Format

Send to

Choose Destination
J Proteome Res. 2014 May 2;13(5):2571-84. doi: 10.1021/pr5000472. Epub 2014 Apr 4.

Reproducible ion-current-based approach for 24-plex comparison of the tissue proteomes of hibernating versus normal myocardium in swine models.

Author information

1
Department of Pharmaceutical Sciences, ‡Department of Biochemistry, §Department of Medicine, ∥Department of Physiology and Biophysics, ⊥The Center for Research in Cardiovascular Medicine, and #Center for Excellence in Bioinformatics and Life Sciences, State University of New York at Buffalo , Buffalo, New York 14214, United States.

Abstract

Hibernating myocardium is an adaptive response to repetitive myocardial ischemia that is clinically common, but the mechanism of adaptation is poorly understood. Here we compared the proteomes of hibernating versus normal myocardium in a porcine model with 24 biological replicates. Using the ion-current-based proteomic strategy optimized in this study to expand upon previous proteomic work, we identified differentially expressed proteins in new molecular pathways of cardiovascular interest. The methodological strategy includes efficient extraction with detergent cocktail; precipitation/digestion procedure with high, quantitative peptide recovery; reproducible nano-LC/MS analysis on a long, heated column packed with small particles; and quantification based on ion-current peak areas. Under the optimized conditions, high efficiency and reproducibility were achieved for each step, which enabled a reliable comparison of 24 the myocardial samples. To achieve confident discovery of differentially regulated proteins in hibernating myocardium, we used highly stringent criteria to define "quantifiable proteins". These included the filtering criteria of low peptide FDR and S/N > 10 for peptide ion currents, and each protein was quantified independently from ≥2 distinct peptides. For a broad methodological validation, the quantitative results were compared with a parallel, well-validated 2D-DIGE analysis of the same model. Excellent agreement between the two orthogonal methods was observed (R = 0.74), and the ion-current-based method quantified almost one order of magnitude more proteins. In hibernating myocardium, 225 significantly altered proteins were discovered with a low false-discovery rate (∼3%). These proteins are involved in biological processes including metabolism, apoptosis, stress response, contraction, cytoskeleton, transcription, and translation. This provides compelling evidence that hibernating myocardium adapts to chronic ischemia. The major metabolic mechanisms include a down-regulation of mitochondrial respiration and an increase in glycolysis. Meanwhile, cardioprotective and cytoskeletal proteins are increased, while cardiomyocyte contractile proteins are reduced. These intrinsic adaptations to regional ischemia maintain long-term cardiomyocyte viability at the expense of contractile function.

PMID:
24697261
PMCID:
PMC4015685
DOI:
10.1021/pr5000472
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for American Chemical Society Icon for PubMed Central
Loading ...
Support Center