Dynamic Profiling of the Post-translational Modifications and Interaction Partners of Epidermal Growth Factor Receptor Signaling after Stimulation by Epidermal Growth Factor Using Extended Range Proteomic Analysis (ERPA)

Abstract

In a recent report, we introduced Extended Range Proteomic Analysis (ERPA), an intermediate approach between top-down and bottom-up proteomics, for the comprehensive characterization at the trace level (fmol level) of large and complex proteins. In this study, we extended ERPA to determine quantitatively the temporal changes that occur in the tyrosine kinase receptor, epidermal growth factor receptor (EGFR), upon stimulation. Specifically A 431 cells were stimulated with epidermal growth factor after which EGFR was immunoprecipitated at stimulation times of 0, 0.5, 2, and 10 min as well as 4 h. High sequence coverage was obtained (96%), and methods were developed for label-free quantitation of phosphorylation and glycosylation. A total of 13 phosphorylation sites were identified, and the estimated stoichiometry was determined over the stimulation time points, including Thr(P) and Ser(P) sites in addition to Tyr(P) sites. A total of 10 extracellular domain N-glycan sites were also identified, and major glycoforms at each site were quantitated. No change in the extent of glycosylation with stimulation was observed as expected. Finally potential binding partners to EGFR were identified based on changes in the amount of protein pulled down with EGFR as a function of time of stimulation. Many of the 19 proteins identified are known binding partners of EGFR. This work demonstrates that comprehensive characterization provides a powerful tool to aid in the study of important therapeutic targets.

Amphiregulin-EGFR signaling regulates PTHrP gene expression in breast cancer cells

“…Although we cannot rule out the possibility that AREG stimulates phosphorylation of other EGFR tyrosine residues such as Tyr974, Tyr1086, Tyr1101 and Tyr1114 [48,49], our data highlights the importance of phosphorylation of EGFR Tyr992 in coupling EGFR to PTHrP gene expression. Phosphorylated Tyr992 binds to PLCγ, RasGAP, SHP2, and PTP1B [50][51][52][53].…”

Precision proteomics: The case for high resolution and high mass accuracy

“…3 A vs. B). Here, peptides longer than Ϸ20 residues (48,49) or large endogenous peptides (42) are sought for analysis to increase the sequence coverage provided by each successfully identified peptide.…”

Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays

“…Subsequently, these antibodies were immobilized on beads to create a bead array (supplemental Table 1). An attempt to classify the set of antibody targets revealed proteins from different classes (29), such as transmembrane proteins (38), G-protein-coupled receptors (4), plasma proteins (8), cancer-related proteins (10), proteins with a signal peptide (30), enzymes (12), and transcription factors (5).…”

“…The introduction of more and more sophisticated instrumentation has increased the sensitivity and throughput of mass spectrometry during the last years (4). One of the advantages with mass spectrometry is that the method also allows for the detection of differences in protein modifications, such as glycosylation or phosphorylation, which have been found useful for some applications (5). Although many potential biomarkers have been discovered using mass spectrometry, the approach is yet limited to the analysis of a relatively small number of patient samples.…”