Reproducible Ion-Current-Based Approach for 24-Plex Comparison of the Tissue Proteomes of Hibernating versus Normal Myocardium in Swine Models

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.

Revascularization of Chronic Hibernating Myocardium Stimulates Myocyte Proliferation and Partially Reverses Chronic Adaptations to Ischemia

“…These can be minimized using large samples, as employed in our study, which reduces the impact of biological and experimental variability. Nearly all protein changes in nonrevascularized animals were previously reported in independent experiments using the same experimental approach in this model, and in an expanded protein analysis using label-free quantification with LC/MS (22). …”

“…Table 2 summarizes the effects of revascularization on proteomic adaptations in hibernating myocardium (5,17,22) and proteins correlating with increases in flow and function after revascularization are summarized in the heat map (Figure 4). Figure 5 summarizes average changes in selected proteins from each category (proteins italicized in Table 2).…”

“…In contrast, revascularization had only modest effects on metabolic enzymes and reductions in contractile proteins persisted. We previously demonstrated that regional metabolic proteins and mitochondrial oxygen consumption progressively declines as stenosis severity increases in viable dysfunctional myocardium (5,17,22,29). Consonant with our findings, Kelly et al reported persistent reductions in the mitochondrial proteome before and after surgical revascularization of hibernating myocardium.…”

Experimental Null Method to Guide the Development of Technical Procedures and to Control False-Positive Discovery in Quantitative Proteomics

“…Similar strategies have been used by our lab to estimate FADR and to determine the optimal cutoff threshold (e.g., folds of changes) for the discovery of altered proteins in various pharmaceutical and clinical applications. 14,18,22,23 Nevertheless, a systematic assessment of this method, for example, whether it provides an accurate estimation of FADR, has not been conducted thus far.…”

Proteomic Profiling of the Retinas in a Neonatal Rat Model of Oxygen-Induced Retinopathy with a Reproducible Ion-Current-Based MS1 Approach

“…18,20,21 Especially, ion-current- or MS1-based strategies are capable of providing a reliable means for in-depth protein profiling and biomarker discovery in complex proteomes. 22–25 The superior performance of ion-current-based (ICB) method compared to other popular label-free approaches such as MS2-TIC and spectral counting, in terms of reproducibility, quantitative accuracy, dynamic range, and biomarker discovery, has been demonstrated when using high-resolution MS data. 22,26 …”