The Study Of Myocardial Protein Degradation At Postmortem By Multi-omics Methods

Background and objective The chemical changes at postmortem are the foundation for various postmortem phenomena,but the most prominent feature of postmortem chemistry is its uncertain changes with postmortem interval,so it is of great significance to explore the postmortem chemical change patterns of various composition of the body in the field of forensic science.The protein degradation is an important part of postmortem chemical changes.There aren’t progress on postmortem protein in forensic medicine for a long time.The development of omics technologies has made it possible to study systematically postmortem protein degradation and its degradation metabolites as a whole.Targeted metabolomics allows the absolute quantification of amino acid.Proteomics allows systematic characterization of protein content,function and biological processes.It can help reveal the characteristics and patterns of postmortem protein degradation in a panoramic manner to combine metabolomics and proteomics;and break the bottleneck in PMI research.Materials and methods1.Experimental rats were executed by decervicalization under anesthesia,and placed in a constant temperature and humidity chamber after death for 14 days.The stages of decomposition were determined based on scoring methods.The left ventricular myocardial tissue was obtained each day and frozen quickly in liquid nitrogen and placed in a-80℃refrigerator for subsequent analysis.2.Ninhydrin chromogenic assay was used to test total amino acid content.Targeted metabolomics technique was used for quantitative analysis of amino acid concentrations in temporal pattern at post-mortem.3.The proteomics technology was used to screen the differential proteins of myocardial tissue at various points after death.The temporal changes of differential protein degradation was analyzed based on GO annotation and Pathway results.4.The content changes of the selected proteins were verified using Western blot immunoblotting technique.5.Three temperature groups were set up.Twelve amino acids were selected to analyze the effect of temperature on protein degradation.The univariate and multivariate correlation regression analysis were used for estimation of postmortem interval.Results1.The stage of decomposition based on scores in constant humidity and constant temperature(temperature fluctuation 25℃,humidity 50%)showed: 0-2 days in the fresh period,3-11 days in the actively putrefactive period,11-14 days in the highly putrefactive period.2.The results of ninhydrin chromogenic assay indicated an overall increasing trend ofα-amino acid concentration during 0-14 days and a linear correlation with PMI in 0-10 days.3.A linear increase in all neutral nonpolar amino acid concentrations from 0-6 days were observed at postmortem.Glutamate,an acidic amino acid,maintained a good linear correlation with PMI during 10 days.Basic amino acids as well as neutral polar amino acids showed a relatively poor regularity at postmortem.4.The protein degradation process showed nine temporal trends,of which regular degradation accounted for more than 60%,and the content of some enzymatic proteins showed an increase then followed by a decrease.Most of the cytomembrane proteins showed continuous degradation after death.5.Proteins selected for validation showed that the protein change trends were basically consistent with those detected by proteomics technology.6.The trends of amino acids at postmortem was similar at different temperature,but the duration was quicken with increase of temperature.The simple and multiple regression equations based on selected amino acids were established at 15℃,25℃ and 35℃ for estimation of PMI.Conclusion1.The protein degradation at postmortem showed a pattern of relative order to disorder.2.Most amino acid concentrations showed linear correlation with PMI within a certain time.And glutamate had the longest duration of regular changes.3.There was significant heterogeneity in postmortem myocardial protein degradation.Some proteases showed an increase trend which is related to its supravital reaction.4.The amino acids that showed regular temporal change have some predictive power for the postmortem interval.The prediction range of the model was shortened with increasing temperature.