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Poor Quantitative Concordance of Proteomic Data between Autopsy Samples and Fresh Frozen Samples from Human Coronary Arteries

ABSTRACT: Proteomic analyses of human tissues are sometimes conducted on autopsy samples. However, no comparative analysis between proteomic data derived from autopsy samples and fresh frozen samples has been undertaken, nor has there been an assessment of the post-mortem interval (PMI) influences on protein quantification. In the current study, 94 human left anterior descending (LAD) coronary artery were collected from deceased patients. Proteins were analysed using nanoflow liquid chromatography-tandem mass spectrometry. Data were processed with Proteome Discoverer and Mascot. The correlations between the protein abundances and the PMI were calculated. DAVID software was used for pathway and GO annotation enrichment. Among consistently quantified proteins, approximately 40% of the protein abundances exhibited significant correlations with PMI, most of which being inverse. Notably, smooth muscle cell markers displayed substantial reduction with prolonged PMI. Conversely, positive correlations with PMI were observed for immunoglobulins, coagulation factors, and complement factors, including coagulation factor XII, plasminogen, and lactotransferrin. Comparative analyses of sex differences between autopsy LAD samples and fresh-frozen LAD samples (n=65) showed no concordance in protein quantification. However, a robust correlation was observed within a sex comparison conducted between fresh-frozen carotid endarterectomies (CEA) from 2 different cohorts (n=104 and n=200). This study represents the first large-scale proteomics investigation into the influence of PMI on the protein composition of human vasculature. We observed significant correlations with PMI for nearly 40% of the consistently quantified proteins. Our findings underscore potential discrepancies in the quantitative accuracy of proteomics data derived from autopsy samples. Consequently, results obtained from post-mortem specimens may not be reproducible in fresh-frozen samples.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Coronary Artery

DISEASE(S): Atherosclerosis

SUBMITTER: XIAOKE YIN

LAB HEAD: Manuel Mayr

PROVIDER: PXD052071 | Pride | 2025-06-09

REPOSITORIES: Pride

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Project description:Attempts to establish a tissue bank from autopsy samples have led to uncovering of the secrets of many diseases. Here, we examined the length of time that the RNA from postmortem tissues is available for microarray analysis and reported the gene expression profile for up- and down-regulated genes during the postmortem interval (PMI). We extracted RNA from fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) brains and livers of three different groups of mice: 1) mice immediately after death, 2) mice that were stored at room temperature for 3 h after death, and 3) mice that were stored at 4Â°C for 18 h after death, as this storage resembles the human autopsy process in Japan. Based on the microarray analysis, we selected genes that were altered by >1.3-fold or <0.77-fold and classified these genes using hierarchical cluster analysis following DAVID (database for annotation, visualization, and integrated discovery) gene ontology analysis. These studies revealed that cytoskeleton-related genes were enriched in the set of up-regulated genes, while serine protease inhibitors were enriched in the set of down-regulated genes. Interestingly, although the RNA quality was maintained, up-regulated genes were not validated by quantitative PCR, suggesting that these genes may become fragmented or modified by an unknown mechanism. We extracted RNA from fresh-frozen (FF) brains and livers from mice under three different conditions: 1) mice just after death as a control, 2) mice that were stored at room temperature for 3 h after death, and 3) mice that were stored at 4Â°C for 18 h after death to resemble the human autopsy process. We also created formalin-fixed paraffin-embedded (FFPE) tissue blocks at the same time using mouse organs obtained under the three conditions described above. We then purified RNA from the FFPE tissue blocks. Furthermore, we performed microarray analysis to examine changes in the gene expression profiles during the postmortem interval in FF samples and to compare ene expression profiles between FF and FFPE samples at three different postmortem times as described for the FF samples.

			Action	DRS
	Distal_1-01.mzML	Mzml
	Distal_1-01.mzid.gz	Mzid
	Distal_1-01.raw	Raw
	Distal_1-02.mzML	Mzml
	Distal_1-02.mzid.gz	Mzid

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Poor Quantitative Concordance of Proteomic Data between Autopsy Samples and Fresh Frozen Samples from Human Coronary Arteries

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