Immobilized Metal Affinity Chromatography (IMAC) [29] : The IMAC column was first capping one end with a 0.5 mm frit disk enclosed in stainless steel column-end fitting. The Ni-NTA resin was extracted from spin column and packed into a 5-cm microcolumn (500 mm id PEEK column). Automatic purification of phosphopeptides was performed using the IMAC microcolumn connected with autosampler and HP1100 solvent delivery system with a flow rate 13 ml/min. First, the Ni2+ ions were removed with 100 ml EDTA (50 mM) in NaCl (1 M). Then the IMAC column was activated with 100 ml FeCl3 (0.2 M) and equilibrated with loading buffer for 30 min before sample loading. For optimization of the phosphopeptide enrichment, the loading/condition buffer (designated as loading buffer) was 6% (v/v) AA and the pH was adjusted to 3.0 with NaOH (0.1 M at pH 12.8). The peptide samples from trypsin digestion were reconstituted in loading buffer and loaded into the IMAC column that had been equilibrated with the same loading buffer for 20 min. Then the unbound peptides were removed with 100 ml washing solution consisting of 75% (v/v) loading buffer and 25% (v/v) ACN, followed by equilibration with loading buffer for 15 min. Finally, the bound peptides were eluted from the IMAC column with 100 ml NH4H2PO4 (200 mM at pH 4.4). Eluted peptide samples were dried under vacuum and reconstituted in 0.1% (v/v) FA for LC-MS/MS analysis.