Citation: 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(4): 492-501. doi: 10.19756/j.issn.0253-3820.221463 shu

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

1.
School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China;
2.
Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China

Corresponding author: TIAN Rui-Jun, tianrj@sustech.edu.cn
Received Date: 19 September 2022
Revised Date: 16 November 2022

Fund Project: Supported by the National Natural Science Foundation of China (Nos. 22125403, 91953118).

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
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