Determining the binding affinity is an important aspect of characterizing protein-ligand complexes. Here, we describe an approach based on covalent labeling (CL)-mass spectrometry (MS) that can accurately provide protein-ligand dissociation constants (Kd values) using diethylpyrocarbonate (DEPC) as the labeling reagent. Even though DEPC labeling reactions occur on a time scale that is similar to the dissociation/reassociation rates of many protein-ligand complexes, we demonstrate that relatively accurate binding constants can still be obtained as long as the extent of protein labeling is kept below 30%. Using two well-established model systems and one insufficiently characterized system, we find that Kd values can be determined that are close to values obtained in previous measurements. The CL-MS-based strategy that is described here should serve as an alternative for characterizing protein-ligand complexes that are challenging to measure by other methods. Moreover, this method has the potential to provide, simultaneously, the affinity and binding site information.

中文翻译：

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使用共价标记质谱法测定蛋白质配体亲和力。

确定结合亲和力是表征蛋白质-配体复合物的一个重要方面。在这里，我们描述了一种基于共价标记 (CL)-质谱 (MS) 的方法，该方法可以使用焦碳酸二乙酯 (DEPC) 作为标记试剂准确提供蛋白质-配体解离常数（Kd 值）。即使 DEPC 标记反应发生的时间尺度与许多蛋白质-配体复合物的解离/重新结合率相似，我们证明只要蛋白质标记的程度保持在 30% 以下，仍然可以获得相对准确的结合常数. 使用两个完善的模型系统和一个未充分表征的系统，我们发现可以确定的 Kd 值与先前测量中获得的值接近。此处描述的基于 CL-MS 的策略应作为一种替代方法来表征难以通过其他方法测量的蛋白质-配体复合物。此外，这种方法有可能同时提供亲和力和结合位点信息。