In-cell isotope-coded labeling and LC-MS/MS analysis probe warfarin inhibition mechanism

Warfarin targets human vitamin K epoxide reductase (hVKOR), a redox enzyme in the membrane of endoplasmic reticulum (ER), to prevent the formation of blood clots. Although warfarin has been a popular medication since 1954, its mechanism of action is still unclear. A fundamental issue is the controversial orientation of transmembrane helices (TM) in hVKOR. Stable isotope-coded reagents were usedto label VKOR in free, mutated, and warfarin-binding states directly in the cellular environment, followed by LC-MS/MS bottom-up approach to investigate the warfarin binding mechanism.