A Structural Variant Approach for Establishing a Detection Limit in Differential Hydrogen Exchange-Mass Spectrometry Measurements
A Structural Variant Approach for Establishing a Detection Limit in Differential Hydrogen Exchange-Mass Spectrometry Measurements
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DOI：
10.1021/acs.analchem.9b01326
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发表时间：
2019-07-02
影响因子：
7.4
通讯作者：
Weis, David D.
中科院分区：
文献类型：
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作者：
Hageman, Tyler S.;Weis, David D.
氢交换-质谱仪(HX-MS)因其能够检测蛋白质结构中的微小扰动而得到广泛推广，但这种扰动会导致FIX的微小差异。然而，HX-MS的检出限还没有得到广泛的研究，也没有一种有用的方法来确定FIX-MS的检出限。在这项工作中，我们设计了一个具有良好特征的结构变异尖峰模型来研究传统的基于多肽的HX-MS的检测限。通过将结构变体(使用麦芽糖结合蛋白W169G突变体模拟)的一小部分添加到参考蛋白(野生型麦芽糖结合蛋白)中，挑战了检测下限。只有5%的结构变异可以被检测到。通过远紫外圆二色谱、差示扫描量热法或尺寸排斥层析不能解决微小的结构扰动。此外，我们还验证了混合统计分析方法在HX-MS测量中可靠识别微小、显著差异的能力，该方法在一篇配对论文(10.1021/acs.analyChem.9b0第1325)中提出。使用我们的结构变异尖峰模型，我们展示了一种基准方法，用于确定FIX-MS的检测下限，用于检测在蛋白质结构相似性和相似性评估应用中可能遇到的高阶结构变化。
Hydrogen exchange-mass spectrometry (HX-MS) is widely promoted for its ability to detect subtle perturbations in protein structure, but such perturbations will result in small differences in FIX. However, the detection limit of HX-MS has not been widely investigated, nor is there a useful approach for defining the detection limit of FIX-MS measurements. In this work, we designed a well-characterized structural variant spiking model to investigate the detection limit of conventional peptide-based HX-MS. The detection limit was challenged by spiking small fractions of a structural variant (modeled using maltose binding protein W169G mutant) into a reference protein (wild-type maltose binding protein). As little as 5% of the structural variant could be detected. The small structural perturbation was not resolvable by far UV circular dichroism, differential scanning calorimetry, or size exclusion chromatography. Furthermore, we validated the ability of the hybrid statistical analysis approach, presented in a companion paper (10.1021/acs.analchem.9b01325), to reliably identify small, significant differences in HX-MS measurements. With our structural variant spiking model, we demonstrate a benchmarking approach for determining a detection limit of FIX-MS for detection of changes in higher-order structure that might be encountered in protein structural comparability and similarity assessment applications.