Large-scale protein phosphorylation analysis by MS is emerging as a powerful tool in plant signal transduction research.However, our current understanding of the phosphorylation regulatory network in plants is still very limited.To establish a more efficient and robust strategy for phosphoprotein identification, we carried out a large scale phosphoprotein profiling in young Arabidopsis (Arabidopsis thaliana) seedlings by using a phosphopeptide enrichment method which combines the titanium (Ti4+)-based IMAC and the reverse phase-strong cation exchange (RP-SCX) biphasictrap column-based online LC.