Development of a powerful quantitative proteomics strategy and its application for better understanding of myocardial stunning in swine model
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Myocardial stunning following brief ischemia was initially described more than 40 years ago. In general terms, myocardial stunning is caused by molecular cellular modulation occurring during ischemia, but contractile function can be restored after hours or days. Not as myocardial infarction (i.e. irreversible ischemia) that is extensively investigated and has established diagnostic biomarker, mechanism of myocardial stunning lack thorough investigation. It has been proposed that oxygen-derived free radicals and calcium overload may be responsible for depressed cardiac function in stunning, but the specific mechanism by which free radicals and Ca2+ produce stunning is poorly understood. Quantitative proteomics and phosphoproteomics is capable to tackle the problem in a high throughput manner, on protein abundance and post translational modification level respectively. To ensure a high-quality proteomics analysis with deep coverage and high accuracy on this project, method developments had been made at several levels: i) development of shotgun proteomics workflow for in-depth, accurate and reproducible quantitation on large-scale sampleset with extremely low-level-of-missing-values, in aspects of sample processing, LC-MS method and bioinformatics tool; ii) Establishment and evaluation of False Altered proteins Discovery Rate(FADR) method; iii) Establishment of RNA-seq directed pig heart protein database with annotation to guide proper protein identification and downstream analysis. With these technique developments, proteomics and phosphoproteomics analysis on stunned myocardium in porcine model demonstrate a substantial improvement in identification depth compared with previous proteomics/phosphoproteomics investigations. Analysis results reveal novel findings need to be further validated biologically and corroborate previously identified mechanisms.