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Dynamic phosphotyrosine-dependent signaling profiling in living cells by two-dimensional proximity proteomics

ABSTRACT: Tyrosine phosphorylation (pTyr)-dependent signaling pathways play a vital role in various biological processes, which are spatiotemporally assembled and dynamically regulated on 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 superbinder-based pTyr peptide enrichment for simultaneously profiling these protein complexes and associated pTyr sites from the same affinity-purified sample. Upon different time-course of EGF stimulation of the living cells stably expressed with APEX2-fused adaptor protein GRB2, we constructed two-dimensional time-course curves for both GRB2 interactome and tyrosine phosphoproteome. Most of the known GRB2 interacting proteins and more than 40 pTyr sites were identified from each time point with acceptable quantification precision. Well-annotated pTyr signaling complexes in EGFR signaling and located at 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 PRM-based targeted MS analysis. Taking advantage of the high sensitivity of the PRM assay, the low abundant pTyr peptide EGFR pY1110 that cannot be identified in the DDA analysis could be well detected and quantified. Collectively, this two-dimensional proximity proteomic strategy is promising for comprehensively characterizing pTyr-mediated protein complexes with high sensitivity in living cells.

INSTRUMENT(S): Q-Extractive HF-X

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ruijun Tian

PROVIDER: MSV000089826 | MassIVE | Wed Jul 06 18:16:00 BST 2022

REPOSITORIES: MassIVE

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Dynamic phosphotyrosine-dependent signaling profiling in living cells by two-dimensional proximity proteomics

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