Open AccessA Cross-Linking-Aided Immunoprecipitation/Mass Spectrometry Workflow Reveals Extensive Intracellular Trafficking in Time-Resolved, Signal-Dependent Epidermal Growth Factor Receptor Proteome
Author(s) -
Yue Chen,
Mei Leng,
Yankun Gao,
Dongdong Zhan,
Jong Min Choi,
Lei Song,
Kai Li,
Xia Xia,
Chunchao Zhang,
Mingwei Liu,
Shuhui Ji,
Antrix Jain,
Alexander B. Saltzman,
Anna Malovannaya,
Jun Qin,
Sung Yun Jung,
Yi Wang
Publication year - 2019
Publication title -
journal of proteome research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.644
H-Index - 161
eISSN - 1535-3907
pISSN - 1535-3893
DOI - 10.1021/acs.jproteome.9b00427
Subject(s) - proteome , microbiology and biotechnology , epidermal growth factor receptor , proteomics , biology , signal transduction , immunoprecipitation , intracellular , quantitative proteomics , receptor , chemistry , biochemistry , gene
Ligand binding to the cell surface receptors initiates signaling cascades that are commonly transduced through a protein-protein interaction (PPI) network to activate a plethora of response pathways. However, tools to capture the membrane PPI network are lacking. Here, we describe a cross-linking-aided mass spectrometry workflow for isolation and identification of signal-dependent epidermal growth factor receptor (EGFR) proteome. We performed protein cross-linking in cell culture at various time points following EGF treatment, followed by immunoprecipitation of endogenous EGFR and analysis of the associated proteins by quantitative mass spectrometry. We identified 140 proteins with high confidence during a 2 h time course by data-dependent acquisition and further validated the results by parallel reaction monitoring. A large proportion of proteins in the EGFR proteome function in endocytosis and intracellular protein transport. The EGFR proteome was highly dynamic with distinct temporal behavior; 10 proteins that appeared in all time points constitute the core proteome. Functional characterization showed that loss of the FYVE domain-containing proteins altered the EGFR intracellular distribution but had a minor effect on EGFR proteome or signaling. Thus, our results suggest that the EGFR proteome include functional regulators that influence EGFR signaling and bystanders that are captured as the components of endocytic vesicles. The high-resolution spatiotemporal information of these molecules facilitates the delineation of many pathways that could determine the strength and duration of the signaling, as well as the location and destination of the receptor.