Preserving protein profiles in tissue samples: Differing outcomes with and without heat stabilization

▶ Snap freezing vs. heat stabilization of mouse brain tissue differentially alters PTMs. ▶ Stability of specific protein phosphorylation varies between hippocampus and cortex. ▶ pERK1/2, pELK and pCREB levels do not differ with snap freezing vs. heat stabilization. ▶ Heat stabilization preserves sumoylation patterns from further post mortem change. ▶ Heat stabilization prevents site-specific cleavage of calcineurin and TRKA. Post translational modification (PTM) and proteolytic processing of proteins contributes to regulation of their stability, intracellular localization and interactions with other proteins, and to direct enhancement or repression of their activity. Both PTM and proteolysis are dynamic; levels and rates change in response to changes in the cellular environment. Tissue excision, post mortem interval and subsequent methods of tissue processing for protein analysis unavoidably alter the cellular environment and therefore features of protein profiles. To aid in understanding the time frame and protein specificity of these changes and the biological and technical contributions to them, we have compared features of protein profiles in mouse hippocampus and cortex following three methods of tissue handling: immediate lysate preparation, and rapid heating to 95 °C and standard snap freezing in liquid nitrogen, prior to lysate preparation. In spite of the very short time frames involved, we observe protein-specific differences in levels of phosphorylation, general differences in patterns of sumoylation, and specific differences in levels of proteolytic cleavage of calcineurin and the neurotrophin receptor, TRKA. These differences vary with brain region and with post excision time to processing, and highlight the challenges inherent in accurately profiling the in vivo proteome.