Introduction/Background:
Accurate estimation of time since death and the postmortem interval (PMI) are crucial in forensic medico-legal investigations to understand case circumstances (e.g. narrowing down list of missing persons or in-/exclude suspects). Current methods, however, heavily depend on the experience of the forensic examiner such as subjective assessment of morphological changes, stiffness and discolouration. The aim of the current study was to utilize an untargeted LC-MS/MS-based proteomics workflow to study time-dependent postmortem changes of peptides/proteins in human muscle tissue to help establish more objective biomarkers for PMI estimation.
Methods:
Minimal-invasive thigh muscle tissue biopsy samples were collected at the Australian Facility for Taphonomic Experimental Research (Sydney) from 9 donors across multiple time-points after death (≤120 days postmortem). Samples were homogenised and proteins extracted. After reduction (TCEP) and alkylation (IAA), protein extracts were cleaned up (SP3) and proteolytically digested (trypsin). Samples (n=179) were analysed in triplicate on a nanoHPLC system coupled to a SynaptXS ion mobility TOF-MS (DIA mode, scan range 50-1500 m/z). Progenesis Qi for Proteomics was used for data processing and peptide/protein identification (reviewed human database). Sample collection time-points were converted to accumulated degree days (ADD) utilizing daily average temperatures (⁰C). Using R, all possible ratios between peptides originating from the same protein were calculated, log2 transformed, filtered according to linear regression results (r2 and slope) and database matches manually confirmed.
Results:
A total of 11 promising peptide ratios were found for potential PMI estimation up to ADD = 655 (≤48 days, n=6 donors). These ratios displayed consistent and robust linear relationships (increasing/decreasing) over time across all donors, originating from myosin-7 and actin (alpha skeletal muscle) proteins. Similarly, 4 promising ratios of peptides from myosin-2 protein were found for PMI estimation up to ADD = 1535 (≤120 days; n=4 donors). Linear, season-specific peptide ratios were found for bodies decomposing in spring/summer (n=4 donors; 3 peptide ratios from myosin-7 protein) but not for bodies decomposing in autumn/winter (n=5 donors).
Discussion/Conclusions:
All peptide ratios with significant linear regressions were calculated from peptides (non-modified and with oxidation posttranslational modifications) that originate from muscle tissue-specific proteins (myosin and actin) and hence are likely related to the in-vivo decomposition of muscle tissue. While identifying peptide ratios with consistent and robust postmortem ADD-dependent changes is a first step towards objective PMI estimation, increasing the number of donors is necessary to conduct an extensive modelling approach for prediction of PMI for unknown forensic case samples.