Abstract
Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.
Highlights
IntroductionThis study investigated the effects of post mortem degradation in pancreas and liver
From the ‡Department of Pharmaceutical Biosciences, Division of Toxicology, BMC, Box 594, SE-75124 Uppsala University, Sweden, §Denator AB, Uppsala Science Park, Dag Hammarskjolds vag 32a, SE-75183, Uppsala, Sweden, ¶Department of Medical Sciences, Clinical Pharmacology, Uppsala University Hospital, SE-751 85 Uppsala, Sweden, ʈCreate Health, Department of Immunotechnology, BMC, Lund University, SE-221 84 Lund, Sweden, **Molecular Biometry Group, Institute for Cell and Molecular Biology, Box 583, BMC, Uppsala University, SE-75123, Uppsala, Sweden, ‡‡Department of Biological Chemistry, Padova University, 3-35131 Padova, Italy, ¶¶Department of Pharmaceutical Biosciences, Division of Medical Mass Spectrometry, Box 583, BMC, Uppsala University, SE-75123 Uppsala, Sweden
We and others have previously explored the effect of focused microwave irradiation with regard to sample quality, demonstrating that this method is more reliable than snap freezing in liquid nitrogen, especially with regard to posttranslational modification (PTM) stability [2, 3, 17,18,19,20]
Summary
This study investigated the effects of post mortem degradation in pancreas and liver. Both tissues are well studied because of their multiple functions in the body and their involvement in different diseases such as diabetes or hepatocarcinoma. Pancreas is especially interesting in this context as it displays endocrine secretion of peptides, and exocrine secretion of digestive enzymes, the later making it a protease rich tissue We used both two-dimensional difference in gel electrophoresis (2D-DIGE) and label free liquid chromatography mass spectrometry (LC-MS) based differential peptide display [2, 18], the later to better investigate changes in small molecular fragment that are not detectable by gel-based methods. We used both two-dimensional difference in gel electrophoresis (2D-DIGE) and label free liquid chromatography mass spectrometry (LC-MS) based differential peptide display [2, 18], the later to better investigate changes in small molecular fragment that are not detectable by gel-based methods. 2D-DIGE is an unrivaled methodology to characterize alterations in isoform patterns, which is an important aspect considering that post-translational modifications (PTMs) such as phosphorylations are especially sensitive to post mortem influence within a few min-
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