Peptides were analyzed on a Quadrupole-Orbitrap mass spectrometer as previously described (39). In brief, peptides were separated in a homemade C18 column (50 cm, particle size; Sera-Mag). A nano-LC fractionation was performed before analysis in the mass spectrometer. For both phosphopeptide and peptide samples, a 2.5-hour gradient was used using a binary buffer system consisting of an aqueous and organic phase: buffer A (0.1% formic acid) and buffer B (80% acetonitrile, 0.1% formic acid). Data-dependent acquisition was performed using the following parameters: Automatic gain control (AGC) target for MS1 spectra was 1 × 106. The maximal injection time was 20 ms, and resolution was 70,000 (mass range, 200 to 1200 m/z). MS/MS spectra of the top 10 most intense peaks were obtained by higher-energy collisional dissociation fragmentation. MS/MS spectra resolution was set to 35,000 at 200 m/z. AGC target was 5 × 105. Maximal injection time was 120 ms. The isolation window was set to 1.3 Th.

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