A Cross-Linking-Aided Immunoprecipitation/Mass Spectrometry Workflow Reveals Extensive Intracellular Trafficking in Time-Resolved, Signal-Dependent Epidermal Growth Factor …

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 …