Digested peptides were desalted by the solid-phase extraction using Sep-Pak C18 Vac 100 mg column (Waters). Briefly, column was activated with methanol and equilibrated with solvent A* [2% (v/v) acetonitrile and 1% (v/v) formic acid]. After loading the sample, the column was washed with solvent A [0.1% (v/v) formic acid], and peptides were eluted with 1.8 ml of 80% (v/v) acetonitrile and 6% (v/v) TFA. Phosphopeptides were enriched by titanium dioxide (TiO2) chromatography. Eluted peptides were incubated with TiO2 spheres (5 μm, 300 Å, ZirChrom) in 1:10 peptide to bead ratio for 10 min for 5 to 10 consecutive rounds. TiO2 spheres were washed twice with 80% (v/v) acetonitrile and 6% (v/v) TFA and loaded onto C8 (Empore) StageTips. The spheres were washed additionally with 80% (v/v) acetonitrile and 1% (v/v) TFA. Phosphopeptides were first eluted with 50 μl of 1.25% (v/v) ammonium hydroxide of pH 10.5 into 20 μl of 20% (v/v) TFA for 15 min at 1200 rpm. In the second elution step, phosphopeptides were eluted with 50 μl of 5% (v/v) ammonium hydroxide in 60% (v/v) acetonitrile (pH 10.5). Acetonitrile was evaporated from eluates by vacuum centrifugation, and samples were acidified to pH 2, if necessary, and purified by StageTips (see below).

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