Ahmed Ezaz, Jain Rohit, Schlatzer Daniela, Tavares Pereira Lopes Filipa Blasco, Kiselar Janna, Lodowski David T, Chance Mark R, Farquhar Erik R
Center for Synchrotron Biosciences, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH, 44106, USA; Department of Nutrition, Case Western Reserve University, School of Medicine, 10900 Euclid Ave., Cleveland, OH, 44106, USA.
Center for Proteomics and Bioinformatics, Case Western Reserve University, School of Medicine, 10900 Euclid Ave., Cleveland, OH, 44106, USA.
Biochem Biophys Res Commun. 2025 May 5;762:151745. doi: 10.1016/j.bbrc.2025.151745. Epub 2025 Apr 1.
Reactive oxygen species play a crucial role in cellular processes, but their effects on protein structure and function in vivo remain challenging to study. Here, we present an approach using synchrotron-based X-ray footprinting methods to probe protein structure, via quantitative LC-coupled mass spectrometry of methionine oxidation (MSOx) in live E. coli. A label-free proteomic analysis identified 2104 proteins from E. coli, with 465 proteins exhibiting MSOx modifications distributed across multiple cellular compartments. Changes in MSOx modification with increasing X-ray dose revealed a correlation between rates of modification and solvent-accessible surface area in vivo for selected proteins responsive to exposure, providing a direct probe of protein structure and its conformational plasticity in the cell. The approach developed here offers a unique in-cell quantitative readout of methionine oxidation and solvent accessibility through radiolytic hydroxyl radical labeling. With this method, the landscape of methionine oxidation in E. coli can be mapped, providing insights into protein behavior under oxidative stress. It represents a first step in developing radiolysis and E. coli as platforms for in vivo protein structure assessment. The potential applications in drug discovery, protein engineering, and systems biology of protein conformations are considerable.
活性氧在细胞过程中起着至关重要的作用，但其对体内蛋白质结构和功能的影响仍难以研究。在此，我们提出一种方法，利用基于同步加速器的X射线足迹法，通过对活的大肠杆菌中甲硫氨酸氧化（MSOx）进行定量液相色谱-串联质谱分析来探测蛋白质结构。一项无标记蛋白质组学分析从大肠杆菌中鉴定出2104种蛋白质，其中465种蛋白质表现出MSOx修饰，分布在多个细胞区室。随着X射线剂量增加，MSOx修饰的变化揭示了体内对辐射有反应的特定蛋白质的修饰速率与溶剂可及表面积之间的相关性，为细胞内蛋白质结构及其构象可塑性提供了直接探测手段。本文开发的方法通过辐射分解产生的羟基自由基标记，提供了一种独特的细胞内甲硫氨酸氧化和溶剂可及性的定量读数。通过这种方法，可以绘制大肠杆菌中甲硫氨酸氧化的图谱，深入了解氧化应激下蛋白质的行为。这代表了将辐射分解和大肠杆菌发展为体内蛋白质结构评估平台的第一步。其在药物发现、蛋白质工程和蛋白质构象系统生物学方面的潜在应用相当可观。
