Phosphorylation of Receptor-Mimic Tyrosine Peptide on Surface Plasmon Resonance Sensor Chip and Its Interaction with Downstream Proteins

Abstract

Abstract Insulin-like growth factor type-I receptor (IGF-1R) signal pathway is critical in several physiological activities and some pathogenesis. IGF-1R derived nonphosphorylated peptide LYASVNPEY was immobilized on the surface of CM5 sensor chip specialized for surface plasmon resonance (SPR) biosensor. The peptide surface was treated with cell lysates containing or not containing IGF-1R kinase. The results show that using SPR biosensor PY20 can discriminate the different levels of peptide tyrosine phosphorylation (PTP) caused by different incubation times and different lysates. The effect of the PTP on peptide-protein interaction was further studied. The results demonstrated that only the phosphorylated peptide can interact with its downstream insulin receptor substrate-1 (IRS-1). The binding affinity (KA) and the association and dissociation kinetics (ka and kd) of the phosphorylated peptide with IRS-1 were measured by SPR biosensor, with the results of KA = (2.02 ± 0.08) × 108 M−1, ka = (2.30 ± 0.15) × 106 M−1 s−1 and kd = (1.14 ± 0.13) × 10−2 s−1, respectively. This new phosphorylation method of peptide that simulates cell receptor has the characteristics of real-time, speediness, label-free, and convenience for detection, which is instrumental for investigations on receptor phosphorylation signal pathways and drug screening against these signal pathways.