Dynamic Phosphotyrosine-Dependent Signaling Profiling in Living Cells by Two-Dimensional Proximity Proteomics

Qian Kong, Mi Ke, Yicheng Weng, Yunqiu Qin, An He, Pengfei Li*, Zongwei Cai*, Ruijun Tian*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

1 Citation (Scopus)


Tyrosine phosphorylation (pTyr)-dependent signaling pathways play a vital role in various biological processes, which are spatiotemporally assembled and dynamically regulated on a minute scale by pTyr in living cells. Studying these pTyr-mediated signaling complexes is therefore challenging due to the highly dynamic nature of the protein complexes and the low abundance of pTyr. In this study, we adopted minute-resolution APEX2-based proximity labeling (PL) in living cells and Src SH2 superbinder-based pTyr peptide enrichment for simultaneously profiling these protein complexes and associated pTyr sites from the same affinity-purified sample. Upon different time courses of EGF stimulation of the living cells stably expressing APEX2-FLAG-GRB2, we constructed two-dimensional time-course curves for both interactome and tyrosine phosphoproteome. Well-annotated pTyr signaling complexes in EGFR signaling and located at the endosome were quantified with tightly correlated time-course curves for both interacting proteins and pTyr sites. Importantly, the correlated time-course curves for EGFR and endosomal HGS were well validated by targeted-parallel reaction monitoring (PRM)-MS analysis. Taking advantage of the high sensitivity of the PRM assay, the low-abundant pTyr peptide EGFR pY1110, which cannot be identified in the data-dependent acquisition (DDA) analysis, could be well quantified. Collectively, this two-dimensional proximity proteomic strategy is promising for comprehensively characterizing pTyr-mediated protein complexes with high sensitivity in living cells.

Original languageEnglish
Pages (from-to)2727-2735
Number of pages9
JournalJournal of Proteome Research
Issue number11
Early online date24 Oct 2022
Publication statusPublished - 4 Nov 2022

Scopus Subject Areas

  • Chemistry(all)
  • Biochemistry

User-Defined Keywords

  • living cell
  • proximity proteomics
  • tyrosine phosphoproteome


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