Postmortem proteomics to discover biomarkers for forensic PMI estimation

Abstract

The assessment of postmortem degradation of skeletal muscle proteins has emerged as a novel approach to estimate the time since death in the early to mid-postmortem phase (approximately 24 h postmortem (hpm) to 120 hpm). Current protein-based methods are limited to a small number of skeletal muscle proteins, shown to undergo proteolysis after death. In this study, we investigated the usability of a target-based and unbiased system-wide protein analysis to gain further insights into systemic postmortem protein alterations and to identify additional markers for postmortem interval (PMI) delimitation. We performed proteomic profiling to globally analyze postmortem alterations of the rat and mouse skeletal muscle proteome at defined time points (0, 24, 48, 72, and 96 hpm), harnessing a mass spectrometry-based quantitative proteomics approach. Hierarchical clustering analysis for a total of 579 (rat) and 896 (mouse) quantified proteins revealed differentially expressed proteins during the investigated postmortem period. We further focused on two selected proteins (eEF1A2 and GAPDH), which were shown to consistently degrade postmortem in both rat and mouse, suggesting conserved intra- and interspecies degradation behavior, and thus preserved association with the PMI and possible transferability to humans. In turn, we validated the usefulness of these new markers by classical Western blot experiments in a rat model and in human autopsy cases. Our results demonstrate the feasibility of mass spectrometry-based analysis to discover novel protein markers for PMI estimation and show that the proteins eEF1A2 and GAPDH appear to be valuable markers for PMI estimation in humans.

Keywords: Degradation; Postmortem interval (PMI); Protein; Proteomics; Skeletal muscle.

Fig. 1

Mass spectrometry–based analysis of postmortem alterations in mouse and rat skeletal muscle proteome. a Hierarchical clustering and heat map of protein intensities at various hpm. Protein intensity changes from the baseline intensity were clustered using an unbiased hierarchical method with a Euclidian distance function resulting in a few different clusters. Row: protein, column: hpm. Green: increase, red: decrease. b Venn diagram showing the number of total (left panel) and consistently decreased (right panel) proteins. Relative abundance of GAPDH (c) and eEF1A2 (d) at various hpm. Data represented as mean ± SD

Fig. 2

Western blot analysis of native standard proteins and degradation products in rats (n = 5) (a-c) and human cases (H1-H3) (d-f). Tropomyosin (TPM) (a) depicted no qualitative change of the characteristic double-band in any of the investigated samples and time points in rats. Desmin (DES) (b) and vinculin (VCL) (c) showed complete degradation of native bands (native DES and meta-VCL) as well as appearance of degradation products with increasing PMI (in hours postmortem, hpm). In human samples, TPM (d) depicted double bands in H1 and H2. In H3, no bands were present. Native DES bands (e) were found in H1 and H2, whereas in H3 the native band was lost. All human samples depicted characteristic DES degradation products of different molecular weights. Native VCL (f) was present in H1 and H2 and absent in H3. Meta-vinculin was only found in H1. Different sized characteristic degradation proteins were found in all human samples

Fig. 3

a Heat map depicting the frequency of band presence in all tested rat groups (n = 5 for each group). Tropomyosin (TPM) and GAPDH bands were detected in all, and native vinculin (VCL) in all but one analyzed sample and, thus, no correlation to the PMI was detected. However, native desmin (DES), meta-VCL, and native eEF1A2 depicted significant losses of the native band in correlation with the PMI. Additionally, significant correlations of the appearance of a 36 kDa desmin degradation product, and of 84 and 75 kDa vinculin degradation products, were detected. Asterisks mark significant (*) and highly significant (**) correlations, n.s. indicates not significant changes. b Relative abundance of GAPDH and eEF1A2 proteins over a PMI of 96 hpm in all tested rat groups (n = 5 for each group). Asterisks (*) indicate significant decreases of band intensity (relative abundance), as determined by Kruskal-Wallis test (p < 0.05). Data represented as mean ± SD

Fig. 4

Western blot analysis of native proteins and degradation products of identified marker proteins GAPDH and eEF1A2 in rats (n = 5) (a, b) and human cases (H1-H3) (c, d).While GAPDH (a) depicted only a minor decrease of band intensity between 24 and 96 hpm, eEF1A2 in rats (b) showed a decrease below a 5% detection threshold (regarded loss of the band) in all rat 72- and 96-hpm samples. In human samples, there was no obvious difference detectable between H1 and H2 in GAPDH (c) and eEF1A2 (d). However, in H3, distinct GAPDH degradation products appeared (c) and no native eEF1A2 (d) was detected