Intact-protein UPLC–HRMS analysis demonstrates differential stability of select DacB2–penem adducts after washing away the free drug and incubating for 24 h. (A) UPLC–HRMS analysis of apo-DacB2 (one square, 27,435 Da). (B) Meropenem fully reacts with DacB2, and the DacB2–meropenem adduct (decarboxylated, one circle, 27,774 Da; intact, two circles, 27,818 Da) is stable after the removal of drug for 24 h. (C) Like meropenem, T422 and T426 form adducts that are stable for at least 24 h after drug removal, while T405 and T428 exhibit the loss of 24% and 14% of drug, respectively. (D) Mass spectra of T405 (decarboxylated, one triangle, 27,778 Da; intact, two triangles 27,822 Da), T422 (decarboxylated, one star, 28,033 Da; intact, two stars, 28,077 Da), T426 (decarboxylated, one cross, 27,708 Da; intact, two crosses, 27,752 Da), and T428 (decarboxylated, one diamonds, 27,708 Da; intact, two diamonds, 27,752 Da) adducts of DacB2 before and after washout. One black square represents apo-DacB2 (27,435 Da).