1 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU2 Faculty of Science, SDU3 Institut de Recherches Servier4 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU
Background Our understanding of complex signaling networks is still fragmentary. Isolated processes have been studied extensively but cross-talk is omnipresent and precludes intuitive predictions of signaling outcomes. The need for quantitative data on dynamic systems is apparent especially for our understanding of pathological processes. In our study we create and integrate data on phosphorylations that are initiated by several growth factor receptors. We present an approach for quantitative, time-resolved phosphoproteomic profiling that integrates the important contributions by phosphotyrosines. Methods A549 lung carcinoma cells were used as a model and stimulated with hepatocyte growth factor, epidermal growth factor or fibroblast growth factor. We employed a quick protein digestion workflow with spin filters without using urea. Phosphopeptides in general were enriched by sequential elution from immobilized metal affinity chromatography (SIMAC) with TiO2 enrichment. Phosphotyrosine containing peptides were enriched by immunoprecipitation. iTRAQ™ labeling enabled multiplexed quantification of changes during stimulation and comparison of different stimuli. Several fragmentation techniques were used in order to maximize identification of peptides as well as localization of phosphorylation sites. Results and Conclusions The combination of SIMAC with phosphotyrosine enrichment leads to a significant increase in identification of potential signaling events in growth factor receptor signaling networks. In a typical set of measurements (time course of one stimulant) 1500 to 2000 unique phosphopeptides are found. 200 of these contain phosphotyrosine. A large fraction of known network components can be identified. Combining data from collision induced dissociation (normal and higher energy) and electron transfer dissociation adds confidence in modification site assignment. The workflow is relatively simple but the integration of complementary techniques leads to a deeper insight into cellular signaling networks and the potential pharmacological intervention thereof.