Purified peptide samples were separated by an EASY-nLC 1000 or 1200 system (Thermo Fisher Scientific) coupled online to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) through a nanoelectrospray ion source (Thermo Fisher Scientific). Chromatographic separation was performed on a 20-cm-long, 75–μm–inner diameter analytical column packed in-house with reversed-phase ReproSil-Pur C18-AQ 1.9 μm particles (Dr. Maisch GmbH). The column temperature was maintained at 40°C using an integrated column oven. Peptides were loaded onto the column at a flow rate of 700 nl/min or 1 μl/min under maximum back pressure of 500 or 850 bar, respectively. The peptides were eluted using either 46-, 76-, 116-, or 216-min segmented gradient of 10 to 50% solvent B at a constant flow rate of 200 nl/min. When measuring proteome digested with chymotrypsin, the gradient started with 5% of solvent B. For measurements of kinase assays, the peptides were eluted using 33-min segmented gradient of 10 to 50% solvent B at a constant flow rate of 300 nl/min. For measurement of samples fractionated by high-pH chromatography on column, different 76-min segmented gradients optimized for each fraction were used.

Peptides were ionized by nanoelectrospray ionization at 2.3 kV and the capillary temperature of 275°C. The mass spectrometer was operated in a data-dependent mode, switching automatically between one full scan and subsequent MS/MS scans of either 12 (Top12 method) or 7 (Top7 method, phosphoproteome measurement) most abundant peaks selected with an isolation window of 1.4 m/z (mass/charge ratio). Full-scan MS spectra were acquired in a mass range from 300 to 1650 m/z at a target value of 3 × 106 charges with the maximum injection time of 25 ms and a resolution of 60,000 (defined at m/z 200). The higher-energy collisional dissociation MS/MS spectra were recorded with the maximum injection time of 45 or 220 ms (for phosphoproteome measurement) at a target value of 1 × 105 and a resolution of 30,000 (defined at m/z 200) or 60,000 for phosphoproteome measurement. The normalized collision energy was set to 27%, and the intensity threshold was kept at 1 × 105 or 5 × 104 for phosphoproteome measurement. The masses of sequenced precursor ions were dynamically excluded from MS/MS fragmentation for 30 s. Ions with single, unassigned, or six and higher charge states were excluded from fragmentation selection. Two phosphoproteome experiments (chromosomal hipA7, experiments 1 and 3) were measured on an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) with previously described parameters (54).