Dynamic Changes of the Phosphoproteome in Postmortem Mouse Brains

Abstract

Protein phosphorylation is deeply involved in the pathological mechanism of various neurodegenerative disorders. However, in human pathological samples, phosphorylation can be modified during preservation by postmortem factors such as time and temperature. Postmortem changes may also differ among proteins. Unfortunately, there is no comprehensive database that could support the analysis of protein phosphorylation in human brain samples from the standpoint of postmortem changes. As a first step toward addressing the issue, we performed phosphoproteome analysis with brain tissue dissected from mouse bodies preserved under different conditions. Quantitative whole proteome mass analysis showed surprisingly diverse postmortem changes in phosphoproteins that were dependent on temperature, time and protein species. Twelve hrs postmortem was a critical time point for preservation at room temperature. At 4°C, after the body was cooled down, most phosphoproteins were stable for 72 hrs. At either temperature, increase greater than 2-fold was exceptional during this interval. We found several standard proteins by which we can calculate the postmortem time at room temperature. The information obtained in this study will be indispensable for evaluating experimental data with human as well as mouse brain samples.

Postmortem Brain: An Underutilized Substrate for Studying Severe Mental Illness

“…Different patterns were also observed when the tissues were stored at 4 1C prior to processing, which is the norm for human brain processing. These data indicate that phosphoproteins are variably sensitive to PMI and depend on how quickly the tissues are stored at 4 1C before processing (Oka et al, 2011).…”

“…Postmortem variables such as PMI may also impact phosphorylation (Funk et al, 2012;Li et al, 1996Li et al, , 2003Li et al, , 2005O'Callaghan and Sriram, 2004;Oka et al, 2011). The often transient nature of phosphorylation is well characterized, and in part led to the development of strategies (such as microwave devices) for rapid 'fixation' of brain tissues particularly for proteins with a rapid turnover cycle for phosphorylation/dephosphorylation (O'Callaghan and Sriram, 2004).…”

“…This approach is of course not possible for collection of human brain tissues, and several studies have examined the effects of PMI and other factors on phosphoproteins using rodent tissues. One particularly well-designed study examined 4126 proteins using wholeproteome mass spectrometry analysis (Oka et al, 2011). Adult mice were killed and stored at 25 1C or 4 1C for 0, 3, 12, and 72 hours.…”

Brain-derived neurotrophic factor and TrkB expression in the “oldest-old,” the 90+ Study: correlation with cognitive status and levels of soluble amyloid-beta

“…However, clinicopathologic groups did not differ either in the level of phosphorylation at this site or in the ratio of phosphorylated 3R/4R isoforms. Despite short post mortem delays of the human samples, we cannot exclude that endogenous rapid dephosphorylation, heavily depending on postmortem interval and temperature of stored samples (Matsuo et al, 1994; Oka et al, 2011; Li et al, 2003), affected the results.…”

Efecto de la degradación post mórtem sobre la detección inmunohistoquímica de antígenos en el cerebro de ratón

“…Los procesos de destrucción celular post mórtem desencadenan daños estructurales, cambios en el pH, edema cerebral, alteraciones en epítopos estructurales, en la expresión de proteínas y en la integridad química de componentes de la matriz (12,(15)(16)(17). Esto representa un problema para la preservación de antígenos (16).…”