Phosphorylation is one of the most important post-translation modifications of proteins,it is extensively involved in various physiological processes,including signal conduction,cell cycling and cancer development etc.Analysis of phosphorylated proteins has proved to be a new challenge for proteomic technologies,due to its extensive expression and low abundance.High performance trapping,enrichment and separation of phosphorylated peptides are key unit operations that demand effective solutions in phosphorylation research.We thus developed a multi-step workflow at microscale,with titanium dioxide pipette tips at the front end for phosphorylated peptides trapping,microbullet solid phase extraction columns for enrichment and a droplet-based microfluidic two dimensional separation system for high resolution separation.Using breast cancer cells as a real sample,we investigated the workflow and realised low-noise zoom-in 2D mapping of phosphorylated peptides therein.