Samples were separated by SDS-PAGE, and bands were excised and digested with trypsin in 50 mM ammonium bicarbonate overnight at 37°C. Peptides were extracted using 5% formic acid/50% acetonitrile and dried. Peptides were analyzed on a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) equipped with an Easy-nLC 1000 (Thermo Fisher Scientific). Raw data were searched using COMET in high-resolution mode (38), with a precursor mass tolerance of 1 Da, trypsin enzyme specificity with up to three missed cleavages, and carbamidomethylcysteine as fixed modification. Oxidized methionine and phosphorylated serine, threonine, and tyrosine were searched as variable modifications. Probability of phosphorylation site localization was determined by PhosphoRS (39). Quantification of LC-MS/MS spectra was performed using MassChroQ with retention time alignment for smart quantification (40).

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