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
SDS–polyacrylamide gel electrophoresis and in-gel digestion
Incorporation and mixing check
Protein purification by StageTips
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
Chymotrypsin-digested sample of one independent experiment of chromosomal hipA7 (Rep1) was fractionated using an offline peptide fractionation at high pH to increase sequence coverage and detect unmodified GltX peptide. Peptides were loaded onto a reversed-phase XBridge BEH130 C18 3.5 μm 4.6 × 250 mm column installed in an UltiMate 3000 HPLC and detected by ultraviolet at λ = 214 nm at 25°C. The system was operated under basic conditions using buffer A (5 mM NH4OH) and buffer B (5 mM NH4OH in 90% acetonitrile) at pH 10. Peptides were eluted using an 80-min gradient at a flow rate of 1 ml/min. The organic portion was ramped from 5 to 25% B in 45 min to 40% in 10 min and finally to 70% in 5 min, followed by column equilibrated. Fractions were collected in 1-min intervals for 60 min and concatenated evenly into 30 pools. Acetonitrile was evaporated by vacuum centrifugation, and samples were acidified to pH 2 and measured by LC-MS/MS.