Comparison of Extraction Methods for the Comprehensive Analysis of

Article pubs.acs.org/jpr

Comparison of Extraction Methods for the Comprehensive Analysis of Mouse Brain Proteome using Shotgun-based Mass Spectrometry Ganna Shevchenko,* Sravani Musunuri, Magnus Wetterhall, and Jonas Bergquist* Department of Physical and Analytical Chemistry, Analytical Chemistry, Uppsala University, Uppsala, Sweden S Supporting Information *

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 (RPnanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQFT 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%). Detergentbased 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. KEYWORDS: brain, proteomics, membrane proteins (MPs), transmembrane proteins (TMPs), mass spectrometry (MS), sample preparation, extraction

1. INTRODUCTION The brain is unquestionably the most fascinating and complex organ of all higher organisms. It controls most of the body's activities, regulates the body’s actions, functions, thoughts, and emotions, and more than 1000 different disorders have been associated with dysfunction of the brain and nervous system.1 Therefore, the study of the central nervous system (CNS) is of the highest interest in medical and biochemical research. Despite that great progress has been made in brain research, comprehensive analysis of brain membrane-bound proteins (MPs) remains a challenge. These proteins play vital roles in various physiological processes in the cell, including signal transduction, intercellular communication, biogenesis, cell motility and molecular transport. Approximately 30% of the mammalian genome encodes for membrane proteins,2−4 whereas 60% of all approved drug targets in neuroscience research are represented by this class of proteins.5 MPs are associated with various human diseases, such as Alzheimer’s disease,6−8 Parkinson’s disease,9 diabetes,10,11 Down’s syndrome8,12,13 and schizophrenia.8,14 These diseases among others highlight the importance of studying membrane proteins and their potential roles in disease pathology to find new targets for diagnostics and therapeutics. © 2012 American Chemical Society

The analysis of the by nature hydrophobic membrane proteins is a difficult challenge and it is therefore of a continuous need to develop techniques allowing extraction, detection, and characterization of the proteins. The predicaments start with the solubilization and extraction of proteins from the membranes in the tissue. The under representation of MPs in proteome studies is mainly attributed to the heterogeneous, hydrophobic, and naturally low abundance of these proteins.8,15−19 To overcome these limitations, various strategies have been applied to extract, enrich, and solubilize MPs using detergents,11,20−27 organic solvents,28−32 organic acids,33 and salt buffers.34 A high concentration (up to 90%) of formic acid has been reported to effectively solubilize membrane proteins.33 In this method, cyanogen bromide was used to cleave the proteins under acidic conditions. Alternative ways to extract MPs is by intermittent vortexing and sonication in 60% organic solvents, followed by trypsin digestion in the organic-aqueous solvent mixture.28−32 Wu et al.34 have demonstrated the application of proteinase K at a high pH Received: November 25, 2011 Published: February 22, 2012 2441

dx.doi.org/10.1021/pr201169q | J. Proteome Res. 2012, 11, 2441−2451

Journal of Proteome Research

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Sigma Aldrich and trypsin (sequencing grade from bovine pancreas 1418475; Roche diagnostic, Basel, Switzerland) were used. Sucrose was purchased from Fisher Scientific Company (Göteborg, Sweden). Sodium dodecyl sulfate (SDS) and Triton X-114 were obtained from KEBO Lab (Stockholm, Sweden). Ultrapure water was prepared by Milli-Q water purification system (Millipore, Bedford, MA).

for direct digestion of proteins from crude membranes. However, among the most commonly used approaches for the studies of MPs are the use of in-solution solubilization of the crude membranes with surfactants11,20−27 followed by enzymatic digestion, separation, and analysis of the peptide mixture using liquid chromatography (LC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Detergents are amphiphilic and contain a polar group at the end of a long hydrophobic carbon chain. The majority of the lipids in CNS membranes contain two hydrophobic groups connected to a polar head, which can be viewed as biological detergents. Added detergents solubilize the membrane proteins by mimicking the lipid-bilayer environment. Micelles and other organized amphiphilic assemblies are increasingly utilized in analytical and bioanalytical chemistry.20−27,35−37 Separation and preconcentration of proteins based on cloud point extraction (CPE) has shown great practical application in the analysis of CNS tissue.26,27,35−37 The CPE technique is based on nonionic polyoxyethylene detergents in aqueous solutions that form micelles and become turbid when heated to their cloud point temperature (CPT). Above the CPT, the micellar solution separates into a surfactant-rich phase of a small volume and in a diluted aqueous phase, in which the surfactant concentration is close to the critical micellar concentration (CMC). Any analyte solubilized in the hydrophobic core of the micelles will separate and become concentrated in the small volume of the surfactantrich phase. Recently, our group successfully demonstrated the use of CPE for the simultaneous extraction and enrichment of both the membrane and hydrophilic proteins from porcine and mouse brain tissue.26,27 In this study, a broad spectrum of previously reported and inhouse developed protocols for the extraction and subsequent analysis of membrane proteins originating from brain tissue were compared. Mouse brain extracts were prepared using different solubilizing solutions containing either different detergents (Triton X-114, Triton X-100, sodium dodecyl sulfate (SDS), n-octyl-β-D-glucopyranoside (β-OG), 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)); or three organic solvents (methanol, isopropanol, acetonitrile); or aqueous phosphate buffers; or high concentrations of formic acid. The extraction protocols were evaluated while keeping all other analytical steps are constant. The extracted brain proteins were visualized on 1D gels or analyzed by a shotgun based “bottom up” nanoLC−MS/MS proteomic approach for protein separation and identification.

2.2. Brain Samples

Mouse brains were purchased from B&K, Sollentuna, Sweden. The mice were anaesthetized with CO2 and killed by cervical dislocation. The brains were dissected out, frozen in liquid nitrogen and stored at −80 °C. Prior to extraction, the brains were homogenized in liquid nitrogen and the brain powder was stored at −80 °C. Experiments with mice brain samples were approved by the local ethical committee (Uppsala University) and by the Swedish Committee for Ethical Experiments on the Laboratory Animals (S93/92 and S77/94, Stockholm, Sweden). 2.3. Extraction of Membrane Proteins from Mouse Brain Tissue

Two sets of lysis buffers denoted as set A and B were prepared for the extraction of the proteins from the brain tissue (Figure 1). Set A buffers were prepared according to our in-house developed protocols (A). Set B buffers were prepared according to the protocols described in different scientific publications (B). The complete lysis buffer compositions are shown in the Table 1. 2.4. Protein Extraction

Aliquots of 50 mg mouse brain powder were homogenized for 60 s in a blender (POLYTRON PT 1200, Kinematica) with 1 mL of lysis buffer. Protease inhibitor cocktail (10 μL) was added during the sample preparation to prevent protein degradation. After homogenization, the samples were incubated for 1 h at 4 °C during mild agitation. The cell lysates were clarified by centrifugation for 30 min (10000× g at 4 °C) using a Sigma 2K15 ultracentrifuge (Sigma Laborzentrifugen GmbH, Osterode, Germany). The supernatant was collected and further processed. 2.5. Delipidation and Protein Precipitation

Aliquots of 100 μL extracted sample with the organic solvents in the lysis buffers were first lyophilized using speedvac and then further subjected to delipidation. A delipidation protocol according to Mastro et al. was used.26,38 Aliquots (100 μL) of the extracts were mixed with 1.4 mL of ice-cold tri-nbutylphosphate/acetone/methanol mixture (1:12:1) and incubated at 4 °C for 90 min. The precipitate was pelleted by centrifugation for 15 min (2800× g at 4 °C) and then washed sequentially with 1 mL of TBP, 1 mL of acetone and 1 mL of methanol, and finally air-dried.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Reagents

2.6. Protein Quantification

Acetonitrile (ACN), methanol (MeOH), acetic acid (HAc), formic acid (FA), ammonium bicarbonate (NH4HCO3), tri-nbutylphosphate (TBP), sodium chloride (NaCl) were obtained from Merck (Darmstadt, Germany). 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was purchased from Fluka (Steinheim, Germany). Triton X-114, acetone, ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA), protease inhibitor cocktail, phosphate buffered saline (PBS), Tris-HCl, 4(-2-hydroxyethyl)- 1 piperazineethanesulfonic acid (HEPES), N-octyl-β-D-glucopyranoside, and trifluoroacetic acid (TFA) were purchased from Sigma Aldrich (St. Louis, MO). For tryptic digestion, iodoacetamide (IAA), urea and dithiothreitol (DTT) were obtained from

The total protein content of delipidated proteins was determined using the DC Protein Assay Kit (BioRad Laboratories, Hercules, CA), which is based on the modified Lowry method with bovine serum albumin as standard.39 The protein pellets were redissolved in 100 μL of 6% SDS. The DC assay was carried out according to the manufacturer’s instructions using 96-well microtiter plate reader model 680 (BioRad Laboratories). The total protein concentrations of different extraction protocols are listed in Table 2. 2.7. One-dimensional Gel Electrophoresis

Samples corresponding to 100 μL of the extracts were delipidated according to section 2.5. The protein pellets were 2442



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Figure 1. Schematic diagram of experimental setup used in the study. The mouse brains were first homogenized and then extracted with the protocols given in Table 1. Extracted protein samples were delipidated and then analyzed by proteins assay, 1D gel electrophoresis and on-filter digestion followed by nanoLC−MS/MS analysis. Finally, the subcellular location and function of the identified proteins were elucidated and compared.

Table 1. Composition of Different Lysis Buffers no 1

name of lysis buffer

Table 2. Total Concentration of Mouse Brain Proteins (μg/μL) Extracted Using Different Detergent Lysis Buffers (n = 3)a

composition

ACN (A)

10 mM Tris-HCl pH 7.4, 0.15 M NaCl, 1 mM EDTA and PBS containing 60% ACN 2 Methanol (A) 10 mM Tris-HCl pH 7.4, 0.15 M NaCl, 1 mM EDTA and PBS containing 60% Methanol 3 Methanol (B)31 60% Methanol, 50 mM NH4HCO3 4 Isopropanol (A) 10 mM Tris-HCl pH 7.4, 0.15 M NaCl, 1 mM EDTA and PBS containing 40% Isopropanol 5 Isopropanol (B)32 40% Isopropanol 6 Formic acid (A) 10 mM Tris-HCl pH 7.4, 0.15 M NaCl, 1 mM EDTA and PBS containing 1% formic acid 7 Potassium 100 mM K2HPO4/KH2PO4 pH 6.7, 5 mM MgCl2, phosphate (B)34 250 mM Sucrose 8 CHAPS (A) 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1 mL PBS containing 1% CHAPS 9 CHAPS (B)11 250 mM sucrose, 10 mM HEPES, 1 mM EDTA, 1 mL PBS containing 1% CHAPS 10 Triton X-100 (A) 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, and PBS containing 1% (v/v) Triton X-100 11 Triton x-100 (B)21 20 mM Tris- HCl pH 7.4, 100 mM NaCl, 0.05% Triton X-100, 1 mM EDTA 12 SDS (A) 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, and PBS containing 1% (v/v) SDS 13 SDS (B)23 4% (w/v) SDS, 100 mM Tris/HCl pH 7.6 14 β-OG (A) 10 mM Tris-HCl pH 7.4, 0.15 M NaCl, 1 mM EDTA and PBS containing 1% β-OG 15 β-OG (B)22 6 M urea, 2 M thiourea, and 1% β-OG in 10 mM Tris-HCl 16 Triton X-114 10 mM Tris-HCl pH 7.4, 0.15 M NaCl, 1 mM EDTA (A)26 and PBS containing 1% (v/v) Triton X-114

set A lysis buffer Organic solvents

Organic acid Salt buffer Detergents

a

Acetonitrile Methanol Isopropanol Formic acid Phosphate buffer CHAPS Triton X-100 SDS β-OG Triton X-114

set B

C, μg/μL

SD

C, μg/μL

SD

0.10 0.22 0.61 0.41 n/a 2.62 2.80 4.22 3.13 3.69

0.02 0.03 0.04 0.05 n/a 0.05 0.02 0.04 0.02 0.02

n/a 0.21 0.51 n/a 1.41 2.50 2.00 3.68 4.89 n/a

n/a 0.02 0.02 n/a 0.03 0.04 0.03 0.04 0.06 n/a

n/a − not available.

was loaded into each lane of a 18-well (30 μL), 4−12% Bis-Tris Criterion XT Precast Gel (BioRad Laboratories). To avoid overloading of the gel, a volume of 5 μL of samples extracted using detergent lysis buffers were loaded into each lane of gel. The separation was performed with MOPS-buffer at 200 V constant for 60 min. The proteins were fixed for 20 min in 40% methanol containing 10% acetic acid and stained with colloidal Coomassie blue R-250 (Bio-Rad) for 40 min. Finally, the gels were destained 40% methanol containing 10% acetic acid and scanned with a HP scanner (HP Scanjet G3010 Photo Scanner).

redissolved in 25 μL of XT sample loading buffer (BioRad Laboratories) and 55 μL of Milli-Q water. A volume of 10 μL of 45 mM aqueous DTT was added and the samples were heated at 95 °C for 5 min to reduce the disulfide bonds. The samples were cooled to ambient temperature and 10 μL of 100 mM aqueous IAA was added and the mixtures were incubated for 15 min in darkness at room temperature to carbamidomethylate the cysteines. A volume of 20 μL of the samples extracted using organic solvents, organic acid and phosphate lysis buffer

2.8. On-filter Digestion Followed by nanoLC−MS/MS Analysis

2.8.1. On-filter Tryptic Digestion of Proteins at Normalized Amounts. A normalized protein amount corresponding to 35 μg was delipidated according to section 2.5. An on-filter digestion protocol adopted from Wisniewski et al. was used for tryptic digestion of the samples23 using 3 kDa filters (Pall Life Sciences, Ann Arbor, MI). Centrifugation was carried out at a centrifugal force of 14000× g throughout the 2443



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protocol. The samples were first redissolved in 100 μL of 50:50 ACN/8 M urea +1% n-octyl-β-D-glucopyranoside. A volume of 10 μL of 45 mM aqueous DTT was added to all samples and the mixtures were incubated at 50 °C for 15 min to reduce the disulfide bridges. The samples were cooled down to room temperature and 10 μL of 100 mM aqueous IAA was added and the mixtures were incubated for an additional 15 min at room temperature in darkness to carabamidomethylate the cysteines. The samples were transferred to spin filters that had been prewashed with 250 μL of 50% ACN for 15 min and then 500 μL of water for 20 min. The samples were then centrifuged for 10 min to remove the added salts, detergents and other interfering substances. An additional volume of 100 μL of 2% ACN in 50 mM NH4HCO3 was added and the filters were spun for 10 min followed by 100 μL of 50:50 ACN/50 mM NH 4 HCO 3 and 100 μL of 50 mM NH 4 HCO 3 , and centrifugation for another 10 min. Finally, a volume of 100 μL of 50 mM NH4HCO3 was added together with trypsin to yield a final trypsin/protein concentration of 2.5% (w/w). The tryptic digestion was performed at 37 °C overnight in darkness. The samples were then centrifuged for 20 min to collect the tryptic peptides in the filtrate while retaining undigested proteins and trypsin in the retentate. An additional volume of 100 μL of 50% ACN, 1% HAc was added and the filters were spun for 10 min and pooled with the first tryptic peptide filtrate. The collected filtrates were vacuum centrifuged to dryness using a Speedvac system ISS110 (Thermo Scientific, Waltham, MA). Prior to nanoLC−MS/MS analysis, the samples were redissolved in 0.1% TFA to yield an approximate tryptic peptides concentration of 0.3 μg/μL. 2.8.2. On-filter Tryptic Digestion of Proteins at Normalized Volumes. Aliquots (100 μL) of mouse brain samples extracted using detergent lysis buffers were delipidated according to the section 2.5 and redissolved in 100 μL of the digestion buffer (50:50 ACN/8 M urea +1% n-octyl-β-Dglucopyranoside). Twenty-five microliters of each sample was subjected to on-filter digestion according to the section 2.8.1. 2.8.3. Solid Phase Extraction (SPE). All extracts were checked on MALDI-TOF MS prior to the LC−MS/MS analysis to test for interfering compounds that could ruin the LC column and separation. MALDI-TOF MS analysis revealed that protein digests after extraction with phosphate lysis buffer contained salts and some interfering compounds. Therefore, the extracts with phosphate lysis buffer were subjected to desalting after the digestion step using an Isolute18 a (EC) (1 mL, 50 mg capacity, Biotage, Uppsala, Sweden) SPE column using the following schedule; The column was first wetted in 300 μL of 100% ACN and equilibrated with 5 × 1 mL 1% HAc. The tryptic peptides were adsorbed to the media using 5 repeated cycles of sample loading. The column was washed using 5 × 1 mL of 1% HAc and finally the peptides were eluted in 250 μL 50% ACN, 1% HAc. After desalting, the eluate was vacuum centrifuged to dryness and redissolved in 0.1% TFA to yield an approximate tryptic peptide concentration of 0.3 μg/μL prior to nanoLC−MS/MS identification. 2.8.4. NanoLC−MS/MS for Protein Identification. The MS experiments were performed using a 7 T hybrid LTQ FT mass spectrometer (ThermoFisher Scientific, Bremen, Germany) fitted with a nanoelectrospray ionization ion source. Online nanoLC separations were performed using a Agilent 1100 nanoflow system (Agilent Technologies, Waldbronn, Germany). Each sample was analyzed by RP-nanoLC−MS/MS

in duplicates. The peptide separations were performed on inhouse packed 15-cm fused silica emitters (75-μm inner diameter, 375-μm outer diameter). The emitters were packed with a methanol slurry of reversed-phase, fully end-capped Reprosil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) using a pressurized packing device operated at 50−60 bar. The separations were performed at a flow rate of 200 nL/min with mobile phases A (water with 0.5% acetic acid) and B (89.5% acetonitrile, 10% water, and 0.5% acetic acid). A 100-min gradient from 2% B to 50% B followed by a washing step with 98% B for 5 min was used. Mass spectrometric analyses were performed using unattended data-dependent acquisition mode, in which the mass spectrometer automatically switches between acquiring a high resolution survey mass spectrum in the FTMS (resolving power 100000 fwhm) and consecutive low-resolution, collisioninduced dissociation fragmentation of up to five of the most abundant ions in the ion trap. Acquired data (.RAW-files) were converted to the .mgf format using an in-house written program (C++) and subjected to protein identification using MASCOT search engine (version 2.2.2, Matrix Science Ltd., U.K.) against the SwissProt database version 51.6. The search parameters were set to Taxonomy: Mus musculus, Enzyme: Trypsin, Fixed modifications: Carbamidomethyl (C), Variable modifications: Oxidation (M) and Deamidated (NQ), Peptide tolerance: 0.03 Da, MS/MS tolerance: 0.8 Da and maximum 2 missed cleavage sites. Proteins were only considered to be positively matched if they passed the more stringent MudPIT MASCOT ion scoring (p ≤ 0.05) and at least one peptide passing the require bold red criteria. The require bold red criteria imply that each identified protein must have at least one top ranked peptide passing the significance criteria assigned to it. This and the harsher MudPIT scoring criteria reduce the redundancy in the protein identification and strengthen the probability that the identified proteins have been correctly assigned. 2.9. Data Analysis

The subcellular location and functions of the identified proteins were elucidated by collection of information from the Uniprot database and pie charts for these classifications of the identified brain proteins were created. The free share program Venn Diagram Plotter (http://omics.pnl.gov/software/ VennDiagramPlotter.php) was used for the creation of Venn diagrams.

3. RESULTS AND DISCUSSION Currently, there is no single analytical approach that can sufficiently address all levels of organization of the proteome, including the membrane proteome. Solubilization and extraction of membrane proteins is one of the key issues in such studies. Other important factors affecting the quality, depth and outcome of a proteomic study are sample selection, collection and preparation. In this study, different protein extraction protocols were evaluated with regards to proteins yield and the number of identified proteins with emphasis on the membrane bound proteins. The mouse brain proteome was extracted using different lysis buffers (detergents, organic solvents, high pH solution, and organic acid). The set A lysis buffers composition was identical for all protocols apart from the extracting constituent, that is, detergents, organic solvents and organic acids (Table 1). The set B buffers were adapted from previous scientific publications but only the extracting constituent was kept similar to the set A buffers. For all sample preparation 2444



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detergent based protocols are shown in Figure 2a. The extraction with high pH phosphate lysis buffer (lane 1) was more efficient compared to organic solvents (lane 2−7) and organic acid (lane 8). This is also in good agreement with the protein assay measurements of the total protein amounts extracted from the brain tissue (Table 2) where the extracted amounts by the phosphate buffer was more than twice compared to all organic solvents and acids. Visual comparison of the extraction and enrichment using organic solvents revealed that the isopropanol lysis buffer (lanes 2 and 3) yielded a higher number of protein bands compared to methanol or acetonitrile (lanes 4−7). This was also obtained in the protein assay measurements where the extracted amount by isopropanol was 3 times higher compared to the other two organic solvents. The extraction with formic acid is shown in lane 8 and is consistent with the assay measurements having an efficiency less than the phosphate buffer and isopropanol but higher than methanol and acetonitrile. A comparison of the detergent based protocols is shown in Figure 2b. All evaluated protocols gave high protein yield. The lowest number of protein bands was observed for the samples extracted with Triton X-100 lysis buffer set B21 (lane 3). Again, the 1D gel results can be compared to the protein assay results where Triton X-100 in lysis buffer B gave the lowest extracted amounts for the detergents. However, it should be mentioned that this extraction protocol still yielded higher total amounts than any of the non detergent based protocols. The extraction protocols with octyl-β-D-glucopyranoside (lane 6 and 7), Triton X-114 (lane 1) and SDS (lane 8 and 9) showed the largest number of protein bands and also the highest yield of extracted protein amounts. The 1D gels clearly demonstrate the power of using detergents as a solubilizing agent for the extraction of proteins from a fatty tissue, such as the mouse brain. It should be noted that despite the fact that the gel sample load for the detergent based protocols were one-fourth compared to the non detergent based ones, the number of protein bands were still higher and a streaking of the bands could be observed, which is an indication of sample overloading. All 1D gel results are consistent with the protein assay measurements confirming the detergents as very effective extraction and solubilizing reagents for proteins from CNS tissue. The 1D gels and protein assays does not give any indications of identity and classification of the extracted proteins, especially regarding the targeted membrane and transmembrane proteins. For instance, the use of high pH phosphate buffer gave a high yield in the extracted protein amounts and number of protein bands in the 1D gel. However, it is expected that this protocol promotes the extraction of hydrophilic proteins rather than the hydrophobic membrane and transmembrane ones and thereby cause a sample preparation bias in which the extracted proteome does not reflect the actual tissue proteome. Therefore, on-filter enzymatic digestion of the extracted proteomes was performed in combination with nanoLC−MS/MS analysis in order to investigate and compare the number of extracted proteins and their subcellular classification.

protocols, the brain samples were homogenized with the lysis buffers, incubated at 4 °C, sonicated, and centrifuged. The obtained protein extracts were purified by a previously evaluated delipidated protocol by the treatment with tri-nbutylphosphate/acetone/methanol (1:12:1).26,38 For organic solvent lysis buffers, 60% methanol and 40% isopropanol were used owing to their efficiency in enrichment of low abundant MPs at this concentration.29,31,32 For formic acid, the reported protein cleavage with CNBr was not conducted due to the toxic nature of CNBr and that protein cleavage should be enzymatic (tryptic) for better comparison of the results. For the high pH phosphate protocol, only a set B buffer was evaluated due to the composition of the buffer that has to yield high pH. For Triton X-114, only a set A buffer was evaluated since the protocol has been developed in-house.26 3.1. 1D Gel Protein Separation

Visual comparison of the extraction efficiencies for the extraction protocols was performed using one-dimensional gel electrophoresis and the resulting 1D gels are shown in Figure 2a and b. The 1D gel

Figure 2. (a) 1D gel electrophoresis of mouse brain proteins extracted with (1) phosphate33 (B); (2) isopropanol (A); (3) isopropanol31 (B); (4) methanol (A); (5, 6) methanol30 (B); (7) acetonitrile (A); (8) formic acid (A) lysis buffers. (b) 1D gel electrophoresis of mouse brain proteins extracted with (1) Triton X-114 (A); (2) Triton X-100 (A); (3) Triton X-10020 (B); (4) CHAPS (A); (5) CHAPS10 (B); (6) octyl-β-D-glucopyranoside (A); (7) octyl-β-D-glucopyranoside21 (B); (8) SDS (A); (9) SDS22 (B) lysis buffers.

3.2. On-filter Digestion Followed by Separation and MS/MS Analysis

Two approaches were utilized to compare the different extraction protocols in combination with on-filter “shotgun” based MS analysis. These approaches differed regarding sample normalization, which was performed either on (1) normalized protein amounts or (2) normalized volumes after delipidation

images clearly demonstrate differences in the protein distribution patterns for the extraction protocols. The non 2445



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Figure 3. Total number of mouse brain proteins extracted using different extraction methods and identified by nano-LC−MS/MS analysis. Normalized protein amount (35 μg) (a) and normalized volume of extract (25 μL) (b) were taken for digestion.

3.2.1. Normalized Protein Amounts. Comparison of the protein identifications obtained for normalization on protein amounts (35 μg) for digestion is shown in Figure 3a. Furthermore, the full comparison of the identified unique mouse brain proteins, number and percentages for membrane and transmembrane proteins obtained for duplicate extractions of each method is given in Table 3. The number of identified proteins clearly strengthens the results from the protein assays and 1D gels. Despite the normalization to the same protein amount for each extraction method, the detergent based protocols and phosphate buffer yields a substantially greater number of identified proteins. The use of organic solvents and organic acids does not provide sufficient extraction of the proteome from such a fatty tissue as the brain, despite the reported success for these protocols for the study of membrane bound proteins.40−42 However, these protocols have primarily been applied on cell cultures and cells in biological fluids and not brain samples where lipids constitute about 60% of entire tissue composition.43 The extraction of proteins with potassium phosphate at high pH (set B) gave approximately the same number of identified proteins (336) as in the case of detergents.

and protein precipitation (only conducted on the detergent based protocols). Comprehensive data regar assigned subcellular localizations for the extracted brain proteomes are given in Supplementary Tables 1−3, Supporting Information. Normalized protein amounts were used since the difference in total protein yield for the extraction protocols varied up to 40 times. Therefore, normalized protein amounts were first analyzed in order to yield an unbiased evaluation how effective and selective the different protocols were in their extraction of the brain proteome. The initial assumption was that if there is no difference between the protocols, then the number of identified proteins as well as number and percentages of membrane and transmembrane proteins would be similar for all extraction methods. The second approach with normalized volumes after delipidation and protein precipitation was only performed for the detergent based protocols. All of these protocols gave similar extracted protein amounts and normalization based on sample volume was carried out to investigate the differences regarding the number of identified proteins as well as number and percentages of membrane and transmembrane proteins. 2446



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Table 3. Number of Mouse Brain Proteins, Number and Percentage of Membrane Proteins, Number and Percentage of Transmembrane Proteins out of Membrane Fraction Extracted using Different Extraction Methods and Identified by nanoLC− MS/MS Analysisa set A lysis buffer Normalized amount

Organic solvents Organic acid Salt buffer Detergents

Normalized volume

a

Detergents

Acetonitrile Methanol Isopropanol Formic acid Phosphate buffer CHAPS Triton X-100 SDS β-OG Triton X-114 CHAPS Triton X-100 SDS β-OG Triton X-114

set B

proteins number

MPs number

MPs percentage

TMPs number

TMPs percentage

proteins number

MPs number

MPs percentage

TMPs number

TMPs percentage

51 124 150 240 n/a

9 25 36 34 n/a

18 20 23 14 n/a

3 5 15 10 n/a

33 20 41 30 n/a

n/a 100 104 n/a 336

n/a 18 23 n/a 55

n/a 18 22 n/a 16

n/a 0 5 n/a 9

n/a 0 22 n/a 16

259 360 410 418 463 423 272 503 541 466

67 101 119 121 134 114 76 141 157 133

26 28 29 29 29 27 28 28 29 29

31 44 50 62 70 51 34 65 82 69

46 44 42 51 52 45 45 46 52 52

339 330 373 392 n/a 445 143 428 507 n/a

85 63 108 110 n/a 111 23 124 142 n/a

25 19 29 28 n/a 25 16 29 28 n/a

27 27 48 57 n/a 39 10 56 74 n/a

32 43 44 52 n/a 35 42 45 52 n/a

n/a − not available.

and B buffers is shown in Figure 3b. For all detergent based protocols except CHAPS, extractions with buffer A gave a higher number of identified proteins. Protein solubilization and extraction with β-OG, SDS, Triton X-100 with set A buffers gave a relative increase of 7, 18, and 90% in the number of identified proteins compared to set B buffers. The CHAPS protocol demonstrates 5% increase of identified proteins for the set B compared to set A. Also, all detergent protocols with the set A buffer yielded a higher number (and in almost all cases %) of identified membrane and transmembrane proteins. Thus, the in-house developed set A buffer seems to be slightly more efficient in the extraction of the brain tissue proteome. However, only the composition of set B buffers was adapted from scientific publications and not the entire protocol of the experimental conditions and sample handling. Therefore, the results might deviate from the original publications for the set B buffer based protocols. β-OG lysis buffer (set A) provided the highest total number of identified unique proteins (541) compared to other methods despite the protein assay results. According the protein assay, β-OG lysis buffer set B gave a higher protein concentration than that of set A lysis buffer. A possible explanation for this could be the use of thiourea in the set B lysis buffer that may facilitate better disruption of the hydrophobic interactions compared to urea used in the set A lysis buffer.45 However, the nanoLC− MS/MS analysis after delipidation and on-filter tryptic digestion revealed a slightly higher number of total proteins as well as numbers and percentages of MPs and transmembrane proteins for set A compared to B. The obtained results indicate that the use of set A buffer yields a better insight into the mouse brain proteome.46

Nonetheless, most of the proteins extracted by the phosphate buffer were of hydrophilic nature. The number and percentages of membrane proteins were half compared to the detergent based protocols and the number and percentages of the highly hydrophobic transmembrane proteins were one-third or less. High pH or high salt buffers facilitates the disruption of non covalent interactions of peripheral membrane proteins as well as their solubilization. Yet, it can not solubilize integral membrane proteins44 and therefore cause an bias in the extracted proteome which does not correctly reflect the brain tissue proteome. The obtained results clearly show the benefit of using detergent-based protocols for the extraction of MPs from fatty brain tissue. For all extraction protocols except CHAPS, extractions with in-house developed set A buffers gave a higher number of identified proteins compared to set B. The extraction with Triton X-114 lysis buffer provided the highest total number of protein identities. It yielded an overall identification of 463 unique proteins out of which 134 (29%) were assigned to be membrane proteins and 70 proteins (52%) out of these were of transmembrane origin. All evaluated detergent-based protocols yielded a representative insight into the brain tissue proteome with expected membrane representation of the reported approximate 30%. The detergents also provided sufficient solubilization of the very hydrophobic transmembrane protein, which often are difficult to analyze and thus underrepresented in tissue proteomic studies. 3.2.2. Normalized Volumes. Normalization to the volume of the extracts after delipidation and protein precipitation was only applied for the detergent based protocols owing to the large difference in total protein yield the extraction methods. Normalized volumes were analyzed to relatively compare the number of identified proteins (both in total and MPs) for each detergent based extraction method. Hence, normalized volumes (25 μL) of the extracts were subjected to on-filter tryptic digestion and the results of analysis are given in Table 3. A comparison of identified proteins for each detergent using set A

3.3. Subcellular Location and Functional Survey of Identified Mouse Brain Proteomes

The identified mouse brain proteins were classified according to their subcellular localization. These classifications are shown in Figure 4a and b for normalized amounts and normalized volumes, respectively. In Figure 4a based on normalized amounts interesting results can be observed for mouse brain 2447



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Figure 4. Percentage of subcellular localized mouse brain proteins extracted using different lysis buffers and identified by nano-LC−MS/MS analysis. Normalized protein amount (35 μg) (a) and normalized volume of extract (25 μL) (b) were taken for digestion.

proteins extracted with acetonitrile and formic acid lysis buffers which gave more percentage of other (unknown) proteins. Another interesting trend observed in figures 4a and b is that SDS lysis buffers produced high percentage of MPs compared

to cytoplasmic proteins. This might be due to high ionic strength of SDS buffer. Extraction with all set A detergent lysis buffers produced 26−29% of membrane proteins which is superior compared to organic solvents and organic acids that 2448



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Figure 5. Subcellular localization (a,c) and biological functions (b,d) of mouse brain proteins extracted using Triton X-114 lysis buffer (a,b) and octyl-β-D-glucopyranoside (c,d). Normalized protein amounts (35 μg) were taken for digestion. The values reflect the identification obtained by nanoLC−ESI−MS/MS.

produced MPs in the range of 14−23%. The percentages of membrane proteins extracted using all detergent protocols produced similar results which are in the same vicinity of the genomic percentages for MPs (25−30%) showing the overall method is highly reproducible. A further in depth classification with both subcellular localization as well as cellular function of the assigned membrane bound proteins was conducted for the two best performing detergent-based extraction protocols: Triton X-114 lysis buffer set A and octyl-β-D-glucopyranoside lysis buffer set A based on normalized amounts. The assigned subcellular localization and cellular function of the identified membrane proteins are shown in Figure 5a,b and c,d for Triton X-114 and octyl-β-D-glucopyranoside, respectively. The comparison of subcellular location of identified proteins and cellular function of the assigned membrane proteins for best two detergentbased extraction protocols gave quite similar results (Figure 5). The most abundant classes of proteins allocated to the cytoplasm (29−30%) and in the membrane (29%). Among the other groups, 11−12% of the proteins were annotated as

mitochondrial and 10% as cytoskeleton. Nuclear (7%) and extracellular (1−2%) proteins that are involved in gene silencing, transcriptional regulation and cellular communication were also well represented for both protocols. Of the remainder, 4−5% were annotated as cytosol, 1−2% as Golgi apparatus, 1−2% as secreted and 3−6% as others which are unknown proteins. The membrane proteins were also further categorized by their cellular functions. Approximately 29−38% of MPs have transporter activity. About 16−25% and 16−18% of MPs were involved in the catalytic and cell structure molecular activity. One to 3% and 2−3% of the identified MPs were ion channels and G-protein coupled receptors, respectively. Three to 7% and 2−9% of the membrane proteins were involved in cell adhesion and signal transduction, respectively. Five to 8% of MPs were categorized as miscellaneous which represents membrane proteins with more than one cellular function. Also 5−10% of identified MPs have unknown functions which are categorized as others. 2449



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4. CONCLUSIONS This work comprises the first large-scale comparative study of sixteen different extraction approaches for the extraction and analysis of the mouse brain proteome with emphasis on membrane proteins. Membrane and transmembrane proteins are of primary interest in the exploration of the CNS and special focus was therefore put on these proteins in the comparison. These proteins are also difficult to analyze due to their low abundance, heterogeneity and very hydrophobic nature. All evaluated detergent based protocols yielded a representative insight into the brain tissue proteome and provided sufficient solubilization of the MPs and transmembrane proteins. In fact, using a detergent based protocol gave up to 40 times higher protein yield compared to organic solvents and acids. Among all of the protocols, the use of the detergents octyl-β-D-glucopyranoside and Triton X-114 protocols provided the highest protein yields, total number of identified proteins, and the highest numbers and percentages of membrane and transmembrane proteins. The percentages of MPs identified for these two best methods were 29%, which is at the reported genome expression levels of 25−30%. Furthermore, more than 50% of the identified MPs were assigned as transmembrane proteins using either octyl-β-Dglucopyranoside or Triton X-114. Transmembrane proteins are often underrepresented in tissue proteomic studies due to solubilization issues. The detergent Triton X-114 has an additional important advantage that it can be used in cloud point extraction and phase separation of hydrophobic MPs in biological matrices as previously reported.26,27

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from the Swedish Institute for GS is gratefully acknowledged. We also thank Dr. Konstantin Artemenko for helpful advice during the preparation of this manuscript.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Table 1. List of mouse brain proteins extracted using different lysis buffers (organic solvents, organic acid and phosphate buffer) and identified by nano-LC−MS/MS analysis. Normalized protein amount (35 μg) was taken for digestion. Supplementary Table 2. List of mouse brain proteins extracted using different detergent lysis buffers and identified by nanoLC−ESI−MS/MS. Normalized protein amount (35 μg) was taken for digestion. Supplementary Table 3. List of mouse brain proteins extracted using different detergent lysis buffers and identified by nano-LC−ESI−MS/MS. Normalized volume of extract (25 μL) was taken for digestion. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Dr. Ganna Shevchenko/Prof. Jonas Bergquist, Department of Physical and Analytical Chemistry, Uppsala University, Box 599, SE-751 24 Uppsala, Sweden. E-mail: ganna.shevchenko@ kemi.uu.se. Telephone: +46 18 471 3680. Fax: +46 18 471 3692. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was performed within Uppsala Berzelii Technology Centre for Neurodiagnostics, financed by the Swedish Governmental Agency for Innovation Systems, the Swedish Research Council, and Uppsala University. J.B. further acknowledges SRC grant 621-2008-3562. Financial support 2450



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Comparison of Extraction Methods for the Comprehensive Analysis of

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