Dataset Information

Characterization of the Cardiac Myosin Binding Protein-C Phosphoproteome in Healthy and Failing Human Hearts

ABSTRACT: 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 control and end-stage failing hearts. Methods: The first approach used to characterize the cMyBP-C phosphoproteome employed a strong-cation exchange chromatography (SCX)-based 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, 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, while Ser286 and Thr290 showed a trend towards a reduced phosphorylation. Conclusion: This study demonstrates that cMyBP-C is more extensively phosphorylated than previously known, with 10 novel sites identified. Most sites were primarily located within the N-terminal side of the protein. The three most highly phosphorylated sites on cMyBP-C were Ser284, Ser286 and Thr290 and these three sites showed decreased phosphorylation in the failing heart, which implicate their 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. Bioinformatics pipeline: All RAW files were searched using the Sorcerer 2 Sequest algorithm. Human samples were searched against the IPI human database version 3.79 and mouse samples were searched against the IPI mouse database version 3.80. All searches were carried out with static modification of +57 on C and differential modifications of +16 on M and +80 on S,T,Y. Parent mass tolerance was set to 50 ppm and fragment mass tolerance was set to 1 Da. Post-search analysis was performed using Scaffold version 3.2.0 and site localization was determined using Scaffold PTM version 1.1.2 (Proteome Software, Inc., Portland, OR, USA).

INSTRUMENT(S): LTQ Orbitrap, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Ronald Holewinski

PROVIDER: PXD000158 | Pride | 2013-05-01

REPOSITORIES: Pride

ACCESS DATA

Publications

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

Kooij Viola V Holewinski Ronald J RJ Murphy Anne M AM Van Eyk Jennifer E JE

Journal of molecular and cellular cardiology 20130422

<h4>Introduction</h4>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 ...[more]

PMID: 23619294

Similar Datasets

Project description:Yugi2014 - Insulin induced signalling (PFKL phosphorylation) - model 1 Insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, which thereby controls a key reaction in glycolysis. This mechanism is revealed using the mathematical model. In this model, the PFKL phosphorylation time courses are obtained from experimental data. Author's Note: Katsuyuki Yugi thank Akira Funahashi (Keio University, Japan) for his kind advice in converting the model from MATLAB to SBML. This model is described in the article: Reconstruction of insulin signal flow from phosphoproteome and metabolome data. Yugi K, Kubota H, Toyoshima Y, Noguchi R, Kawata K, Komori Y, Uda S, Kunida K, Tomizawa Y, Funato Y, Miki H, Matsumoto M, Nakayama KI, Kashikura K, Endo K, Ikeda K, Soga T, Kuroda S. Cell Rep 2014 Aug; 8(4): 1171-1183 Abstract: Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis has not been explored. We have developed a reconstruction method for the signal flow based on time-course phosphoproteome and metabolome data, using multiple databases, and have applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin signal flows through a network, through signaling pathways that involve 13 protein kinases, 26 phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites. Analysis of the network reveals that insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, thereby controlling a key reaction in glycolysis. We thus provide a versatile method of reconstruction of signal flow through the network using phosphoproteome and metabolome data. This model is hosted on BioModels Database and identified by: BIOMD0000000540. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Dataset Information

Characterization of the Cardiac Myosin Binding Protein-C Phosphoproteome in Healthy and Failing Human Hearts

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure