In vitro kinase assay measured by MS

Kinase (1 μM) (His₆-HipA or His₆-HipA7) was incubated with 6 μM His-tagged substrate in a kinase buffer [50 mM tris-HCl (pH 8.0), 10 mM MgCl₂, 1 mM DTT, and 16 μM ZnSO₄] with or without 5 mM ATP. Each reaction contained 4.5 μg of a total protein amount. Samples were incubated at 37°C for 45 min and stopped by the addition of nine volumes of denaturation buffer, followed by the protein digestion using chymotrypsin or Lys-C endoproteinase, as previously described (see above). Digested peptides were purified using StageTips (see above), and 0.2 μg of each sample was measured by LC-MS/MS (see above).