A combinational approach provides high-resolution signaling data. (A) Schematic overview of the combined phosphoproteomic and FCB-phospho flow methods that provide a high-resolution map of intracellular signaling and stimulus-specific signaling networks. (B) Summary depicting results obtained from kinase prediction, phosphoproteomics, and FCB-phospho flow (from left to right). Kinase prediction shows a probable connection between PKA and its substrates. All 3 depicted substrates (VASP, GSK3α, and heat shock protein 27 [Hsp27]) have been detected by mass spectrometry, and the relative changes in phospho-status at the specified sites have been determined. In addition, the detected phosphosites were studied with the use of FCB-phospho flow to show high-resolution kinetic information over extensive time courses in 5 lymphocyte cell populations, both in the absence and presence of a PKA inhibitor (H89). Color coding of histograms is according to a scale of 2 for VASP and GSK3α, and to a scale of 1 for Hsp27.