Understanding the signaling pathways governing pluripotency and self-renewal is 
a prerequisite for better controlling stem cell differentiation to specific 
fates. Reversible protein phosphorylation is one of the most important 
posttranslational modifications regulating signaling pathways in biological 
processes. Global analysis of dynamic changes in protein phosphorylation is, 
therefore, key to understanding signaling at the system level. Here, we describe 
a generic mass spectrometry (MS)-based phosphoproteomics strategy applied to 
monitor phosphorylation dynamics after bone morphogenetic protein 4 
(BMP4)-induced differentiation of human embryonic stem cells (hESCs). Our method 
combines the use of strong cation exchange (SCX) and titanium dioxide (TiO(2)) 
for phosphopeptide enrichment, high-resolution MS for peptide and protein 
identification, and stable isotope labeling by amino acids in cell culture 
(SILAC) for quantification. This approach allows us to identify thousands of 
phosphorylation sites and profile their relative abundance during 
differentiation. This systems-biology-based approach provides new insights into 
how human pluripotent stem cells exit the pluripotent state.