Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts

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• 作者：Viola Kooij^a ; Ronald J. Holewinski^a ; Anne M. Murphy^b ; Jennifer E. Van Eyk^a ; ^{Jvaneyk1@jhmi.edu}
• 关键词：Cardiac myosin binding protein-C ; cMyBP-C ; Phosphorylation ; Phosphoproteome ; Mass spectrometry ; Titanium dioxide
• 刊名：Journal of Molecular and Cellular Cardiology
• 出版年：2013
• 出版时间：July, 2013
• 年：2013
• 卷：60
• 期：Complete
• 页码：116-120
• 全文大小：458 K

文摘

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 (<em>nem> = 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 <em>in vivoem>, 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 <em>in vivoem>. 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 <em>in vitroem> kinase incubation. The data have been deposited to the ProteomeXchange with identifier PXD000158.