Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics

Abstract

After tissue or body fluid sampling, proteases and other protein-modifying enzymes can rapidly change composition of the proteome. As a direct consequence, analytical results will reflect a mix of in vivo proteome and ex vivo degradation products. Vital information about the presampling state may be destroyed or distorted, leading to variation between samples and incorrect conclusions. Sample stabilization and standardization of sample handling can reduce or eliminate this problem. Here, a novel tissue stabilization system which utilizes a combination of heat and pressure under vacuum was used to stop degradation in mouse brain tissue immediately after sampling. It was found by biochemical assays that enzymatic activity was reduced to background levels in stabilized samples. Western blot analysis confirmed that post-translational phosphorylations of analyzed proteins were stable and conserved for up to 2 h at room temperature and that peptide extracts were devoid of abundant protein degradation fragments. The combination of reduced complexity and proteolytic inactivation enabled mass spectrometric identification of several neuropeptides and endogenous peptides including modified species at higher levels compared to nonstabilized samples. The tissue stabilizing system ensures reproducible and rapid inactivation of enzymes. Therefore, the system provides a powerful improvement to proteomics by greatly reducing the complexity and dynamic range of the proteome in tissue samples and enables enhanced possibilities for discovery and analysis of clinically relevant protein/peptide biomarkers.

High Identification Rates of Endogenous Neuropeptides from Mouse Brain

“…5,14 The application of rapid tissue stabilization has been shown to substantially reduce protein fragmentation interference, and importantly, to maintain the levels of neuropeptides in the brain tissue. 5,7,14,37 Our study allowed identifying a large number of unreported peptides, which included peptides with N-and C-terminal modifications and/or dibasic cleavage sites as well as peptide fragments such as truncated peptides. The identification of these novel peptides reflected the overall increased detection capability of our approach.…”

“…2,14 Male mice of 8− 10 weeks old were sacrificed, and then their brains were removed immediately and heat stabilized using Denator irradiation (Denator AB, Gothenburg, Sweden) as described elsewhere. 7 Three mouse brains were pooled and homogenized as entire brain (EB) samples for method comparison. Three samples prepared using the MOC method were further subjected to LC-FT-MS/MS analysis for identification of peptides from EB.…”

Chronic Nicotine Treatment Impacts the Regulation of Opioid and Non-opioid Peptides in the Rat Dorsal Striatum

“…For peptide identification, three naive adult rats were sacrificed by decapitation after anesthetizing with ketamine (100 mg/ kg, Streuli Pharma AG, Uznach, Switzerland)); the brains were then removed and stabilized by using Denator heat irradiation (Denator AB, Gothenburg, Sweden) as described elsewhere (18). Dorsal striatum, hypothalamus, and pre-frontal cortex were dissected from the denaturized brains.…”

“…The main advantage of this approach is that it allows the simultaneous monitoring of many peptides from the same brain tissue derived from a single drug protocol. We used a combination of a robust sample preparation method (18), high accuracy LC-MS analysis (19,20), and the use of multiple synthetic internal standards (21) to compare peptide levels in the DS between chronic nicotine and control animals. Our peptidome analysis determined 14 peptides exhibiting significant changes following chronic nicotine administration.…”

Environmental Stress Affects the Activity of Metabolic and Growth Factor Signaling Networks and Induces Autophagy Markers in MCF7 Breast Cancer Cells

“…A potential caveat in the interpretation of such experiments is that protein phosphorylation is a dynamic modification that can be affected by variables difficult to control including cell confluence, circadian rhythms, shear stress and other types of environmental stresses including exposure to ambient conditions (16 -22). Thus, during the course of an experiment variations or delays in sample retrieval and processing can potentially alter the quantitative characteristics of the phosphoproteome (17,18,22). Similar problems could in principle occur in a clinical environment where several hours may elapse from patient sample collec-tion to processing or preservation (16,17,23).…”