Cell system perturbation for time-resolved quantification of tyrosine phosphorylation in complex samples

Signal transduction mediated by protein phosphorylation regulates many cellular biological processes. In order to effectively monitor protein phosphorylation events governing signaling cascades, we have developed a methodology enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins in a time-resolved manner. In our initial implementation, we have applied this technology to the analysis of cellular signaling pathways initiated by epidermal growth factor (EGF) stimulation of the epidermal growth factor receptor (EGFR), identifying and quantifying temporal phosphorylation profiles of tyrosine phosphorylation sites on 77 proteins. Application of bioinformatics tools, such as self-organizing maps, has resulted in identification of several cohorts of tyrosine residues exhibiting self-similar temporal phosphorylation profiles, operationally modules in the EGFR defining signaling dynamic network. In order to identify key signaling components which regulate downstream biological response, we have constructed a model using partial least-squares regression (PLSR). Information obtained through our proteomic studies was represented in an MtimesN matrix. By combining these modeling approaches, it was possible to associate EGFR-family dimerization at the cell surface to activation of specific phosphorylation sites which appear to most critically regulate proliferation and/or migration. Thus, we have characterized quantitative relationships between diverse ligand stimulation, network-wide signaling, and cell functional responses that may help explain how HER2 over-expression dysregulates network signals governing important downstream cell behaviors