T-cell receptor-CD3 complex(TCR)is one of the most versatile signaling machines,which contains in total twelve different phosphorylatable tyrosines from six immunoreceptor tyrosine-based activation motif(ITAM)in the cytoplasmic domains of CD3 chains to orchestrate T cell responses under different stimulations.However,no systematic and absolutely quantitative study has been conducted to investigate the phosphorylation program of the twelve tyrosines.Here,we developed a new Quantitative Reference-Guided Target Mass Spectrometry method to address this question,in which different reference peptide samples and different immunoprecipitated TCR samples from stimulated T cells were labeled by 10*TMT and mixed together.We found that although different tyrosines may be phosphorylated in similar kinetics,they were phosphorylated in distinct amounts by absolute quantification,even two tyrosines from the same ITAM.Different stimulation will also induce totally different phosphorylation program.To study the mechanism under the phosphorylation program,additional biochemistry,structure biology and cell biology assays were performed,and we found that a novel electrostatic interaction between CD3ε and LCK kinase played an important role.This study thus provides a systematic and mechanistic explanation to the versatile TCR signal.Besides,the Quantitative Reference-Guided Target Mass Spectrometry method can be generally applied to all other proteins or complexes.