Investigating the role of FGF-2 in stem cell maintenance by global phosphoproteomics profiling

Human embryonic stem cells (hESCs) are of immense interest for regenerative medicine as a source of tissue replacement. Expansion of hESCs as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. One of the key growth factors that maintains this balance is fibroblast growth factor-2 (FGF-2). However, the underlying molecular mechanisms are poorly understood. We recently profiled specifically tyrosine phosphorylation events that occur during FGF-2 stimulation of hESCs (Ding et al., J. Cell. Physiol. 2010, 225, 417-428). Here, we complement this phosphoproteome profiling by analyzing temporal dynamics of mostly serine and threonine protein phosphorylation events. Our multi-dimensional strategy combines strong cation exchange chromatography to reduce the sample complexity followed by titanium dioxide off-line for the enrichment of phosphopeptides and dimethylation-based stable isotope labeling for quantification. This approach allowed us to identify and quantify 3261 unique proteins from which 1064 proteins were found to be phosphorylated in one or more residues (representing 1653 unique phosphopeptides). Approximately 40% of the proteins (553 unique phosphopeptides) showed differential phosphorylation upon FGF-2 treatment. Among those are several members of the canonical pathways involved in pluripotency and self-renewal (e.g. Wnt and PI3K/AKT), hESC-associated proteins such as SOX2, RIF1, SALL4, DPPA4, DNMT3B and 53 proteins that are target genes of the pluripotency transcription factors SOX2, OCT4 and NANOG. These findings complement existing pluripotency analyses and provide new insights into how FGF-2 assists in maintaining the undifferentiated state of hESCs.