Comparison of Extraction Methods for the Comprehensive Analysis of Mouse Brain Proteome using Shotgun-based Mass Spectrometry

Abstract

This study compares 16 different extraction methods for the comprehensive extraction of mouse brain proteome in combination with "shotgun"-based mass spectrometry (MS). Membrane proteins (MPs) are responsible for a large part of the regulatory functions of the cell and are therefore of great interest to extract and analyze. Sixteen protein extraction protocols were evaluated in regards to protein yield and number of identified proteins with emphasis on MPs. The extracted proteins were delipidated, on-filter digested, and analyzed by reversed phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQ-FT mass spectrometer. Detergent-based lysis buffers showed higher efficiencies and yields in the extraction of proteins from the brain tissue compared to solubilization with organic solvents or organic acids. The detergent octyl-β-D-glucopyranoside gave the highest number of identified proteins (541) as well as numbers and percentages of identified MPs (29%). Detergent-based protocols are the best sample preparation tools for central nervous system (CNS) tissue and can readily be applied to screen for candidate biomarkers of neurological diseases.

New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers

“…Taking into account the reported distinct subcellular localizations of C9ORF72 isoforms with localization of C9-S to the nuclear membrane and C9-L in the cytoplasm (Xiao et al, 2015), lysis protocols were investigated. We found that Triton X-100 facilitated the extraction of the C9ORF72 proteins, in agreement with higher extraction yield reported with detergent based protocols in comparison to organic solvents or chaotropic reagents such as urea, in fatty tissue such as the brain (Shevchenko et al, 2012). In addition, the Triton X-100 protein extraction protocol probably enhanced the subsequent trypsin digestion as previously reported (An et al, 2015).…”

The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

“…For this comparative study we selected an in gel digest method adapted from 19 , conducted in combination with an SDS-based and native protein extraction, two filter-aided ( FASP (Filter Aided Sample Preparation) adapted from 32 and a recent enhancement termed eFASP adapted from 33 , and two in solution procedures (RapiGest, adapted from 22 , and RapidACN adapted from 16 ). Their characteristics are summarized in Figure 1.…”

“…For both protocols the digestion was performed directly on filters (Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-3 membrane, Millipore). The FASP procedure ( Supplementary protocol 3) involved a treatment with endoproteinase Lys-C (Promega) prior to digestion with trypsin 20 , while the eFASP protocol ( Supplementary protocol 4) required protein precipitation using tri-n-butylphosphate/acetone/methanol mix (1:12:1) for lipid removal before digestion 21 . For in solution digest protocols ( Supplementary protocol 5 and Supplementary protocol 6) protein extraction was performed either in 200 µl lysis buffer (0.1 M NaOH, 0.05 M EDTA, 2% SDS, 2% β-mercaptoethanol) (RapiGest) or 0.05 M ammonium bicarbonate (RapidACN) 20 and using heat or glass-bead lyses on a Fast-Prep 24 instrument (MP Biomedicals), respectively.…”

“…We compare popular sample protocols that are based on in gel 19 , filter-aided 20, 21 and in solution 9, 22 digestion procedures. Processing identical proteome samples obtained from budding yeast, and acquiring proteomic data without further prefractionation on two LC-MS/MS platforms, these methods were compared by their performance in sample preparation, their precision in label-free quantification experiments and their effectiveness in terms of time and reagents.…”

Comprehensive proteome analysis of fresh frozen and optimal cutting temperature (OCT) embedded primary non-small cell lung carcinoma by LC–MS/MS

“…The average of median peak widths from two technical replicates was approximately 38 s with 75% identified peptides eluted within 50 s. Based on this data, we tested the effect of 4 different DE duration settings: 15 s, 30 s, 50 s, and 90 s, on the number of identified peptides ( Table 2). The number of peptide identifications rose by nearly 18% when the DE duration increased from 15 s to 50 s, but There are other parameters in our protocol that were not necessarily fully optimized including the selection of protein exaction lysis buffers [21,24], enzymes and protocols for protein digestion [25][26][27], and peptide fractionation methods [28][29][30].…”