Bacterial strains and plasmids
Media and antibiotics
SILAC labeling
Cell lysis and protein extraction
Protein digestion in solution
Phosphopeptide enrichment
High-pH reversed-phase peptide fractionation on commercial spin columns
Offline high-pH reversed-phase peptide fractionation
SDS–polyacrylamide gel electrophoresis and in-gel digestion
Incorporation and mixing check
LC-MS/MS measurement
MS data processing and analysis
Protein production for His-tag affinity purification
His-tag affinity purification
In vitro kinase assay measured by MS
Dynamics of HipA and HipA7 in vitro (auto)phosphorylation measured by autoradiography
Determination of cell viability on SMG plates
Measurement of persistence
Before each LC-MS/MS measurement, all peptide samples were desalted and purified on C18 StageTips (53). Reversed-phase C18 discs (Empore) were activated with methanol and equilibrated with solvent A*. Up to 10 μg of peptides was loaded onto the membrane and washed with solvent A. Peptides were eluted with 50 μl of solvent B [80% (v/v) acetonitrile and 0.1% (v/v) formic acid] and concentrated by vacuum centrifugation. The sample volume was adjusted with solvent A and final 10% (v/v) of solvent A*.