A Cleavable Trifunctional Probe for Systematically Profiling Tyrosine Phosphorylation-mediated Protein Complexes

Qin, Yun-Qiu1,2; Zheng, Zhen-Dong1,2; Tian, Rui-Jun2

2023

10.19756/j.issn.0253-3820.221463

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY   影响因子和分区

0253-3820

1872-2040

Phosphotyrosine (pTyr) signaling plays an important role in regulation of many pathways in cell signaling. Protein complexes regulated by pTyr signaling are key molecular machines in the early processes of signal transduction and therefore the analysis of dynamic protein complexes is critical. Photo-pTyr-scaffold is a chemical proteomics strategy that combines chemical probes with pTyr-recognizing domains to enable the large- scale analysis of pTyr signaling protein complexes in the epidermal growth factor receptor (EGFR) pathway. However, this approach is unable to resolve the one-to-one organization of the identified complexes, increasing the complexity of subsequent complex analysis. In this study, a novel cleavable trifunctional probe was designed and synthesized based on the design of photo-pTyr-scaffold probe, with a cleavable group being introduced into the biotin end of the probe, which enabled controllable elution of the enriched product. Based on the difference in molecular weight of the pTyr signaling protein complexes after cross-linking, sodium dodecyl sulfate-acrylamide gel electrophoresis was used to achieve the separation of the eluted pTyr protein complexes, and the in-gel digestion and mass spectrometry analysis were further performed to identify proteins in different molecular weight regions. Theoretically, the crosslinked pTyr protein complexes would be identified in the regions higher than the original protein molecular weight. With this strategy, the pTyr signaling protein complexes in the EGFR signaling pathway were systematically investigated and the molecular weight migration profiles of 20 important proteins were obtained. And the migration phenomenon was further validated by western blot, demonstrating the feasibility of this approach for analysis of pTyr signaling protein complexes.

Tyrosine phosphorylation Protein complexes Epidermal growth factor receptor signaling pathway Proteomics analysis

SCI

英语

通讯

National Natural Science Foundation of China["22125403","91953118"]

Chemistry

Chemistry, Analytical

WOS:000971129200001

SCIENCE PRESS

CHEMISTRY

Web of Science

1.Harbin Inst Technol, Sch Chem & Chem Engn, Harbin 150001, Peoples R China
2.Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China

Qin, Yun-Qiu,Zheng, Zhen-Dong,Tian, Rui-Jun. A Cleavable Trifunctional Probe for Systematically Profiling Tyrosine Phosphorylation-mediated Protein Complexes[J]. CHINESE JOURNAL OF ANALYTICAL CHEMISTRY,2023,51(1).

Qin, Yun-Qiu,Zheng, Zhen-Dong,&Tian, Rui-Jun.(2023).A Cleavable Trifunctional Probe for Systematically Profiling Tyrosine Phosphorylation-mediated Protein Complexes.CHINESE JOURNAL OF ANALYTICAL CHEMISTRY,51(1).

Qin, Yun-Qiu,et al."A Cleavable Trifunctional Probe for Systematically Profiling Tyrosine Phosphorylation-mediated Protein Complexes".CHINESE JOURNAL OF ANALYTICAL CHEMISTRY 51.1(2023).