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

Zheng, Zhendong1,2; Chu, Bizhu2; Kong, Qian2; Chen, Xiong2; Ke, Mi2; Qin, Yunqiu2; Lu, Yi1; Feng, Shun3; Tian, Ruijun2,4

2019-08-06

10.1021/acs.analchem.9b01845

ANALYTICAL CHEMISTRY   影响因子和分区

0003-2700

1520-6882

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, photo-affinity 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 (R-2 = 0.99) and high sensitivity by which only 5 mu g 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 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.

SCI ; EI

英语

NI论文

通讯

Guangdong Provincial Grants[2016A030312016] ; Guangdong Provincial Grants[2017B030301018]

Chemistry

Chemistry, Analytical

WOS:000480499200094

AMER CHEMICAL SOC

20193307307573

Binding energy ; Proteins ; Signaling

Biomedical Engineering:461.1 ; Physical Chemistry:801.4 ; Organic Compounds:804.1

CHEMISTRY

Web of Science

1.Xinjiang Univ, Minist Educ & Xinjiang Uyghur Autonomous Reg, Coll Chem & Chem Engn, Key Lab Oil Gas & Fine Chem, Urumqi 830046, Peoples R China
2.Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China
3.Southwest Jiaotong Univ, Sch Life Sci & Engn, Chengdu 610031, Sichuan, Peoples R China
4.Guangdong Prov Key Lab Cell Microenvironm & Dis R, Shenzhen 518055, Peoples R China

Zheng, Zhendong,Chu, Bizhu,Kong, Qian,et al. High-Throughput Phosphotyrosine Protein Complexes Screening by Photoaffinity-Engineered Protein Scaffold-Based Forward-Phase Protein Array[J]. ANALYTICAL CHEMISTRY,2019,91(15):10026-10032.

Zheng, Zhendong.,Chu, Bizhu.,Kong, Qian.,Chen, Xiong.,Ke, Mi.,...&Tian, Ruijun.(2019).High-Throughput Phosphotyrosine Protein Complexes Screening by Photoaffinity-Engineered Protein Scaffold-Based Forward-Phase Protein Array.ANALYTICAL CHEMISTRY,91(15),10026-10032.

Zheng, Zhendong,et al."High-Throughput Phosphotyrosine Protein Complexes Screening by Photoaffinity-Engineered Protein Scaffold-Based Forward-Phase Protein Array".ANALYTICAL CHEMISTRY 91.15(2019):10026-10032.