Dionex ultimate 3000 nano liquid chromatography system (Thermofisher Dionex) was used for online reversed phase chromatographic separation of the phosphopeptide samples prior to nanoelectrospray ion source and mass spectrometric detection. 5 μL of peptide samples (dissolved in 98% H2O/ 2% acetonitrile / 0.1% formic acid) were loaded onto a C18 trap column (0.1 mm × 2 cm, 5 μm, Thermofisher Dionex) using a flow rate of 10 μL/min. The oven temperature was set at 50 °C. Separation of the peptides was performed on a C18 analytical column (75 μm × 150 mm, 3 μm, Thermofisher Dionex) with a flow rate of 400 nL/min. The mobile phase of the binary gradient elution consisted of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid, and separation was performed using the following gradient: 2–8% B over 0–2 min, 8–20% B over 2–75 min, 20–35% B over 75–93 min, 35–80% B over 93–98 min, the composition was held at 80% B for 10 min. All the samples were kept at 4 °C during analysis. The mass spectrometric (MS) data were collected using a maxis impact Q-TOF (Bruker) equipped with an electrospray ionization (ESI) source operating in nanospray positive ion mode. The mass spectrometer was set as follows: Scan mode, full-scan MS and MS/MS scan; MS spectra rate, 3 HZ; MS/MS spectra rate, 5~10 HZ; Acquisition modes, data dependent analysis; Mass and MS/MS range, 50~2500 amu; lockmass, 445 Da; Precursor ion list, 350~1500 m/z; Source Capillary, 1800–1900 v; Dry gas, 2.0 L/min; Dry temperature, 120 °C.