Assessing quantitative post‐mortem changes in the gray matter of the human frontal cortex proteome by 2‐D DIGE

Abstract

The number of proteomics studies concerning human brain samples has been increasing in recent years, in particular in the discovery of biomarkers for neurological diseases. The human brain samples are obtained from brain banks, which are interested in providing high quality human nervous tissue. In order to provide brain banks as well as scientists working in the proteomics field with measures for tissue quality, the critical factors after death, the effect of post-mortem interval (PMI) and storage temperature on the human brain proteome were investigated. This study was focused on the gray matter of the frontal cortex. The PMI was artificially prolonged from the time of autopsy (2 h after death) by storing samples at 4 degrees C or room temperature over 18, 24, and 48 h. The samples were analyzed by 2-D DIGE using a pH 4-7 gradient, revealing a time course of quantitative protein changes. The degradation of three proteins, peroxiredoxin-1, stathmin, and glial fibrillary acidic protein were further confirmed by Western-blot analysis. Proteins vulnerable to PMI were analyzed by the 2-D DIGE analysis of cortex samples from three donors, and were derived from a variety of functional groups, including metabolic, structural, stress response, antioxidants, synaptosomal, and neuronal proteins.

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

“…For example, the enkephalin neuropeptides (met-enk-RF and met-enk-RSL) were clearly detected in the stabilized samples and almost undetectable in the snap-frozen samples. Interestingly, the isotopically labeled neuropeptides added as internal standards were only detected in stabilized tissue, aside from the labeled protein fragment stathmin (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) which was detected independent of sample treatment, indicating ex vivo proteolytic activity and the relative stability of the stathmin (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) fragment.…”

“…Mice in a fourth sample group (n ) 4) were killed by focused microwave irradiation (1.4 s at 4.5-5 kW, Muromachi Kikai, Tokyo, Japan) which denature the proteins before dissection and homogenization. A microtip ultrasonicator (Vibra-Cell, Sonics & Materials) was used to homogenize the tissue in 5× the sample weight of 0.25% HAc extraction solution containing 40 mM of isotopically labeled stathmin (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), met-enkephalin, neurotensin, substance P (1-11), and substance P (1-7) as internal standards. The crude extract was centrifuged at 20 000g for 30 min at 4°C to pellet cell debris and ultrafiltered using a 10 kDa centrifugal cutoff filter (Microcon YM-10, Millipore) to isolate the peptide content.…”

“…However, it has been shown that, following tissue sampling, substantial alterations of the proteome occurs. [3][4][5][6][7] Removal of a sample from its natural surrounding leads to major disturbance of the tissue homeostasis which rapidly causes release of mediators of degradation 8 which alters the levels and composition of proteins and posttranslational modifications (PTMs). Until recent advances in proteomics and mass spectrometry (MS), some issues regarding tissue preservation prior to sample preparation and analysis have been overlooked.…”

High Identification Rates of Endogenous Neuropeptides from Mouse Brain

“…It served as a data alignment and analysis tool with following parameters: m/z range from 300 to 1500 Da, time frame 5 min, m/z frame 0.01 Da with a peak intensity threshold 100 000. [DTyr, 27,36 D-Thr 32 ]-Neuropeptide Y (AA27−36) was used as internal standard for quality control. The normalized intensities of the 2000 most intensive features were plotted against their corresponding retention time (Rt).…”

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

“…Post mortem degradation in during PMI is a well known compromising problem when studying endogenous peptides (2,3) and has also been proven to affect the results of polypeptide (here defined as proteins larger than 10 kDa) studies (3)(4)(5)(6)(7)(8). PMI degradation has mainly been studied on human or mouse brain tissue, using two-dimensional electrophoresis (2-DE), SDS-PAGE, and immunoblotting (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). There are also a few proteomic studies on muscle tissue degradation in livestock (13)(14)(15)(16).…”