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Submitted on March 28, 2006
Barnett Institute, Northeastern University, Boston, MA 02115
Corresponding Author: b.karger{at}neu.edu
In a recent paper, 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 (fmole level) of large and complex proteins. In this paper, we have extended ERPA to determine quantitatively the temporal changes that occur in the tyrosine kinase receptor, EGFR, upon stimulation. Specifically, A431 cells were stimulated with EGF, following which EGFR was immunoprecipitated at stimulation times of 0, 0.5, 2 and 10 minutes, as well as 4 hours. 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 determined over the stimulation time points, including pT and pS sites, in addition to pY sites. A total of 10 extracellular domain N-glycan sites were also identified, and major glycoforms at each site 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 partner to EGFR. This work demonstrates that comprehensive characterization provides a powerful tool to aid in the study of important therapeutic targets. The detailed molecular information will prove useful in future studies in tissue.
Revised on June 23, 2006
Accepted on June 23, 2006
Dynamic profiling of the post-translational modifications and interaction partners of epidermal growth factor receptor signaling after stimulation by EGF using extended range proteomic analysis (ERPA)
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