Immunoreceptor tyrosine-based activation motif (ITAM) is a conserved sequence which occurs in a variety of proteins associated with activation, survival, and differentiation.The phosphorylated tyrosine residues of ITAM will function during cell signaling pathway by binding to the SH2 domain of other receptor proteins.Kinase family is one of the most important types of SH2 domain containing proteins, which will bind to consensus phosphotyrosine containing sequence to execute signal transduction functions.Therefore, it is very important to characterize the affinity between the ITAM peptide and its binding partners.Herein, we developed an integrated proteomics method was developed to characterize binding affinity of an ITAM peptide to its interacting proteins in a complex sample.