Using a chemical proteomics approach, we efficiently enriched for the generally low abundant cAMP signaling proteins, and their interactors, directly from mouse ventricular tissue. The presence of undesired contaminating (noncyclic) nucleotide-binding proteins was diminished using a tailored sequential elution protocol. Through further optimization of this affinity purification and elution protocol, we were able to detect all known protein kinase A regulatory isoforms (PKA-R). Furthermore, 11 different A-kinase anchoring proteins (AKAPs) were detected. A proposed fusion protein of paralemmin 2 and AKAP2 could be decisively established as a novel AKAP at the protein level in ventricular tissue. When comparing this dataset of cAMP-affinity purified proteins with earlier data obtained with immobilized cGMP from rat ventricular tissue, we observe a large overlap in the retained proteins but also some clear differences. Furthermore, implementation of an in-depth analysis of in vivo phosphorylation sites on PKA-R revealed the presence of several differentially phosphorylated PKA-R isoforms. This illustrates yet another layer of functional regulation in cyclic nucleotide signaling. In general, our improved chemical proteomics screen offers a broad, but detailed, view on nature's complex diversity in cyclic nucleotide signaling mechanisms. Possibly different AKAP-isoforms may direct differentially phosphorylated PKA-R isoforms to different cellular compartments, providing a multifaceted platform for just this kinase.