title: 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.
authors.affiliation: Denator AB, Uppsala Science Park, Dag Hammarskjölds väg 32a, 751 83, Uppsala, Sweden. marcus@denator.com
authors.forename: Marcus
authors.initials: M
authors.lastname: Svensson
authors.forename: Mats
authors.initials: M
authors.lastname: Boren
authors.forename: Karl
authors.initials: K
authors.lastname: Sköld
authors.forename: Maria
authors.initials: M
authors.lastname: Fälth
authors.forename: Benita
authors.initials: B
authors.lastname: Sjögren
authors.forename: Malin
authors.initials: M
authors.lastname: Andersson
authors.forename: Per
authors.initials: P
authors.lastname: Svenningsson
authors.forename: Per E
authors.initials: PE
authors.lastname: Andren
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D006358:Hot Temperature; D051379:Mice; D010455:Peptides / Q000032:analysis; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D046888:Tissue Array Analysis / Q000295:instrumentation / Q000379:methods; D014618:Vacuum
chemical_list: D010455:Peptides; D011506:Proteins; D020543:Proteome