High-Throughput Phosphotyrosine Protein Complexes Screening by Photoaffinity-Engineered Protein Scaffold-Based Forward-Phase Protein Array

Low-abundance phosphotyrosine (pTyr)-mediated signaling protein complexes play critical roles in cancer signaling. The precise and comprehensive profiling of these pTyr-mediated protein complexes remains challenging because of their dynamic nature and weak binding affinity. Taking advantage of the SH2 domains modified with trifunctional chemical probes and genetic mutations (termed Photo-pTyr-scaffold), we developed a Photo-pTyr-scaffold-based forward-phase protein array that can be used to specifically capture complexes by developing an engineered SH2 domain, photoaffinity cross-linking, and antibody-based measuring weak pTyr-mediated protein complexes from complex biological samples in a 96-well microplate format. This platform demonstrated good precision for quantitation (R2 = 0.99) and high sensitivity by which only 5 μg of whole cell lysates is needed. We successfully applied the technology for profiling the dynamic EGF-stimulation-dependent EGFR signaling protein complexes across four different time...