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Phosphorylation modification is one of the most important posttranslational protein modifications used to modulate protein activity and propagate signals within cellular [1,2].Therefore,acquiring an inventory of phosphoproteins and the phosphorylation sites is crucial to an in-depth understanding of regulatory and signaling processes in cells [3].Although a large percentage of cellular proteins can be phosphorylated,the abundance of the individual phosphorylated forms and the large dynamics range in protein concentration results in the difficult identification especially for a large-scale phosphopmteomics analysis.Additionally,phosphorylation of a particular site in vivo is often substoichiometry [4] making the aforementioned problem even more difficult.In view of these above,a preliminary separation step of phosphopeptides is important to reduce sample complexity and increase their relative concentration.