Structural analysis of proteins in a conformationally heterogeneous mixture has long been a difficult problem in structural biology. In structural analysis by covalent labeling mass spectrometry, conformational heterogeneity results in data reflecting a weighted average of all conformers, complicating data analysis and potentially causing misinterpretation of results. Here, we describe a method coupling size exclusion chromatography (SEC) with hydroxyl radical protein footprinting using inline fast photochemical oxidation of proteins (FPOP). Using a controlled synthetic mixture of holomyoglobin and apomyoglobin, we demonstrate that we can achieve accurate footprints of each conformer using LC-FPOP when compared to off-line FPOP of each pure conformer. We then applied LC-FPOP to analyze the adalimumab heat-shock aggregation process. We found that the LC-FPOP footprint of unaggregated adalimumab was consistent with a previously published footprint of the native IgG. The LC-FPOP footprint of the aggregation product indicated that heat shock aggregation primarily protected the hinge region, suggesting this region is involved with the heat shock aggregation process of this molecule. LC-FPOP offers a new method to probe dynamic conformationally heterogeneous mixtures such as biopharmaceutical aggregates, and obtain accurate information on the topography of each conformer.

中文翻译：

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在线液相色谱-蛋白质的快速光化学氧化允许对构象异质混合物进行目标结构分析

构象异质混合物中蛋白质的结构分析长期以来一直是结构生物学中的难题。在通过共价标记质谱进行结构分析中，构象异质性导致数据反映所有构象异构体的加权平均值，从而使数据分析复杂化并可能导致结果的错误解释。在这里，我们描述了一种使用内嵌快速光化学氧化蛋白质（FPOP）的尺寸排阻色谱（SEC）与羟基自由基蛋白质足迹耦合的方法。使用全血红蛋白和载脂蛋白的受控合成混合物，我们证明与每个纯构象剂的离线FPOP相比，使用LC-FPOP可以实现每个构象剂的准确足迹。然后，我们应用LC-FPOP分析了阿达木单抗热休克聚集过程。我们发现未聚集的阿达木单抗的LC-FPOP足迹与先前发表的天然IgG足迹一致。聚集产物的LC-FPOP足迹表明，热激聚集主要保护铰链区，表明该区域与该分子的热激聚集过程有关。LC-FPOP提供了一种新方法来探测动态构象异质的混合物，例如生物制药聚集体，并获得有关每个构象异构体形貌的准确信息。提示该区域与该分子的热激聚集过程有关。LC-FPOP提供了一种新方法来探测动态构象异质的混合物，例如生物制药聚集体，并获得有关每个构象异构体形貌的准确信息。提示该区域与该分子的热激聚集过程有关。LC-FPOP提供了一种新方法来探测动态构象异质的混合物，例如生物制药聚集体，并获得有关每个构象异构体形貌的准确信息。