A standard protocol for the analysis of postmortem muscle protein degradation: process optimization and considerations for the application in forensic PMI estimation

Pittner, Stefan; Merold, Veronika; Anders, Sven; Lohner, Larissa; Amendt, Jens; Klinger, Miriam; Hausmann, Roland; Kissling, Steffen; Monticelli, Fabio; Geißenberger, Janine; Zissler, Angela; Steinbacher, Peter

doi:10.1007/s00414-022-02849-3

Cited by 8 publications

(5 citation statements)

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“…Compared to muscle, thawed non-muscle samples were found to be characterized by higher levels of amino acids and other metabolites, likely the result of enhanced protein degradation, and a higher susceptibility to oxidation [ 37 , 38 ]. In line with this, recent work from our own lab had shown muscle proteins including vinculin, alpha tubulin, and GAPDH exhibit similar degradation patterns irrespective of whether analyzed fresh or after a brief freeze–thaw cycle of 1 week [ 29 ], thus supporting target isotype choice for the present work.…”

Section: Discussionsupporting

confidence: 76%

“…To address this task, this study uses a standardized protein degradation model that was implemented in previous work [ 18 , 29 ]. We compare two sets of dismembered pig hind limbs ( n = 6 each), one of which was left to degrade in a climate chamber at 30 °C immediately after slaughter, whereas the second set was freeze-stored at − 20 °C for 4 months before being subjected to degradation under the same conditions.…”

Section: Introductionmentioning

confidence: 99%

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Effect of temporary freezing on postmortem protein degradation patterns

et al. 2023

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Background A precise determination of time since death plays a major role in forensic routine. Currently available techniques for estimating the postmortem interval (PMI) are restricted to specific time periods or cannot be applied for individual case-specific reasons. During recent years, it has been repeatedly demonstrated that Western blot analysis of postmortem muscle protein degradation can substantially contribute to overcome these limitations in cases with different background. Enabling to delimit time points at which certain marker proteins undergo distinct degradation events, the method has become a reasonable new tool for PMI delimitation under various forensic scenarios. However, additional research is yet required to improve our understanding of protein decomposition and how it is affected by intrinsic and extrinsic factors. Since there are temperature limits for proteolysis, and investigators are confronted with frozen corpses, investigation of the effects of freezing and thawing on postmortem protein decomposition in the muscle tissue is an important objective to firmly establish the new method. It is also important because freezing is often the only practical means to intermittently preserve tissue samples from both true cases and animal model research. Methods Sets of dismembered pig hind limbs, either freshly detached non-frozen, or thawed after 4 months of freeze-storage (n = 6 each), were left to decompose under controlled conditions at 30 °C for 7 days and 10 days, respectively. Samples of the M. biceps femoris were regularly collected at predefined time points. All samples were processed via SDS-PAGE and Western blotting to identify the degradation patterns of previously characterized muscle proteins. Results Western blots show that the proteins degrade predictably over time in precise patterns that are largely unaffected by the freeze-and-thaw process. Investigated proteins showed complete degradation of the native protein band, partly giving rise to degradation products present in distinct time phases of the decomposition process. Conclusion This study provides substantial new information from a porcine model to assess the degree of bias that freezing and thawing induces on postmortem degradation of skeletal muscle proteins. Results support that a freeze–thaw cycle with prolonged storage in frozen state has no significant impact on the decomposition behavior. This will help to equip the protein degradation–based method for PMI determination with a robust applicability in the normal forensic setting.

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Section: Discussionsupporting

confidence: 76%

Section: Introductionmentioning

confidence: 99%

Effect of temporary freezing on postmortem protein degradation patterns

et al. 2023

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“…Nevertheless, a significant limiting factor is the ability to stop the tissue degradation process at the time of sampling. One way to circumvent this limitation is by snap-freezing and storing the samples in liquid nitrogen, but this procedure is impractical for routine application [ 74 ]. Further studies are thus needed to evaluate the applicability of these new techniques for day-to-day forensic cases.…”

Section: Discussionmentioning

confidence: 99%

New Trends in Immunohistochemical Methods to Estimate the Time since Death: A Review

et al. 2022

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The identification of a reliable and accurate post-mortem interval (PMI) is a major challenge in the field of forensic sciences and criminal investigation. Several laboratory techniques have recently been developed that offer a better contribution to the estimation of PMI, in addition to the traditional physical or physico-chemical (body cooling, lividity, radiocarbon dating, rigor mortis), chemical (autolysis), microbiological (putrefaction), entomological, as well as botanical parameters. Molecular biology (degradation pattern of macromolecules such as proteins, DNA, RNA), biochemical analysis of biological fluids (such as blood, cerebrospinal fluid, and vitreous humor), and immunohistochemistry are some of the most recent technological innovations. A systematic review of the literature was performed with the aim of presenting an up-to-date overview on the correlation between the immunohistochemical (IHC) expression of specific antigenic markers at different PMIs. The systematic review was performed according to PRISMA guidelines. Scopus and PubMed were used as search engines from January 1, 1998 to March 1, 2022 to evaluate the effectiveness of immunohistochemistry in estimating PMI. The following keywords were used: (immunohistochemical) OR (immunohistochemistry) AND (time since death) OR (post-mortem interval) OR (PMI). A total of 6571 articles were collected. Ultimately, 16 studies were included in this review. The results of this systematic review highlighted that IHC techniques, in association with traditional methods, add, in Bayesian terms, additional information to define a more accurate time of death and PMI. However, current IHC results are numerically limited and more data and studies are desirable in the near future.

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“…Several biochemical techniques that analyze protein degradation have been suggested for assessing PMI. Proteins undergo specific changes postmortem, some of which follow a regular and predictable pattern (Sacco et al, 2022;Pittner et al, 2022;Zissler et al, 2020). Many studies have investigated protein degradation in various tissues with different techniques, such as Western blotting, liquid chromatography-mass spectrometry, and immunohistochemistry (Zhang et al, 2020;Choi et al, 2019;Alibegović et al 2020).…”

Section: Introductionmentioning

confidence: 99%

Comparison of the Visual Scoring Method and Semi-Automatic Image Analysis for Evaluating Staining Intensity of Human Cartilage Sections

Alibegović,

Umek,

Pušnik

et al. 2024

Image Anal Stereol

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Accurate estimation of postmortem interval (PMI) is crucial in forensic medicine. The hyaline cartilage, being predominantly composed of a dense extracellular matrix and partly resistant to factors influencing protein degradation, can be utilized for analyzing PMI intervals. Various staining methods are available for cartilage staining for PMI evaluation; however, the conventional visual scoring method for assessing staining intensity is susceptible to evaluator bias. This study compared the visual scoring method with a modified Bern score with semi-automatic image analysis. The cartilage samples were obtained from three cadavers with known time of death. Forty-five histological slices were prepared and stained using Alcian blue, Safranin-O with Fast green, Safranin-O without Fast green, Masson trichrome, and Sirius red. Ten evaluators visually scored each sample on a scale of 0 to 3. A semi-automatic analysis was conducted on the same images using the deconvolution plugin of the ImageJ software. Linear regression was used to assess the correlation between the mean grey value and the mean Bern score from all evaluators. The results showed strong correlations across all evaluated staining techniques, with Masson trichrome staining exhibiting the highest correlation. Accordingly, semi-automatic image analysis can be a suitable replacement for the visual scoring method, particularly when no procedural artifacts are present.

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A standard protocol for the analysis of postmortem muscle protein degradation: process optimization and considerations for the application in forensic PMI estimation

Cited by 8 publications

References 28 publications

Effect of temporary freezing on postmortem protein degradation patterns

Effect of temporary freezing on postmortem protein degradation patterns

New Trends in Immunohistochemical Methods to Estimate the Time since Death: A Review

Comparison of the Visual Scoring Method and Semi-Automatic Image Analysis for Evaluating Staining Intensity of Human Cartilage Sections

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