Identification of Phosphorylation and Other Post-Translational Modifications in the Central C4C5 Domains of Murine Cardiac Myosin Binding Protein C.

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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.", "claims": [{"id": "claim-method", "statement": "Comparisons of protein phosphorylation levels for the 3SA and 4SA samples were performed using a two-tailed t test.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:35573219", "locator": "sections[25] \"Data and Statistical Analysis\" paragraph 2", "quote": "Comparisons of protein phosphorylation levels for the 3SA and 4SA samples were performed using a two-tailed t test."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository91,92 with the data set identifier PXD031262 and 10.6019/PXD031262. All other data are available from the corresponding author upon request.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:35573219", "locator": "sections[32] \"Notes\" paragraph 2", "quote": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository91,92 with the data set identifier PXD031262 and 10.6019/PXD031262. All other data are available from the corresponding author upon request."}], "state": "complete"}, {"id": "claim-target-result", "statement": "The study identified 13 phosphorylation sites in the C4C5 domains of mouse cardiac myosin binding protein C, including 10 novel sites, and identified S690 as the predominant target of PKA and PKG1.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:35573219", "locator": "sections[6] \"Identification of Specific Phosphorylated Residues\" paragraph 2", "quote": "From our mass spectrometry results, we confirmed phosphorylation at S546, T603, and S690 but did not identify phosphorylation on the Y544 and T602 (h) sites."}, {"asset": "source:pmid:35573219", "locator": "sections[6] \"Identification of Specific Phosphorylated Residues\" paragraph 2", "quote": "We identified 10 additional phosphorylation sites that have not been characterized in the literature and appear to be novel."}, {"asset": "source:pmid:35573219", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Parallel reaction monitoring and subsequent mutagenesis experiments revealed that the S690 site (UniProtKB O70468) was the predominant target of PKA and PKG1."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "The study identified 13 phosphorylation sites in the C4C5 domains of mouse cardiac myosin binding protein C, including 10 novel sites, and identified S690 as the predominant target of PKA and PKG1.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:35573219"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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