Zheng Zhendong, Chu Bizhu, Kong Qian, Chen Xiong, Ke Mi, Qin Yunqiu, Lu Yi, Feng Shun, Tian Ruijun
||Analytical chemistry, 2019
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 due to their dynamic nature and weak binding affinity. Taking the 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 for specifically capturing by engineered SH2 domain, photoaffinity crosslinking, and antibody-based measuring weak pTyr-mediated protein complexes from complex biological samples in 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 are needed. We successfully applied the technology for profiling the dynamic EGF-stimulation depended EGFR signaling protein complexes across four different time courses (i.e. 0, 2, 5, 10 and 30 min) in a high-throughput manner. We further evaluated the modulation of EGFR-GRB2-SHC1 protein complexes by FDA-approved EGFR kinase inhibitor erlotinib, demonstrating the feasibility of this approach for high-throughput drug screening. The Photo-pTyr-scaffold-based forward-phase protein array could be generically applicable for exploring the dynamic pTyr signaling complexes in various biological systems and screening for related drugs in a high-throughput manner.