are maintained in the gas-phase can provide insight into a protein’s solution-phase energy landscape. The gas-phase conformations of proteins and complexes can be investigated using ion mobility (IM) spectrometry. Specifically, drift tube (DT)-IM utilizes uniform electric fields to propel a population of gas-phase ions through a region containing a neutral gas. By measuring the mobility (K) of gas-phase ions, users are able to calculate an average momentum transfer cross section (DTCCS), which provides structural information on the ion. Conversely, in traveling-wave ion mobility spectrometry (TWIMS),TWCCS values cannot be derived directly from an ion’s mobility but must be determined following calibration. Though the required calibration adds uncertainty, it is common to report only an average and standard deviation of the calculatedTWCCS, accounting for uncertainty associated with replicate measurements, which is a fraction of the overall uncertainty. Herein, we calibrate a TWIMS instrument and deriveTWCCSN2andTWCCSN2→Hevalues for four proteins: cytochromec, ubiquitin, apo-myoglobin, and holo-myoglobin. We show that compared to reporting only the standard deviation ofTWCCS, propagating error through the calibration results in a significant increase in the number of calculatedTWCCS values that agree within experimental error with literature values (DTCCS). Incorporating this additional uncertainty provides a more thorough assessment of a protein ion’s gas-phase conformations, enabling the structures sampled by native IM-MS to be compared against other reported structures, both experimental and computational.