Phosphorylation of receptor-mimic tyrosine peptide on surface plasmon resonance sensor chip surface and its interaction with downstream proteins

Insulin-like growth factor receptor-1 (IGF-IR) signal pathway is critical in many physiological activities and occurance of some diseases. IGF-IR derived nonphosphorylated peptide LYASVNPEY was immobilized on the surface of CM5 sensor chip specialized for surface plasmon resonance (SPR) biosensor. The IGF-IR derived peptide surface was treated with cell lysates containing or not containing IGF-IR kinase. The results show that the antibody PY20 can sensitively detect the level of tyrosine phosphorylation using SPR biosensor, i. e. PY20 can discriminate the different level of peptide tyrosine phosphorylation treated with different incubation time and different lysates. The effect of the peptide tyrosine phosphorylation on peptideprotein interaction was further studied. The results demonstrated that only the phosphorylated peptide can interact with its downstream insulin receptor substrate-1 (IRS-I). The binding affinity (K_A) and association and dissociation kinetics constants (k_a and k_d) of the phosphorylated peptide with IRS-I were measured by SPR biosensor, with the results of K_A = (2.02 ± 0.08) × 10⁸ L mol^-1, k_a., = (2.30 ±0. 15) × 10⁶ L mol^-1 s^-1 and k_d = (1.14 ± 0.13) × 10_-2 s^-1 , respectively. This new phosphorylation method of tyrosine peptide that simulates cell receptor has the characteristics of real-time, speediness, no labeling and convenience for detection , which is instrumental for the investigations on receptor phosphorylation signal pathways and the drug screening against these signal pathways.