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PXD000158

PXD000158 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleCharacterization of the Cardiac Myosin Binding Protein-C Phosphoproteome in Healthy and Failing Human Hearts
DescriptionIntroduction: 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).
HostingRepositoryPRIDE
AnnounceDate2017-10-24
AnnouncementXMLSubmission_2017-10-24_00:12:30.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterRonald Holewinski
SpeciesList scientific name: Mus musculus (Mouse); NCBI TaxID: 10090; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListphosphorylated residue; monohydroxylated residue; iodoacetamide derivatized residue
InstrumentLTQ Orbitrap; LTQ Orbitrap Elite
Dataset History
RevisionDatetimeStatusChangeLog Entry
02013-02-25 12:34:41ID requested
12013-05-01 03:33:25announced
12014-07-24 03:47:02announced
22013-05-01 03:37:35announcedAdd reference
22017-06-21 00:30:37announcedUpdated project metadata.
32017-10-24 00:12:31announcedUpdated project metadata.
Publication List
Kooij V, Holewinski RJ, Murphy AM, Van Eyk JE, Characterization of the cardiac myosin binding protein-C phosphoproteome in healthy and failing human hearts. J Mol Cell Cardiol, 60():116-20(2013) [pubmed]
Keyword List
curator keyword: Cardiovascular
submitter keyword: cardiac, cardiac myosin binding protein C, cMybpC, phosphoprotein, phosphoproteome, mass spectrometry,
Contact List
Ronald Holewinski
contact affiliationJohns Hopkins University
contact emailrholewi1@jhmi.edu
dataset submitter
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Dataset FTP location
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