New Citation 30 August 2019

A cross-linking-aided IP/MS workflow reveals extensive intracellular trafficking in time-resolved, signal-dependent EGFR proteome
Publication Date:August 23, 2019https://doi.org/10.1021/acs.jproteome.9b00427 Copyright © 2019 American Chemical Society
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Abstract

Ligand binding to the cell surface receptors initiates signaling cascades（信号级联） that are commonly transduced through protein-protein interaction (PPI) network to activate a plethora of（大量的） response pathways. However, tools to capture membrane PPI network are lacking. Here, we describe a cross-linking-aided mass spectrometry workflow for isolation and identification of signal-dependent EGFR proteome. We performed protein cross-linking in cell culture at various time points following EGF treatment followed by immunoprecipitation(免疫沉淀法) of endogenous EGFR and analysis of the associated proteins by quantitative mass spectrometry（定量质谱）. We identified 140 proteins with high confidence during a 2h-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（不同时间的行为）; 10 proteins that appeared in all timepoints 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(内吞作用的囊泡). The high-resolution spatiotemporal（时空） information of these molecules facilitates the delineation描述 of the many pathways that could determine the strength and duration of the signaling, as well as the location and the destination of the receptor.