Phosphoprotein profiling of erythropoietin receptor‐ dependent pathways using different proteomic strategies

Proteomic techniques provide new tools for the global analysis of protein profiles but also for the investigation of specific protein functions. The analysis of signaling cascades has traditionally been performed by the determination of enzymatic or transcription factor activities representing a certain pathway. Functional proteomics now allows more comprehensive approaches to study cellular responses induced during ligand/receptor interactions. In this study we evaluated proteomic strategies for the investigation of structure‐function relationships in the erythropoietin receptor signalling complex. After expression of epidermal growth factor/erythropoietin receptor mutant molecules in an identical cellular background we characterized their potential to induce cellular activities. Using this system we focused our efforts on post‐translational modifications of signalling proteins reflecting a substantial part of receptor‐dependent signaling events. Although tyrosine phosphorylated proteins were enriched by immunoprecipitation the analysis using the classical approach combining two‐dimensional gel electrophoresis and identification by matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry revealed that low expressed signaling proteins cannot be detected by this technique. An alternative strategy using one‐dimensional gel separation of phosphoproteins and liquid chromatography‐tandem mass spectrometry, however, allowed us to identify multiple proteins involved in intracellular signalling representing already established pathways but also proteins which have not been linked to EPO‐induced signaling so far. This approach offers the potential to extend functional proteomic studies to complex signaling processes.