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Phosphoproteome mapping of cardiomyocyte mitochondria in a rat model of heart failure

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Abstract

Mitochondria are complex organelles essential to cardiomyocyte survival. Protein phosphorylation is emerging as a key regulator of mitochondrial function. In the study reported here, we analyzed subsarcolemmal (SSM) mitochondria harvested from rats who have received 4 weeks of aldosterone/salt treatment to simulate the neurohormonal profile of human congestive heart failure. Our objective was to obtain an initial qualitative inventory of the phosphoproteins in this biologic system. SSM mitochondria were harvested, and the phosphoproteome was analyzed with a gel-free bioanalytical platform. Mitochondrial proteins were digested with trypsin, and the digests were enriched for phosphopeptides with immobilized metal ion affinity chromatography. The phosphopeptides were analyzed by ion trap liquid chromatography–tandem mass spectrometry, and the phosphoproteins identified via database searches. Based on MS/MS and MS3 data, we characterized a set of 42 phosphopeptides that encompassed 39 phosphorylation sites. These peptides mapped to 26 proteins, for example, long-chain specific acyl-CoA dehydrogenase, Complex III subunit 6, and mitochondrial import receptor TOM70. Collectively, the characterized phosphoproteins belong to diverse functional modules, including bioenergetic pathways, protein import machinery, and calcium handling. The phosphoprotein panel discovered in this study provides a foundation for future differential phosphoproteome profiling toward an integrated understanding of the role of mitochondrial phosphorylation in heart failure.

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Fig. 1
Fig. 2

Abbreviations

ALDOST:

Aldosterone/salt treatment

CHF:

Congestive heart failure

ETC:

Electron transport chain

FAO:

Fatty acid oxidation

IMAC:

Immobilized metal ion affinity chromatography

LC-MS/MS:

Liquid chromatography–tandem mass spectrometry

MRM:

Multiple reaction monitoring

MSTE:

Mitochondriocentric signal-transducer-effector pathway

SSM:

Subsarcolemmal mitochondria

TFA:

Trifluoroacetic acid

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Acknowledgments

This work was supported by the UTHSC College of Pharmacy Seed Grant (SBG) and by NIH grants R01-HL73043 and R01-HL90867 (KTW). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Funds for the LTQ mass spectrometer were provided by the NIH Shared Instrumentation Grant S10RR16679, and by the UTHSC College of Pharmacy.

Conflict of interest

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 881 Madison Avenue, Room 445, Memphis, TN, 38163, USA

    Francesco Giorgianni & Sarka Beranova-Giorgianni

  2. Division of Cardiovascular Diseases, University of Tennessee Health Science Center, Memphis, TN, 38163, USA

    M. Usman Khan & Karl T. Weber

  3. Division of Endocrinology, University of Tennessee Health Science Center, Memphis, TN, 38163, USA

    Ivan C. Gerling

Authors
  1. Francesco Giorgianni
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  2. M. Usman Khan
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  3. Karl T. Weber
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  4. Ivan C. Gerling
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  5. Sarka Beranova-Giorgianni
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Corresponding author

Correspondence to Sarka Beranova-Giorgianni.

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Giorgianni, F., Usman Khan, M., Weber, K.T. et al. Phosphoproteome mapping of cardiomyocyte mitochondria in a rat model of heart failure. Mol Cell Biochem 389, 159–167 (2014). https://doi.org/10.1007/s11010-013-1937-7

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  • DOI: https://doi.org/10.1007/s11010-013-1937-7

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