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Heat stabilize or snap freeze – the story behind heat-stabilization technology
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Biological changes begin from the moment that tissue is removed from its native environment. This traumatic event can have drastic effects at the molecular level within a very short timeframe. Changes range from alterations to phosphorylation states to the start of general protein cleavage through the activity of enzymes such as proteases, phosphatases and kinases. The scientific founders of Denator AB encountered just such a problem when studying endogenous neuropeptides. They recognized that post-mortem changes could be distorting their analysis. They reasoned that rapid heating, rather than conventional snap-freezing, could permanently denature the enzymes causing these changes thus eliminating potential artefacts. |
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Heat stabilization stops enzyme activity that causes post-mortem change. Activity remains in snap-frozen samples, even in the presence of inhibitors. |
Biomarkers or post-mortem changes – the story continues
Recently scientists reported that the same proteins, regardless of tissue or species, are often found expressed differentially in disease states, bringing into question the significance of these proteins as potential biomarkers.1, 2
Scientists at Denator have drawn on data from studies investigating post-mortem changes in proteomic profiles to reveal a remarkable overlap between the proteins commonly identified as changing in these studies and those proteins found to change post-mortem.3
1.Deja vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins. Petrak et al., Proteomics. 2008;8:1744-9
2. Generally detected proteins in comparative proteomics--a matter of cellular stress response?. Wang P. et al., Proteomics. 2009;9:2955-66
3. Biomarkers of disease and post-mortem changes — Heat stabilization, a necessary tool for measurement of protein regulation. Kultima K. et al., J Proteomics. 2011
What do these proteins have in common?When compared with typical levels reported in the literature4 they show a clear over-representation of phosphorylation sites, indicating that the detected regulation of these proteins is due to changes in post-translational modifications after tissue sampling. Full details of this investigation were presented in a scientific poster presented at HUPO 2011 (download) 4. The protein kinase complement of the human genome. Manning G, et al.,Science. 2002 Dec 6;298 (5600):1912-34. Review.
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POSTER Changes in phosphorylation states post-mortem may distort our view of in vivo proteomic profiles Download
(PDF, 1.4 MB)
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The solution
Rapid heat stabilization instantly and permanently stops enzymatic activity, preventing post-mortem changes and thereby revealing results closer to the actual in vivo profile. This approach may help to differentiate true biomarkers from those proteins found in any situation where cells are under stress.
Proteins indicating the risk of post-mortem change
To view a comprehensive list of proteins that may indicate post-mortem changes, click here