Study on Tissue Homogenization Buffer Composition for Brain Mass Spectrometry-Based Proteomics

Abstract

Mass spectrometry-based proteomics aims to study the proteome both qualitatively and quantitatively. A key step in proteomic analysis is sample preparation, which is crucial for reliable results. We investigated the effect of the composition of the homogenization buffer used to extract proteins from brain tissue on the yield of protein extraction and the number and type of extracted proteins. Three different types of buffers were compared—detergent-based buffer (DB), chaotropic agent-based buffer (CAB) and buffer without detergent and chaotropic agent (DFB). Based on label-free quantitative protein analysis, detergent buffer was identified as the most suitable for global proteomic profiling of brain tissue. It allows the most efficient extraction of membrane proteins, synaptic and synaptic membrane proteins along with ribosomal, mitochondrial and myelin sheath proteins, which are of particular interest in the field of neurodegenerative disorders research.

Cfap91-Dependent Stability of the RS2 and RS3 Base Proteins and Adjacent Inner Dynein Arms in Tetrahymena Cilia

“…After incubation, samples were centrifuged for 15 min at 20,000× g and supernatants were discarded. In-solution protein digestion was performed as described [ 43 ] with minor modifications. Briefly, protein pellets were resuspended in 50 µL of 0.1% RapiGest in 500 mmol/L Triethylammonium bicarbonate buffer and reduced with 1,4-dithiothreitol (final concentration 10 mmol/L) for 45 min at 56 °C, then alkylated with iodoacetamide (final concentration 30 mmol/L) for 30 min.…”

A Standardized Protocol for Assuring the Validity of Proteomics Results from Liquid Chromatography–High-Resolution Mass Spectrometry

“…Mass spectrometry-based proteomics has contributed significantly to the detection of drug target identification [ 3 ], and alteration of expression patterns in response to different environmental factors such as diet enriched in selenium compounds [ 4 , 5 ] or fluorinated drugs administration [ 6 , 7 ]. Modern proteomics methods have also recently become useful in neurodegenerative diseases in developing methods for the analysis [ 8 ] and identification of new biomarkers for early diagnosis and disease progression [ 9 , 10 , 11 , 12 ]. Many proteomics studies are also being conducted to identify novel proteins and biological functions in plants [ 13 , 14 ] and on model organisms such as Tetrahymena thermophila to elucidate the involvement of many key proteins in the basis of biological processes [ 15 ].…”