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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)
- Source :
- Molecular & Cellular Proteomics. 5:1610-1627
- Publication Year :
- 2006
- Publisher :
- Elsevier BV, 2006.
-
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. The detailed molecular information will prove useful in future studies in tissue.
- Subjects :
- Proteomics
Glycosylation
Proteome
Immunoprecipitation
Molecular Sequence Data
Stimulation
Biochemistry
Mass Spectrometry
Receptor tyrosine kinase
Analytical Chemistry
chemistry.chemical_compound
Epidermal growth factor
Cell Line, Tumor
Protein Interaction Mapping
Humans
Amino Acid Sequence
Epidermal growth factor receptor
Phosphorylation
Molecular Biology
Epidermal Growth Factor
biology
Cell biology
ErbB Receptors
chemistry
Carcinoma, Squamous Cell
biology.protein
Peptides
Protein Processing, Post-Translational
Chromatography, Liquid
Signal Transduction
Subjects
Details
- ISSN :
- 15359476
- Volume :
- 5
- Database :
- OpenAIRE
- Journal :
- Molecular & Cellular Proteomics
- Accession number :
- edsair.doi.dedup.....a7b6e0bfdd6c1bd7911be8c61e1f7db9