A multi-tissue longitudinal proteomics study to evaluate the suitability of post-mortem samples for pathophysiological research

Abstract

Recent developments in mass spectrometry-based proteomics have established it as a robust tool for system-wide analyses essential for pathophysiological research. While post-mortem samples are a critical source for these studies, our understanding of how body decomposition influences the proteome remains limited. Here, we have revisited published data and conducted a clinically relevant time-course experiment in mice, revealing organ-specific proteome regulation after death, with only a fraction of these changes linked to protein autolysis. The liver and spleen exhibit significant proteomic alterations within hours post-mortem, whereas the heart displays only modest changes. Additionally, subcellular compartmentalization leads to an unexpected surge in proteome alterations at the earliest post-mortem interval (PMI). Additionally, we have conducted a comprehensive analysis of semi-tryptic peptides, revealing distinct consensus motifs for different organs, indicating organ-specific post-mortem protease activity. In conclusion, our findings emphasize the critical importance of considering PMI effects when designing proteomics studies, as these effects may significantly overshadow the impacts of diseases. Preferably, the samples should be taken in the operation room, especially for studies including subcellular compartmentalization or trans-organ comparison. In single-organ studies, the planning should involve careful control of PMI.

Fig. 1. Multi-tissue longitudinal proteomics approach reveals drastic post-mortem proteome alterations.

Assessment of the post-mortem interval (PMI) on protein alteration from Ping et al. (a) and Aryal et al. (b). c Selection of proteins exhibiting distinct protein level alteration post-mortem from Aryal et al. d Experimental design and workflow for proteomics measurement of post-mortem tissues in isogenic mice. e Heatmap of proteins with significant post-mortem alterations across the different tissues and subcellular fractions (p < 0.05 from one-sided ANOVA, absolute log2FC > 1). f Selection of protein with tissue-specific post-mortem protein alterations in mice. N = 5 for all mouse proteomics samples.

Fig. 2. Organ-specific post-mortem difference.

a Volcano plot highlights differentially abundant protein in the spleen between fresh samples (group 1) and samples incubated for 4 days. b Relative protein abundance changes of most significant changing protein in the spleen post-mortem. c Gene ontology analysis of upregulated proteins in the spleen in group 4 compared to group 1 (fresh). d Volcano plot highlights differentially abundant protein in the liver between fresh samples (group 1) and samples incubated for 4 days (group 4). e Scatter plot assessing the correlation of post-mortem changes between the spleen and liver. f Number of differentially regulated proteins post-mortem in the liver (yellow), spleen (blue), and heart (green). g Correlation analysis of protein alterations across the various tissues post-mortem. P values were calculated using a two-sided Student t-test using equal variance and a p < 0.05 was considered as significant. The horizontal line in the boxplots represents the median, 25th, and 75th percentiles and whiskers represent measurements to the 5th and 95th percentiles. N = 5 for all mouse proteomics samples.

Fig. 3. Post-mortem changes in the heart and its soluble and ECM subcellar compartment.

a Volcano plot highlights differentially abundant protein in the heart between fresh samples (group 1) and samples incubated for 4 days. b Number of differentially regulated proteins post-mortem in the ECM, soluble fraction, and the total heart. c Distribution of protein alterations across various time points compared to fresh samples. d Scatter plot of protein changes in the subcellular fraction of the heart comparing group 2 and fresh samples. P values were calculated using a two-sided Student t-test using equal variance and a p < 0.05 was considered as significant. N = 5 for all mouse proteomics samples.

Fig. 4. Analysis of the post-mortem degradome.

a Concept of protein degradation post-mortem. b Number of significantly changing peptides post-mortem classified into their cleavage specificity. c Volcano plot highlights differentially abundant peptides in the spleen between fresh samples (group 1) and samples incubated for 4 days. d Semi-tryptic cleave motif enrichment analysis, reveals specific and time-dependent cleavage motifs. P values were calculated using a two-sided Student t-test using equal variance and a p < 0.05 was considered as significant. N = 5 for all mouse proteomics samples.