The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: stathmin 2-20 and peptides as sample quality indicators.
Comparisons of transcriptional and translational expression in normal and abnormal states are important to reach an understanding of pathogenesis and pathophysiology. Maintaining the biochemical, molecular, and structural sample integrity is essential for correct sample comparisons. We demonstrate that both proteins and neuropeptides, including their PTMs, are subjected to massive degradation in the brain already 1 min postmortem. Further, markers for determining the integrity and status of a biological sample were identified. The protein fragment stathmin 2-20 correlated well with the general level of postmortem degradation and may serve as a sample quality indicator for future work, both in animal and human postmortem brains. Finally, a novel method for preventing degradation of proteins and peptides in postmortem tissue is presented using rapid and uniform conductive heat transfer on tissue prior to the actual sample preparation procedures, which enables the relatively low-abundant neuropeptides to remain intact, minimizes degradation of proteins by proteolysis, and conserves the PTMs of the neuropeptides.
Sköld
Karl
K
Department of Pharmaceutical Biosciences, Medical Mass Spectrometry, Uppsala University, Biomedical Centre, Uppsala, Sweden.
Svensson
Marcus
M
Norrman
Mathias
M
Sjögren
Benita
B
Svenningsson
Per
P
Andrén
Per E
PE
D000818:Animals; D015415:Biomarkers; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005629:Frozen Sections; D006801:Humans; D008297:Male; D008872:Microwaves; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009479:Neuropeptides / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis*; D010455:Peptides / Q000032:analysis*; D010766:Phosphorylation; D011180:Postmortem Changes; D040901:Proteomics / Q000379:methods*; D050777:Stathmin / Q000032:analysis*; D013997:Time Factors; D016707:Tissue Fixation
D015415:Biomarkers; D009479:Neuropeptides; D010446:Peptide Fragments; D010455:Peptides; D050777:Stathmin; D020928:Mitogen-Activated Protein Kinases