Determination of the postmortem interval (PMI) is one of the most challenging and difficult tasks in daily forensic casework due to limitations of accurate and reliable methods. Precise estimation of the time of death is crucial for criminal law as it often validates a witness’s statement, possibly limits the number of suspects and assesses alibies. Especially in cases with multiple dead bodies within temporal proximity, information about the succession of events is often of utmost importance. Although there is a lot of research focused on new attempts for PMI estimation and on the improvement of existing approaches [
1‐
6], novel methods for time of death estimation that comply with the requirements of practice are still of high demand. In early decomposition stages, first physical changes, effects of cellular breakdown, autolytic activity and structural alterations in tissue can be detected [
7]. In this postmortem period characteristic changes such as
rigor mortis (stiffening and relaxation of skeletal muscle),
livor mortis (accumulation of blood and blood products at lower parts of the body),
algor mortis (changes in body core temperature) as well as supravital reactions [
8,
9], such as response to electrical or pharmacological excitation [
10] are applied for the estimation of the time since death in every day forensic work. These parameters, however, are highly dependent upon individual and environmental factors, such as temperature, humidity, age, body size, cause of death, or pathological precondition [
11,
12] and are generally restricted to early postmortem time periods (i.e., the first 36 h postmortem, hpm) [
11]. PMI estimation in later stages is usually even more difficult as the range of available methods is far smaller. In postmortem periods, starting from approximately 72 hpm, forensic entomology, the analyses of developmental stages of cadaver-feeding insects, can sometimes provide information about the minimum PMI [
13]. But also, this method is limited in many cases, e.g., by insect accessibility (bodies discovered indoor, drowned, or covered bodies, etc...), or certain threshold temperatures for the development of different species [
12,
14]. PMI estimation in even later stages postmortem, such as in mummified corpses is even more difficult. This is only rarely possible solely on behalf of biomedical data such as autopsy findings [
15], but is most often limited to other sources of information, like evidence found at the scene (call register, transport tickets or newspaper), testimonies or confessions. These data are however, highly case dependent and cannot be transferred from one to another. New biomedical approaches to account for cases like these, and thus additional research in this field is highly required.
An important consideration is to achieve highest possible precision, but reliability, cost, and time effort as well as handling play a central role in the establishment of new methods for forensic routine work and often prevent successful application [
16]. Recently, an approach based on skeletal muscle protein degradation was reported for PMI estimation. Various alterations over a postmortem period of 10 days were observed in an animal model [
2] and in further consequence, verified in human forensic cases [
17]. Comparable changes were obtained in humans that significantly correlated with time and temperature [
17]. A first successful application of this approach already proved the viability in a comparison of two cases, in which major influencing factors (e.g., temperature) could be excluded [
18]. In general, samples obtained in daily forensic practice are difficult to compare to each other due to varying individual influencing factors (e.g., age, BMI, cause of death, etc.) and variable conditions within the PMI (temperature, humidity, etc...). To describe the basic principles of biological processes and certain influencing factors, animal models are often inevitable [
19‐
22]. However, obtained results cannot be applied to human cases without further considerations and appropriately comparative studies, which represent a very common task, in bridging basic and applied research. Especially in forensic science, pigs are commonly used as model organisms due to their comparable body size and physiology to humans [
23,
24], but require large scale experimental setups and according facilities. Rodents, on the other hand, are cheap and easy in availability and handling, but provide fewer sample material. To evaluate the legitimacy of both of these models, and to eventually benefit from the advantages of both, we conducted an experimental series, comparing protein degradation events in humans, pigs, and mice. We aimed to identify qualitative alterations in three different species as well as to compare eventual differences and similarities and thus validate the legitimacy for further research on the respective animal model. Additionally we expanded the set of possible target proteins to detect further degradation patterns and thereby identify additional viable postmortem interval markers.