Systems Analysis of EGF Receptor Signaling Dynamics with Micro-Western Arrays

We describe micro-western arrays, which enable quantitative, sensitive and high-throughput assessment of protein abundance and modifications following electrophoretic separation of micro-arrayed cell lysates. This method allowed us to measure 91 phosphosites on 67 proteins at six time points following stimulation with five EGF concentrations in A431 human carcinoma cells. We inferred the connectivities among 15 phosphorylation sites across 10 receptor tyrosine kinases (RTK) and 2 sites from Src kinase using Bayesian network modeling and two mutual information-based methods; the three inference methods yielded significant agreement on the network topology. These results imply multiple distinct RTK coactivation mechanisms and support the notion that small amounts of experimental data collected from phenotypically diverse network states may enable network inference.