Quantitative readout of methionine residue solvent accessibility in E. coli cells using radiolytic hydroxyl radical labeling and mass spectrometry

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.