Ylva Elias (Presenter)
Denator AB
Bio: Ylva Elias, M.Sc, is the Director of Sales, US west for Denator. She has a long, and broad, scientific experience both in proteomics, molecular biology and sample handling.
Authorship: Ylva Elias, Karl Sköld, Marcus Söderquist, Mats Borén
Denator AB, Sweden
| Short Abstract Active enzymes rapidly change the composition of biomolecules after sampling. Subsequent analytical results reflect a mix of the in vivo status and degradation products with increased inter-sample variation. The proteome, metabolites, and existing or potential biomarkers are affected. We describe efficient enzyme inactivation and standardization of sample handling using a heat-stabilization technology to eliminate degradation. Comparisons were made to standard snap-freezing and inhibitors. The results show that heat inactivated samples reflect the in vivo status as closely as possible which enables analyses to differentiate true biomarkers from those degradation products found in any situation where cells are under stress. |
Long Abstract
Background
The enzymes in a sample remain active long after sampling and rapidly change the composition of biomolecules. Subsequent analytical results reflect a mix of the in vivo status and degradation products with increased inter-sample variation. Proteins, peptides and post translational modifications (PTM), lipids, metabolites, and existing or potential biomarkers are affected. Effective enzyme inactivation and standardization of sample handling eliminate this problem.
Methods
A heat-stabilization system has been used to generate rapid, homogenous thermal denaturation of proteins, including enzymes, to stop enzymatic degradation in the sample. Comparisons were made to snap-freezing and inhibitors, and in time study manner, compared with different post-mortem intervals. Using mass spectrometry, Western blot, RPPA and activity assays, the protein, peptide, PTMs, and free fatty acid (FFA) content, were examined.
Results
The results show rapid changes in phospho-states on a variety of different proteins detected only minutes after excision whereas after heat-stabilization the phospho-levels remain unchanged during 2 hours in room temperature. In three minutes post-mortem both proteins and endogenous peptides/neuropeptides, including PTM’s, are subjected to substantial degradation. Conversely, amounts and identities of the detected proteins/peptides in heat-stabilized samples show maintained integrity. Similarly, levels of pCREB, pGSK3β and pERK1/2 were unchanged for 2 hours, whereas snap-frozen samples showed a dramatic decrease in levels after 10 min in room temperature. There was a significant increase in FFAs already at 2 min, which continued over time. Heat stabilization was shown to efficiently reduce phospholipase A2 (PLA2) activity and ex vivo lipolysis. Post-sampling effects due to tissue thawing and sample preparation induced a massive release of FFAs (up to 3700%) from non-stabilized liver and brain tissues compared to heat stabilized tissue.
Conclusion
Post-mortem changes may distort our view of in vivo sample molecule composition. Adequate suppression of enzymatic activity is important not only for accurate proteome analyses. Metabolites, phospho-states, FFA and other highly transient molecules in the sample are heavily affected. Heat-stabilization stops activity thereby enables sample analysis to reflect the in vivo status as closely as possible. This approach may be of great help in clinical research to differentiate true biomarkers from those degradation products found in any situation where cells are under stress.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | |
| Board Member | no | |
| Stock | no | |
| Expenses | yes |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |