Cardiac myosin binding protein C (cMyBPC) is a critical multidomain protein that modulates myosin cross bridge behavior and cardiac contractility. cMyBPC is principally regulated by phosphorylation of the residues within the M-domain of its N-terminus. However, not much is known about the phosphorylation or other post-translational modification (PTM) landscape of the central C4C5 domains. In this study, the presence of phosphorylation outside the M-domain was confirmed in vivo using mouse models expressing cMyBPC with nonphosphorylatable serine (S) to alanine substitutions. Purified recombinant mouse C4C5 domain constructs were incubated with 13 different kinases, and samples from the 6 strongest kinases were chosen for mass spectrometry analysis. A total of 26 unique phosphorylated peptides were found, representing 13 different phosphorylation sites including 10 novel sites. Parallel reaction monitoring and subsequent mutagenesis experiments revealed that the S690 site (UniProtKB O70468) was the predominant target of PKA and PKG1. We also report 6 acetylation and 7 ubiquitination sites not previously described in the literature. These PTMs demonstrate the possibility of additional layers of regulation and potential importance of the central domains of cMyBPC in cardiac health and disease. Data are available via ProteomeXchange with identifier PXD031262.

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