The identification of FGF-dependent phosphorylation events in embryonic stem cells using mass spectrometry

Fibroblast growth factor-four (FGF4) signalling is essential for embryonic stem (ES) cells to become competent to undergo differentiation and enter lineage commitment pathways. The signalling pathway whereby this occurs is as yet not fully characterized. In this study, high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis was used in conjunction with phosphopeptide enrichment using TiO₂ affinity chromatography to detect peptides phosphorylated in response to FGF stimulation of mouse ES cells. Many phosphorylation events were observed in FGF-treated ES cells and some phosphopeptides were unambiguously identified that associate with four different proteins: Lig1, Eif3b, 6430527G18Rik and Sin3a. We propose a novel pathway in which FGF signalling enables differentiation of ES cells via activation of Sin3a, a transcription repressor, which regulates expression of pluripotency genes.