Assessing the use of thermal treatment to preserve the intact proteomes of post‐mortem heart and brain tissue

Abstract

Protein degradation that occurs in tissue during post-mortem interval or sample preparation is problematic in quantitative analyses as confounding variables may arise. Ideally, such artefacts should be prevented by preserving the native proteome during sample preparation. We assessed the efficacy of thermal treatment (TT) to preserve the intact proteome of mouse heart and brain tissue in comparison to standard snap-freezing with liquid nitrogen (LN). Tissue samples were collected, either snap frozen (LN), subjected to TT, or snap frozen followed by thermal treatment, and subsequently analysed by 2-DE. In heart tissue, following quantitative image analysis, we observed 77 proteins that were significantly altered across the three treatment groups (ANOVA, p<0.05). Principal component and clustering analyses revealed LN and TT to be equally beneficial. These findings were confirmed by MS identification of the significantly altered proteins. In brain tissue, 189 proteins were significantly differentially expressed across the three treatment groups (ANOVA, p<0.05). Brain tissue appeared to be more responsive to TT than heart and distinct clusters of differentially expressed proteins were observed across treatments. Overall, TT of brain tissue appears to have beneficial effects on protein stabilisation during sample preparation with preservation of high-molecular-weight proteins and reduction in protein fragmentation.

Improved identification and quantitation of mature endogenous peptides in the rodent hypothalamus using a rapid conductive sample heating system

“…25,30,45,62 The advantages were observed for both fresh and frozen tissues collected from different animal models. We were also interested in knowing whether or not full-length endogenous neuropeptides can be as effectively recovered from tissues frozen for transportation or storage, followed by ST1 treatment.…”

The Impact of Biosampling Procedures on Molecular Data Interpretation

“…Although there are differences between species, there very likely remains some similarity in underlying stress-adaptive mechanisms. Proteins identified in the hypoxic/anoxic tissue of ectothermal organisms (41,42) overlap with those identified in BT methodology studies (10,12,14,17), for example glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase, triose-phosphate isomerase, dihydropyrimidinase-related protein 3, and voltage-dependent anionselective channels.…”

“…This image is not as clear with larger proteins (Ͼ10 kDa). Proteomic profiles derived from SFIϩMCI neural tissue and in vitro yeast biosamples demonstrate an increase in low molecular weight proteins and a reduction in high molecular weight proteins (some of the former being fragments of the latter), a trend absent in SFI-handled biosamples from heart, liver, and pancreatic tissue (8,10,28). The neural tissue degradation profile is possibly a reflection of its high sensitivity to ischemia and/or the dissipation of ion gradients and subsequent rise in Ca 2ϩ concentration (causing an elevation of Ca 2ϩ protease activity) (77,82).…”

“…Overall, there are major differences between SFIϩMCI and heat stabilization at the neuroproteomic level (Figs. 1B and 2A) (8,10,12). In less anoxia-sensitive human uterine biopsies (2), a number of signaling pathways (including PI3K-Akt and Ras-ERK) have increased phosphorylation levels during the first 30 min of BTI before eventually decreasing.…”

“…The neuroprotective effects invoked by anesthesia against ischemia/reperfusion damage (5, 6) also indicate that cells initially try to adapt to the hypometabolism and/or hypothermia associated with biosampling. The mechanisms underlying such physiological changes need to have an extremely short response time and are therefore heavily dependent on enzymatic processes, singling out the proteome as being particularly sensitive to the choice of both sampling methodology and type of tissue (7)(8)(9)(10)(11)(12). A few minutes difference in sampling time can, for instance, have profound effects on the peptidome (here defined as all proteins Ͻ10 kDa) (13,14), yet it easily remains within the acceptable time frame for sampling in most biomedical research.…”