The commercial TiO2 beads (GL Sciences, Tokyo, Japan) were used to enrich phosphorylated peptides according to the manufacturer’s instructions. Briefly, 200 μL of loading buffer (65% acetonitrile/ 2% trifluoroacetic acid/ saturated by glutamic acid) was used to equilibrium of TiO2 beads, and then 200 μL of peptide samples in loading buffer were incubated with TiO2 beads (peptides/beads = 1/4, w/w) for 20 min at room temperature. For consecutive incubations, the peptide-beads slurry was incubated and centrifuged, then the supernatant was incubated with another aliquot of freshly prepared TiO2 beads for the next enrichment. The enrichment procedures were repeated two times. The incubated beads were then washed with 800 μL of wash buffer I (65% acetonitrile/0.5% trifluoroacetic acid) and buffer II (65% acetonitrile/ 0.1% trifluoroacetic acid). and the bound peptides were eluted once with the 200 μL elution buffer I (300 mM aqua ammonia/50% acetonitrile) and twice with 200 μL elution buffer II (500 mM aqua ammonia/60% acetonitrile). All the incubation, washing as well as elution procedure, was rotated end-over-end for 20 min at room temperature. The eluates were dried by a vacuum dryer (RE-52AA, Shanghai Zhenjie Instrument Co. Ltd) to obtain the phosphorylated peptides. The enrichment efficiency of commercial TiO2 beads for phosphopeptide was evaluated and the results were shown in Supplementary Figs S3 and S4 and Table S5.