Reversible protein phosphorylation plays vital roles in signal transduction, cell cycle, gene expression and enzymatic regulation.Thus, characterization of phosphorylation status is critical to the elucidation of signal transduction pathways and understanding mechanisms of disease and drug actions.However, the low abundance of phosphopeptides and the signal suppression by nonphosphopeptides make the large-scale analysis of phosphorylation at proteome-wide scale remains a substantial challenge.Therefore, it is essential to employ a phosphopeptides enrichment strategy prior to MS analysis.