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

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.