The enriched phosphopeptides were analyzed with a Finnigan Surveyor HPLC system coupled online with a LTQ-Oribitrap XL (Thermo Fisher Scientific, Waltham, MA) equipped with a nanospray source. The phosphopeptides were firstly loaded on a strong cation exchange (SCX) column using an autosampler, and the peptides were eluted by NH 4 Cl with different concentrations (1 mM, 10 mM, 100 mM, 200 mM, 1 M). Then, each fraction peptide was respectively loaded onto a C18 column (100 mm i.d., 10 cm long, 5 mm resin from Michrom Bioresources, Auburn, CA) using an autosampler. Peptides were eluted during a 0-35% gradient (Buffer A, 0.1% formic acid, and 5% ACN; Buffer B, 0.1% formic acid and 95% AcN) over 90 min and online detected in LTQ-Orbitrap using a data-dependent method [47] . The general mass spectrometric conditions were: spray voltage, 1.80 kV; no sheath and auxiliary gas flow; ion transfer tube temperature, 200uC. Ion selection thresholds were: 1000 counts for MS 2 and 500 counts for MS 3 . An activation q = 0.25 and activation time of 30 ms were applied in MS 2 acquisitions. The mass spectrometers were operated in positive ion mode, employing a data-dependent automatic switch between MS and MS 2 acquisition modes. For each cycle, one full MS scan in the Orbitrap at 1610 6 AGC target was followed by five MS 2 in the LTQ at 5000 AGC target on the five most intense ions. Selected ions were excluded from further selection for 90 s. Maximum ion accumulation time was 500 ms for full MS scans and 100 ms for MS 2 scans. All MS/MS spectra were collected using normalized collision energy (a setting of 35%), an isolation window of 3 m/z, and 1 micro-scan. The resolution used in the MS step in the Orbitrap is 60000. An extra DDNL (data-dependent neutral loss) MS 3 method was applied for phosphopeptide detection [17] . MS 3 was triggered if a neutral loss peak at 298.0, 249.0, 232.7 or 224.5 Da was observed in the MS 2 and that peak was one of the three most intense ions of the MS 2 spectra. Application of mass spectrometer scan functions and HPLC solvent gradients were controlled by XCalibur data system (Thermo Fisher Scientific, Waltham, MA).