Protein-based analysis for time since death estimation has emerged as a promising tool in recent years, providing a, yet limited, spectrum of target proteins that are known to undergo predictable proteolysis in the early and mid-postmortem phase. Aiming to expand the insights into systemic postmortem protein alterations in skeletal muscle tissue, the present study demonstrates an efficient MS-based proteomics approach to characterize additional marker proteins for future use in PMI delimitation to extend the toolset for protein degradation–based time since death estimation method. By means of this target-based and unbiased system wide protein analysis, we were able to identify the new markers GAPDH and eEF1A2. By establishing a standardized rat degradation model, we generated a suitable model for testing the proteins under laboratory conditions and created the basis for testing the forensic applicability of the marker proteins in a pilot experiment using three autopsy cases. By performing SDS-PAGE and Western blot experiments, we proved the potential of both of these proteins to act as future markers for postmortem alterations in skeletal muscle and, thus, recommend this approach for the characterization of further reliable marker proteins applicable in practical forensic PMI delimitation.
GAPDH, one of the two newly discovered markers, is a widely used housekeeping protein, and antibodies against GAPDH are often used as a loading control in Western blot experiments because of its ubiquitously and constitutively expression in mammalian body cells [28, 29]. Antibodies against GAPDH are thus commonly available, and the protein is easily detectable by the Western blot technique. In contrast to other common housekeeping proteins, GAPDH is reported to be relatively stable in postmortem tissue of humans (brain tissue, 48 hpm) , which is in accordance with our qualitative evaluation of the results of GAPDH in H3 (PMI > 14 dpm). However, from the low number of human samples in this study, it cannot be concluded whether there is a statistical decrease of the native band in later postmortem stages, as it was the case in rat vastus lateralis muscle. The significant decomposition of GAPDH protein in rats over a PMI of 96 hpm is confirmed by the proteomic analysis and by a study reporting postmortem GAPDH decrease in rat psoas muscle  over a similar period of time. Despite the trend to decrease, the presence of the native band of GAPDH band at a PMI of 96 hpm and the occurring degradation products at a larger PMI in the human sample H3 indicate possible applicability as a marker in later postmortem stages. Especially, the degradation products depict a qualitative change of band pattern that, in other proteins, has been shown to be able to significantly contribute to estimate the time since death . Additional experiments in wider time frames and a large sample size are required to determine whether there is a significant loss of the native band at later postmortem stages and whether the degradation products can be reproduced in human samples and may also appear in rat muscles in later stages. It has to be further evaluated whether their appearance is influenced by individual or environmental factors.
In contrast to the steadily decreasing character of GAPDH, the (early) loss of the translation factor eEF1A2 was found to be in strong correlation with the PMI, demonstrating the importance of this marker. Although eEF1A is found in high concentrations within body cells (3% of the total cellular protein) [31, 32], this is, to our knowledge, the first study on postmortem eEF1A2 decomposition. Proteomic investigations resulted in a successive decrease in the rat and mouse proteome, which was confirmed in Western blot experiments. Additionally, band intensities decreased below a 5% threshold from 48 hpm onwards and were, thus, considered a qualitative change (loss of a band). In contrast, in the human cases, we detected no obvious difference in eEF1A2 band intensity between H1 and H2, underlining the requirement of strict criteria for postmortem changes of protein band patterns for the use in forensic casework. Therefore, we generally suggest that qualitative changes (loss of a band, or appearance of a degradation product) should be preferred over quantitative measurements (e.g., merely a decrease in band intensities). However, additional experiments are required to characterize the precise timeframe in which the eEF1A2 band loss occurs in humans, and whether or how it is influenced by certain individual or environmental factors. Additional caution has to be taken when analyzing samples from cancer patients, as eEF1A2 can be overexpressed in such cases . Additional research has to evaluate whether this might be an exclusion criteria for the use of this marker.
When comparing the results to already established markers (tropomyosin, desmin, and vinculin), it was detected that the loss of the native eEF1A2 band in rats depicted the strongest correlation values with the PMI. This resulting correlation could though be influenced by the specific temporal degradation characteristics of the native eEF1A2 band (initial band loss at 48 hpm, complete band loss at 72 hpm) and the selected statistic test in the present study. Comparable alterations, such as the loss of meta-vinculin, and the occurrence of the 84 kDa vinculin degradation product are, nevertheless, equally important markers for time since death estimation although resulting in lower correlation values. In the case of the 84 kDa vinculin band within the rat model, which was negative (−, no measureable band) in all 0-hpm samples and positive (+, measurable band) in all samples with larger PMI, lower correlation values resulted from a less balanced overall ± ratio. Still, in accordance with previous studies , all results of the animal model were found to significantly correlate with the PMI.
Changes, such as the transient appearance of the 41 kDa band in the cytoskeletal protein desmin, could not be described using Spearman correlations. However, if certain timeframes can be excluded in future application, such transiently appearing protein bands can be of potential interest. The transient character of the 41 kDa desmin degradation product in this experimental series was in accordance with earlier studies [16, 34]. Together with the loss of the native band and the appearance of the 34 kDa band (both significant correlations with the PMI), it could clearly be confirmed that desmin is a reliable marker for early phase time since death estimation. Tropomyosin results were as well in accordance with previous studies reporting postmortem stability of this protein until 240 hpm . Although a real time passage (analysis of multiple sampling time points of one individual) is lacking for humans, selected cases with different PMI/ADD clearly demonstrated predictable degradation pattern of all tested standard proteins which are comparable to those in the rat model and also largely in accordance with previous studies on human muscle samples . Notably, there were no tropomyosin bands detected in H3. Whether this represents a characteristic degradation pattern for larger PMIs remains to be determined.
It can be concluded that LC-MS/MS analysis of postmortem skeletal muscle samples is a valuable method for the identification of biomarkers for forensic time since death estimation by analysis of protein degradation. With the cost-effective method we presented here, we were able to characterize promising marker candidates for further research in an appropriate timeframe. By running additional routine experiments, we confirmed significant correlations of the new identified marker proteins GAPDH and eEF1A2 with the PMI in an animal model. Although the animal models used in this study are distinct to humans, we were able to demonstrate similar degradation pattern of these proteins in humans. With this, we prove their relevance in future forensic application and generally strengthen the applicability of animal models for basic research on PMI estimation as indicated by a previous work . For future characterization of additional markers using proteomics, we recommend to consider additional experiments focusing as well on different species, extended time frames, or varying environmental conditions. Especially, investigations on the human postmortem proteome could, in future experiments, crucially contribute to the characterization of important additional biomarkers.