TY - JOUR
T1 - Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts
AU - Kooij, Viola
AU - Holewinski, Ronald J.
AU - Murphy, Anne M.
AU - Van Eyk, Jennifer E.
N1 - Funding Information:
We would like to thank Dr. S. Sadayappan for his kind gift of the cMyBP-C antibodies and the recombinant C0–C2 cMyBP-C fragment and Dr. C Dos Remedios for providing us with the banked human explanted hearts. This work was in part supported by National Institutes of Health grants R01 HL63038 (A.M.M.) and P01 HL077180 (J.E.V.E.), National Heart Lung and Blood Institute HHSN268201000032C (J.E.V.E.), and by an American Heart Association post-doctoral fellowship (V.K., 12POST11520006 ). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the dataset identifier PXD000158.
PY - 2013/7
Y1 - 2013/7
N2 - Introduction: Cardiac myosin binding protein-C (cMyBP-C) becomes dephosphorylated in the failing heart and reduced phosphorylation-dependent regulation of cMyBP-C has been implicated in contractile dysfunction. To date, several phosphorylation sites have been identified for human cMyBP-C; however, a comprehensive characterization of the cMyBP-C phosphoproteome is lacking. This study aimed to characterize the cMyBP-C phosphoproteome using two different proteomic-based methods in explanted donor and end-stage failing hearts. Methods: The first approach used to characterize the cMyBP-C phosphoproteome employed a strong-cation exchange chromatography (SCX) fractionation method (10 pooled samples, technical replicates=4) and the second employed a sodium dodecylsulfate polyacrylamide gel electrophoresis method (n=10; technical replicates=2). Each subsequently underwent titanium dioxide (TiO2) affinity chromatography to enrich for the tryptic phosphopeptides, which were analyzed using an LTQ-Orbitrap mass spectrometer. Moreover, recombinant C0-C2 fragment of mouse cMyBP-C incubated with PKA, PKC, CaMKII and CK2 was analyzed to identify the kinases involved with phosphorylation of cMyBP-C. Results: Seventeen phosphorylation sites on cMyBP-C were identified in vivo, with the majority localized in the N-terminal domains C0-C2. The three most abundant phosphorylated sites, Ser284, Ser286 and Thr290, are located in the regulatory M-domain of cMyBP-C. Ser284 showed a significant reduction in phosphorylation in HF. Conclusion: This study demonstrates that cMyBP-C harbors more phosphorylation sites than previously known, with a total of 17 (9 novel) identified phosphorylation sites in vivo. Most sites were primarily located within the N-terminal side of the protein. The most highly phosphorylated site on cMyBP-C was Ser284 and this site showed decreased phosphorylation in the failing heart, which implicates importance for fine-tuning contractility. To date, the functional importance of Ser286 and Thr290 is unknown. In addition, 16 sites were identified after in vitro kinase incubation. The data have been deposited to the ProteomeXchange with identifier PXD000158.
AB - Introduction: Cardiac myosin binding protein-C (cMyBP-C) becomes dephosphorylated in the failing heart and reduced phosphorylation-dependent regulation of cMyBP-C has been implicated in contractile dysfunction. To date, several phosphorylation sites have been identified for human cMyBP-C; however, a comprehensive characterization of the cMyBP-C phosphoproteome is lacking. This study aimed to characterize the cMyBP-C phosphoproteome using two different proteomic-based methods in explanted donor and end-stage failing hearts. Methods: The first approach used to characterize the cMyBP-C phosphoproteome employed a strong-cation exchange chromatography (SCX) fractionation method (10 pooled samples, technical replicates=4) and the second employed a sodium dodecylsulfate polyacrylamide gel electrophoresis method (n=10; technical replicates=2). Each subsequently underwent titanium dioxide (TiO2) affinity chromatography to enrich for the tryptic phosphopeptides, which were analyzed using an LTQ-Orbitrap mass spectrometer. Moreover, recombinant C0-C2 fragment of mouse cMyBP-C incubated with PKA, PKC, CaMKII and CK2 was analyzed to identify the kinases involved with phosphorylation of cMyBP-C. Results: Seventeen phosphorylation sites on cMyBP-C were identified in vivo, with the majority localized in the N-terminal domains C0-C2. The three most abundant phosphorylated sites, Ser284, Ser286 and Thr290, are located in the regulatory M-domain of cMyBP-C. Ser284 showed a significant reduction in phosphorylation in HF. Conclusion: This study demonstrates that cMyBP-C harbors more phosphorylation sites than previously known, with a total of 17 (9 novel) identified phosphorylation sites in vivo. Most sites were primarily located within the N-terminal side of the protein. The most highly phosphorylated site on cMyBP-C was Ser284 and this site showed decreased phosphorylation in the failing heart, which implicates importance for fine-tuning contractility. To date, the functional importance of Ser286 and Thr290 is unknown. In addition, 16 sites were identified after in vitro kinase incubation. The data have been deposited to the ProteomeXchange with identifier PXD000158.
KW - CMyBP-C
KW - Cardiac myosin binding protein-C
KW - Mass spectrometry
KW - Phosphoproteome
KW - Phosphorylation
KW - Titanium dioxide
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U2 - 10.1016/j.yjmcc.2013.04.012
DO - 10.1016/j.yjmcc.2013.04.012
M3 - Article
C2 - 23619294
AN - SCOPUS:84878650247
SN - 0022-2828
VL - 60
SP - 116
EP - 120
JO - Journal of Molecular and Cellular Cardiology
JF - Journal of Molecular and Cellular Cardiology
IS - 1
ER -