We also used LC-MS to directly observe the formation of covalent complexes with recombinant BTK and measure their dissociation kinetics (Figure , D, Supp. Fig. ). LC-MS measurements with 2 µM BTK and 3 µM compound indicated covalent labeling by all compounds except NC-1, with RC-3 forming the complex extremely fast, followed by RC-2 and IR-2, in agreement with the data from the kinase activity assay. We should note that the preincubation of the compounds with 5 mM GSH did not significantly affect protein binding of neither reversible nor irreversible covalent binders (Supp. Fig. ).