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

Kong，Qian1,2,4; Ke，Mi1,2; Weng，Yicheng1,2; Qin，Yunqiu1,2; He，An1,2; Li，Pengfei1,2,3; Cai，Zongwei4; Tian，Ruijun1,2,3

2022-11-04

10.1021/acs.jproteome.2c00418

JOURNAL OF PROTEOME RESEARCH   Impact Factor & Quartile

1535-3893

1535-3907

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.

living cell proximity proteomics tyrosine phosphoproteome

SCI

English

First ; Corresponding

China State Key Basic Research Program Grants["2020YFE0202200","2021YFA1301601","2021YFA1301602"] ; National Natural Science Foundation of China["22125403","91953118","32171433","22104047","22004056"] ; Shenzhen Innovation of Science and Technology Commission["JCYJ20200109141212325","JCYJ20210324120210029","JCYJ20200109140814408"] ; Guangdong province[2019B151502050]

Biochemistry & Molecular Biology

Biochemical Research Methods

WOS:000877563100001

AMER CHEMICAL SOC

BIOLOGY & BIOCHEMISTRY

2-s2.0-85140968982

Scopus

1.Department of Chemistry,College of Science,Southern University of Science and Technology,Shenzhen,1088 Xueyuan Road,518055,China
2.Research Center for Chemical Biology and Omics Analysis,College of Science,Southern University of Science and Technology,Shenzhen,1088 Xueyuan Road,518055,China
3.Shenzhen Grubbs Institute,Southern University of Science and Technology,Shenzhen,1088 Xueyuan Road,518055,China
4.State Key Laboratory of Environmental and Biological Analysis,Department of Chemistry,Hong Kong Baptist University,Hong Kong,Kowloon Tong,999077,Hong Kong

Kong，Qian,Ke，Mi,Weng，Yicheng,et al. Dynamic Phosphotyrosine-Dependent Signaling Profiling in Living Cells by Two-Dimensional Proximity Proteomics[J]. JOURNAL OF PROTEOME RESEARCH,2022,21(11):2727-2735.

Kong，Qian.,Ke，Mi.,Weng，Yicheng.,Qin，Yunqiu.,He，An.,...&Tian，Ruijun.(2022).Dynamic Phosphotyrosine-Dependent Signaling Profiling in Living Cells by Two-Dimensional Proximity Proteomics.JOURNAL OF PROTEOME RESEARCH,21(11),2727-2735.

Kong，Qian,et al."Dynamic Phosphotyrosine-Dependent Signaling Profiling in Living Cells by Two-Dimensional Proximity Proteomics".JOURNAL OF PROTEOME RESEARCH 21.11(2022):2727-2735.