These experiments directly compared the effects of heat stabilization using the Stabilizor T1 vs. snap freezing and fresh processing for the preservation of protein features in mouse brain tissue. PTMs in general are dynamic and reversible, and changes in phosphorylation and sumoylation are expected to occur in brain as a result of ischemia and hypoxia following death as cells alter the activity levels and targets of their endogenous phosphatases, kinases and sumo ligases and proteases. In all three protocols, the time between sacrifice and tissue preservation (by heat, freezing or lysis) was short (). Common to all protocols is the ~30 seconds required to remove the brain from the skull. Completion of whole brain heat stabilization required a further 50 seconds, during which time the brain was being heated to 95°. Dissection of brain regions in the other two protocols, whether to freezing in liquid nitrogen or to lysis buffer for fresh processing, required 90 seconds, during which time the brains were held on ice. Given differences in time and temperature, it is therefore reasonable that cellular and enzymatic responses will differ during processing and result in different endpoint protein profiles. Notably, none of these protocols, not even FSH, can be claimed to faithfully reflect the in vivo state for every protein. One general interpretation, for example, is that phosphorylation rapidly increases in FZ and FSH tissues in the initial two minutes post euthanasia due to the time required for dissection (on ice) and an imbalance in enzyme activities that favors kinases over phosphatases. These changes are either different (favoring phosphatases over kinases) or more limited in stabilized tissues, due to the shorter time frame and higher temperatures, giving rise to the significantly lower levels of specific phosphorylation at time 0 in stabilized vs. non-stabilized tissues shown in . It is not possible to distinguish between these two scenarios. During a 30 minute room temperature incubation, in non-stabilized tissues, enzyme activity levels continue to change. In stabilized tissues, however, enzymes are largely inactivated, and further phosphorylation profile changes are very limited. Consistent with the latter observation, Svensson et al (2009)
, in evaluation of the Stabilizor T1, demonstrated residual phosphatase activity of approximately 10% relative to non-stabilized tissue in the presence of phosphatase inhibitors. While this seems negligible, if it is tissue and protein-specific, it could give rise to the decreases in phosphorylation levels of RSK, AKT and JNK listed in .
Analysis of protein feature preservation in other studies (Svensson et al 2008; O'Callahan and Sriram 2004) has focused on phosphorylation profiles. Here, we have shown that sumoylation profiles also change with post mortem interval and tissue handling. This is not inconsistent with the observations of dramatic changes in sumolyation profiles in response to oxidative stress (Manza et al., 2004
). We also show apparent site-specific processing of a subunit of the protein phosphatase, calcineurin, and of the nerve growth factor receptor, TRKA. It remains to be determined if these are biologically programmed responses to post mortem intervals and temperature changes, and indeed, the same cleavages as reported under other conditions (Lee et al 2007
; Diaz-Rodriguez et al 199
), or if one or both are technical artifacts due to release of normally sequestered proteases.
Time delays between death (and attendant cell molecular responses to ischemia and hypoxia) and protein preservation are unavoidable. The data presented here make clear that unambiguous determination of in vivo levels of protein modification and processing is a challenge and that different protocols for tissue handling and protein preparation will result in significantly different protein profiles. In evaluating the relative efficacy of these protocols, three recent observations from analysis of human and mouse tissues are relevant. First, Ferrer et al (2007)
showed that tissue samples from human autopsies stored at 0°C had higher levels of specific phosphorylation that remained stable for longer time periods (several hours to several days), in comparison with tissues stored at 4°C. Second, Espina et al (2008)
, in analysis of eleven different normal or tumor tissue biopsy samples, showed that kinases remained active, and contrary to an expected rapid loss of phosphorylation at room temperature, for most protein phosphorylation examined, levels increased significantly over tens of minutes before decreasing after a few hours. Lastly, Snyder et al (2007)
showed that treatment of mice with common general anesthetics altered phosphorylation of numerous neurologically relevant proteins, among them ERK2, DARRP32 and the NR1 subunit of the NMDA receptor, in a brain region-specific fashion. Notably, they used focused microwave irradiation to euthanize the mice; given the potential for inducing stress responses in mice in application of this method, use of preliminary anesthesia might be attractive, but clearly would add to variation in the protein profiles.
Heat stabilization appears to rapidly and effectively inactivate a broad range of protein modifying and processing enzymes. However, there are additional practical considerations to its use. We noted protein-specific differences between hippocampus and cortex in responses to heat stabilization, and therefore additional differences may exist in similarly treated non-brain tissues. Multiple tissues from a single animal cannot be stabilized within the same time frame. The alternative is rapid freezing of these tissues in liquid nitrogen, to be followed at a later time by stabilization. A second consideration is that heat stabilization, whether with the Stabilizor T1 or focused microwaves, in denaturing proteins, destroys intracellular structures (although NOT tissue morphology); while this does not affect tissue dissection, either manually as used here or by laser capture microdissection, it precludes subcellular fractionation, thus limiting some types of studies. Lastly, the short time delays, <2 minutes, used here are practical only for laboratory animals. For analysis of human samples, collected at biopsies or autopsies, time delays will be longer, variable and uncontrollable. Rather than reliable measurement of in vivo levels, a more reasonable goal will be consistent timing and treatment of tissue samples to generate consistent post mortem or post excision alterations. Maintenance of samples on ice during delays required by pathology followed by stabilization should provide more consistent results. It must be recognized that post mortem and post excision delays may have altered some patterns of disease vs. normal differences. This may be of particular note if the disease phenotype includes mutation or variation in protein modification or processing enzymes. These abnormalities could in theory be obscured or exacerbated by time frame and method of protein lysate preparation.