Mass spectrometry-based footprinting is an emerging approach for studying protein structure. Because integral membrane proteins are difficult targets for conventional structural biology, we recently developed a mass spectrometry (MS) footprinting method to probe membrane protein–drug interactions in live cells. This method can detect structural differences between apo and drug-bound states of membrane proteins, with the changes inferred from MS quantification of the cysteine modification pattern, generated by residue-specific chemical labeling. Here, we describe the experimental design, interpretation, advantages, and limitations of using cysteine footprinting by taking as an example the interaction of warfarin with vitamin K epoxide reductase, a human membrane protein. Compared with other structural methods, footprinting of proteins in live cells produces structural information for the near native state. Knowledge of cellular conformational states is a necessary complement to the high-resolution structures obtained from purified proteins in vitro. Thus, the MS footprinting method is broadly applicable in membrane protein biology. Future directions include probing flexible motions of membrane proteins and their interaction interface in live cells, which are often beyond the reach of conventional structural methods.

中文翻译：

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活细胞中的膜蛋白结构：通过基于质谱的足迹研究药物相互作用的方法

基于质谱的足迹法是研究蛋白质结构的新兴方法。由于完整的膜蛋白对于常规结构生物学而言是困难的靶标，因此我们最近开发了一种质谱（MS）足迹法来探测活细胞中的膜蛋白与药物的相互作用。该方法可以检测膜蛋白的载脂蛋白和药物结合状态之间的结构差异，并通过残留定量化学标记产生的半胱氨酸修饰模式的MS定量推断出变化。在这里，我们以华法林与人膜蛋白维生素K环氧还原酶的相互作用为例，描述了使用半胱氨酸足迹的实验设计，解释，优点和局限性。与其他结构方法相比，活细胞中蛋白质的足迹产生了接近天然状态的结构信息。了解细胞构象状态是从纯化蛋白获得的高分辨率结构的必要补充体外。因此，MS足迹法可广泛应用于膜蛋白生物学。未来的方向包括探测膜蛋白及其在活细胞中的相互作用界面的柔性运动，而这通常是常规结构方法无法实现的。