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Stop activity and preserve the molecular integrity of tissue samples by heat stabilization (779.1)
Author(s) -
Göransson Charlotta,
Söderquist Marcus,
Borén Mats,
Sköld Karl
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.779.1
Subject(s) - in vivo , chemistry , western blot , enzyme , proteome , denaturation (fissile materials) , biochemistry , peptide , endogeny , enzyme assay , biology , microbiology and biotechnology , nuclear chemistry , gene
The action of proteolytic and other protein‐modifying enzymes rapidly change the composition of the proteome and post translational modifications (PTM) after sampling. Subsequent analytical results reflect a mix of the in vivo molecular status and degradation products and display increased inter‐sample variation. Effective enzyme inactivation and standardization of sample handling eliminate this problem. A heat‐stabilization system has been used to generate rapid, homogenous thermal denaturation of enzymes to stop degradation in tissues. 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 and peptide content, including PTM’s were examined. The results show rapid changes in phospho‐states on a variety of different proteins detected only minutes after excision whereas after heat‐stabilization, 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. On the contrary, amounts and identities of the detected proteins/peptides in heat‐stabilized samples show maintained integrity. Similarily, 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. Post‐mortem changes may distort our view of in vivo protein expression. Adequate suppression of both phosphatases and kinases is important for phospho‐state analysis. 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 research on neurodegenerative disease such as Alzheimer and Parkinson to differentiate true biomarkers from those proteins found in any situation where cells are under stress.