Summary
Embryonic stem cells (ESCs) are unique in their pluripotency providing a great promise for human chronic diseases like Parkinson and Diabetes. Understanding the basis of self-renewal and differentiation processes is key in order to grow efficiently hESC populations to specific cell fates. We propose to study hESCs and other stem cells by using a quantitative proteomic approach focusing on signaling cascades invoked before and after induction of differentiation. Phosphorylation events will be studied by a combination of stable isotopic labeling techniques for accurate quantitation and specific phospho-tyrosine antibodies and SCX/TiO2 chromatography for efficient phosphopeptide enrichment. The time-course approach will enable to decipher the dynamics of the identified phosphorylation sites as well. The use of several hESCs lines (HUES7, B1, etc.) and different mechanisms to induce differentiation (BMP4, Activin, FGF, etc.) which will enable us to find specific and common molecular effectors. Different isotope labeling strategies such as SILAC and dimethyl-labeling will provide several platforms to perform in-depth analyses. The acquired data sets will also be an excellent resource for defining profiling protein networks and unravel kinase motifs that are enriched upon stem cell stimulation. The large amount of data generated in these analyses will require an exhaustive computational analysis so bioinformatic tools will be developed as well.