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To understand the fundamental mechanisms underlying ErbB signaling pathway, we present here an approach to fully characterize phosphorylation on those unique sites in ErbB2 and their dynamic changes upon EGF treatment. To gain an efficient immunoprecipitation (IP) of ErbB2, several anti-ErbB2 antibodies were screened by the GeLC-MS analysis. The analytical procedure was used for determine the phosphorylation profiles in ErbB2 upon EGF stimulation in human cancer cell line (SKBR3).To validate identified phosphosites, selected reaction monitoring (SRM) and multiple staged MSn were implemented with a liner ion trap hybrid a high resolution FTMS.The ErbB2 phosphorylation profiles upon EGF treatment at varied 5 time points were determined by the targeted LC-MS method and label-free quantitative analysis. As a result, an optimized IP procedure by using highly selective anti-ErbB2 monoclonal antibodies allowed the effective method to dissociate the heterodimer of EGFR-ErbB2 and thus to explore the phosphorylation events in ErbB2 only.All of tryptic peptides from cytoplasmic region were identified, which allow us to map phosphosites in ErbB2 completely.21 phosphosites (16 new sites and 5 known sites) from 11 peptide fragments were assigned and further confirmed manually by MS spectra. Trace amount of phosphorylated peptides (estimated stoichiomertry, %ES<1%), including di- and tri- phosphopeptides, could be detected and quantified using above label-free quantitative strategy.We observed the remarkable changes of %ES of phosphosites in ErbB2, especially the sites on tyrosine that are closed to the autocatalysis domain (pY1139-pY1248). Our study introduced a highly sensitive and selective approach to monitor the phosphorylation changes in ErbB2 upon ligand binding to EGFR-ErbB2 dimerized RTK. The analytical workflow enabled dynamic quantitation of phosphorylation changes in ErbB2 upon EGF treatments and constituted, to the best of our knowledge, the most comprehensive data set of phosphosites in ErbB2 acquired to date from human cancer cells.