Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules

Zhang, Y., Wolf-Yadlin, A., Ross, P. L., Pappin, D. J., Rush, J., Lauffenburger, D. A., White, F. M. (September 2005) Time-resolved mass spectrometry of tyrosine phosphorylation sites in the epidermal growth factor receptor signaling network reveals dynamic modules. Mol Cell Proteomics, 4 (9). pp. 1240-50. ISSN 1535-9476 (Print)1535-9476 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/15951569
DOI: 10.1074/mcp.M500089-MCP200

Abstract

Ligand binding to cell surface receptors initiates a cascade of signaling events regulated by dynamic phosphorylation events on a multitude of pathway proteins. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues, may play a role in determining cellular response, but experimental data required for analysis of these features have not previously been available. To understand the dynamic operation of signaling cascades, we have developed a method enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins in a time-resolved manner, downstream of epidermal growth factor receptor (EGFR) activation. Tryptic peptides from four different EGFR stimulation time points were labeled with four isoforms of the iTRAQ reagent to enable downstream quantification. After mixing of the labeled samples, tyrosine-phosphorylated peptides were immunoprecipitated with an anti-phosphotyrosine antibody and further enriched by IMAC before LC/MS/MS analysis. Database searching and manual confirmation of peptide phosphorylation site assignments led to the identification of 78 tyrosine phosphorylation sites on 58 proteins from a single analysis. Replicate analyses of a separate biological sample provided both validation of this first data set and identification of 26 additional tyrosine phosphorylation sites and 18 additional proteins. iTRAQ fragment ion ratios provided time course phosphorylation profiles for each site. The data set of quantitative temporal phosphorylation profiles was further characterized by self-organizing maps, which resulted in identification of several cohorts of tyrosine residues exhibiting self-similar temporal phosphorylation profiles, operationally defining dynamic modules in the EGFR signaling network consistent with particular cellular processes. The presence of novel proteins and associated tyrosine phosphorylation sites within these modules indicates additional components of this network and potentially localizes the topological action of these proteins. Additional analysis and modeling of the data generated in this study are likely to yield more sophisticated models of receptor tyrosine kinase-initiated signal transduction, trafficking, and regulation.

Item Type: Paper
Uncontrolled Keywords: Blotting, Western Cell Culture Techniques Cell Line Chromatography, Liquid Computational Biology Epithelial Cells/ cytology Female Humans Kinetics Ligands Mammary Glands, Human/ cytology Mass Spectrometry Phosphorylation Precipitin Tests Receptor, Epidermal Growth Factor/ metabolism Reproducibility of Results Signal Transduction Tyrosine/chemistry/ metabolism
Subjects: Investigative techniques and equipment
Investigative techniques and equipment > spectroscopy > mass spectrometry
Investigative techniques and equipment > spectroscopy
CSHL Authors:
Communities: CSHL labs > Pappin lab
Depositing User: Matt Covey
Date: September 2005
Date Deposited: 01 Mar 2013 21:56
Last Modified: 01 Mar 2013 21:56
Related URLs:
URI: http://repository.cshl.edu/id/eprint/27679

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