For binding experiments in the presence of glutathione, freshly dissolved reduced glutathione was incubated at 6.14 mM with 4 µM compounds for 30 minutes at room temperature in Tris 20 mM pH = 8, 50 mM NaCl. At this point BTK was added to a concentration of 2 µM (diluting the GSH to 5 mM and the compounds to 3.25 µM) and LC-MS was used as described previously to follow the covalent labeling of BTK.