Separation And Identification Of Differentially Expressed Myocardial Proteins In The Late Phase Of Noninvasive Limb Ischemic Preconditioning

Objective: The aim of this study is to investigate the changes of all the myocardial protein expression profiles in the late phase of noninvasive limb ischemic preconditioning(NLIP) by the isobaric tags for relative and absolute quantitation(i TRAQ) technique, to observe the effects of the late phase induced by NLIP on protein expression in normal rat myocardium, and to explore the molecular mechanisms of endogenous effects induced by NLIP.Method: Twelve male SD rats were randomly assigned into the pre-treatment group(group NLIP) and the control group(group C), n=6 each. Group NLIP consisted of three cycles of 5-minute ischemia followed by 5-minute reperfusion in right hind limb by rubber band. Group C was only a sham treatment. After 24 h within the late phase of NLIP, the apical tissues of left ventricle were taken from two groups respectively and stored in liquid nitrogen. A starting material, which was prepared for protein extraction, was mixed by equal weight tissue taken from six samples in each group. i TRAQ technique was applied to detect the differences of protein expression profiles between two groups with the steps such as proteolysis, peptide labeling, high PH reverse separation, reverse liquid chromatography-tandem mass spectrometry, etc. Mass spectral analysis data were searched in the Uniprot database to identify peptides and proteins by the search engine Mascot. Quantitative information of proteins was calculated from the reported ion peak strength value by the software Protein Pilot. Take Ratio>20% and P-Value<0.05 as a standard to screen differentially expressed proteins. The biological functions of those differentially expressed proteins were searched in Gene Ontology database and their subcellular localization information was predicted by the software Wolfpsort. Then, some of those proteins were detected by western blotting to verify their expression levels in each group.Result: After those detection and database searching, the results showed that there were 3280 categories of proteins identified, and 55 of them were regarded as significantly differentially expressed proteins. Compared with group C, there were 35 up-regulated proteins and 20 down-regulated proteins in group NLIP. Based on GO analysis and subcellular localization prediction, most of the 55 proteins were located in the cytoplasm,nucleus or mitochondria, and most are involved in metabolic process, biological regulation, response to stimulus and signaling pathways. Some of them were predicted to play roles as binding proteins, catalysts, antioxidants and enzyme regulators. The results of western blotting and i TRAQ on the differentially expressed proteins ACOX1, PDK4 and PRDX2 showed no significant difference, which confirmed that all of them were up-regulated in group NLIP.Conclusion: There were significant alterations of protein expression profiles in rat myocardium in the late phase of NLIP. The results of function analysis to those proteins suggested that the delayed effects of NLIP might be related to their involvement in the processes such as improving energy metabolism, regulating oxidative stress, and regulating gene expression, protein folding and degradation.