Abstract
Cyclic adenosine monophosphate (cAMP) signaling activates multiple downstream cellular targets in response to different stimuli. Specific phosphorylation of key target proteins via activation of the cAMP effector protein kinase A (PKA) is achieved via signal compartmentalization. Termination of the cAMP signal is mediated by phosphodiesterases (PDEs), a diverse group of enzymes comprising several families that localize to distinct cellular compartments. By studying the effects of inhibiting individual PDE families on the phosphorylation of specific targets it is possible to gain information on the subcellular spatial organization of this signaling pathway.We describe a phosphoproteomic approach that can detect PDE family-specific phosphorylation changes in cardiac myocytes against a high phosphorylation background. The method combines dimethyl labeling and titanium dioxide-mediated phosphopeptide enrichment, followed by tandem mass spectrometry.
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