Assessment of post‐mortem‐induced changes to the mouse brain proteome

Abstract

This study was designed to assess the influence of high-energy head-focused microwave irradiation and the post-mortem interval on measurements of the mouse brain proteome. Difference gel electrophoresis was used to compare mouse brain protein levels in animals killed by decapitation, where the tissue was held at 25 degrees C for selected time intervals post-mortem, and by high-energy head-focused microwave irradiation followed by immediate resection. Microwave-mediated killing was used because it comprehensively snap-inactivates enzymes while largely retaining brain cytoarchitecture. Of the 912 protein spots common to at least eight of 10 gels analyzed, 35 (3.8%) showed significant differences in levels (t-test; p < 0.05) depending on whether animals were killed by microwave irradiation or decapitation. When animals were killed by decapitation, 43 protein spots (4.7%) showed changes in levels over the post-mortem interval (anova; p < 0.05). The vast majority of the near 1000 proteins evident on a 2D gel were stable for up to 4 h. These data have important implications for studies of proteins in the brain, whether based on analysis of tissue derived from animal models or from humans.

Molecular genetic analysis of Down syndrome

“…If properly done, one can detect a statistically signiWcant diVerence in the levels of particular proteins in the order of 10-20%. Clearly, this sensitivity is suYcient to detect changes proportional to gene dosage (Unlü et al 1997;Hunsucker et al 2008).…”

“…For example, for some proteins, especially phosphoproteins, time postmortem can be critically important. Hunsucker et al (2008) compared the brain proteome of mice euthanized by decapitation and rapid dissection with euthanasia using high energy focused microwave application to the brain, which is thought to snap-inactivate enzyme activities in the brain. They found that while most proteins were stable for 4 h, a subset of 3.8% of proteins showed changes depending upon the method of euthanasia, and 4.8% showed changes over the postmortem interval studied (4 h).…”

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

“…The sensitivity of total lysate 2D-DIGE is too low and too few proteins were identified in order to be certain that these spot families encompass all isoforms of any given protein, which seems unlikely anyway. It seems more reasonable that the spot families represent an altered PTM status such as phosphorylations, as described for dynamin-1 in post mortem mouse brain tissue (12). The general level of sample procedure induced differential intensity between the samples, with the exception of some few proteins, lies within Ϯ 100% for the proteomes (Fig.…”

“…5 and 6). Muscle and brain are the most characterized tissues with regard to post mortem degradation, with most proteomics studies focused on brain tissue (1,(3)(4)(5)(11)(12)(13)(14)(15)(16). Several proteins found in the brain degradome were also detected in the liver or pancreas degradome (Table III, IV).…”

“…Post mortem degradation in during PMI is a well known compromising problem when studying endogenous peptides (2,3) and has also been proven to affect the results of polypeptide (here defined as proteins larger than 10 kDa) studies (3)(4)(5)(6)(7)(8). PMI degradation has mainly been studied on human or mouse brain tissue, using two-dimensional electrophoresis (2-DE), SDS-PAGE, and immunoblotting (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). There are also a few proteomic studies on muscle tissue degradation in livestock (13)(14)(15)(16).…”