Quantitative phosphoproteomic mass spectrometry identifies the global PGE₂-regulated phosphoproteome. K-means clustering analysis grouping phosphopeptides with similar phosphorylation profiles after 10 and 60 minutes of PGE₂ (10μM) stimulation (B). All detected phosphopeptides were depicted and color coded according to the temporal change in phosphorylation ratio in response to PGE₂ (10μM) after 10 and 60 minutes, A, C, D, and A', C', D', respectively; green indicates nonregulated phosphopeptide (ratio > 0.5 and < 2); yellow, ratio > 2 regulated phosphopeptide; red, ratio > 5 regulated phosphopeptide; blue, ratio < 0.5 regulated phosphopeptide; purple, predicted kinase. Individual networks were produced showing all identified phosphopeptides (A,A'), regulated phosphopeptides only (C,C') with kinase-predicted phosphopeptides highlighted, and PKA-predicted substrates (D,D'). All networks were visualized with the use of Cytoscape (www.cytoscape.org).