2 D-NanoLC-ESI-MS / MS for Separation and Identification of Mouse Brain Membrane Proteins

© 2012 Van Chi and Dung, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2D-NanoLC-ESI-MS/MS for Separation and Identification of Mouse Brain Membrane Proteins


Introduction
Comprehensive proteomics analysis has the potential to provide new knowledge on cellular responses in development, aging, drug action, environmental stress, and disease pathogenesis (carcinogenesis, cardiovascular disease, etc). However, the separation and identification of proteomes/proteins is a challenging task due to their heterogeneous constituents or complex structures and closely related physico-chemical behaviors. It is clear that the combination of many analytical techniques is necessary to fulfill this complex task. At the start of proteomics research, two-dimensional electrophoresis (2DE) was routinely used to separate complex proteomic sample because of its high resolving power. In this technique, proteins are separated in a two-step process (two dimensions) based on their different physical properties. The first dimension is isoelectrofocusing in which proteins are separated based on their isoelectric points (pI, the pH where a protein's net charge is zero) using immobilized pH-gradient strips. Proteins then are separated according to their mass using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. With 2DE, thousands of proteins can be detected in a single experiment depending on the used staining techniques (Coomassie blue, silver, fluorescent dyes staining) [11]. Mass spectrometry (MS), using either electrospray ionization (ESI) or matrixassisted laser desorption/ionization (MALDI), is the key technology for the identification of protein spots including membrane proteins, for which differential expression has been demonstrated [16,30].
2DE, however, has some major drawbacks/disadvantages. It is time-consuming, difficult to reproduce and automation is hard to achieve. Furthermore, 2DE faces with many difficulties in analyzing several groups of proteins, such as low-abundance proteins, hydrophobic proteins (membrane proteins/membrane-bound and membrane-associated proteins), very large as well as very small proteins and proteins with extreme pI values. Unfortunately, these proteins have high proportion in comparison to total cellular proteins and are usually the most promising targets for drug development or disease diagnostics. About 30% of the mammalian genome encodes integral membrane proteins [27]. However, the comprehensive proteomic analysis of these proteins by mass spectrometry is difficult due to the amphipathic (containing regions that are hydrophobic and hydrophilic) nature in integral membrane proteins and their general low abundance levels [23]. Since the analysis of membrane proteins remains a significant challenge in proteomics, other techniques need to be established to address these problems. There have been many strategies developed for enriching, isolating and separating membrane proteins for proteomic analysis that have moved this field forward.
In recent years, two-dimensional liquid chromatography (2D-LC) has been employed as a complementary or alternative separation technique to 2DE. The combination of liquid chromatography as a separation tool for proteins and peptides with tandem mass spectrometry as an identification tool referred to as LC-MS/MS has generated a powerful and broadly used technique in the field of proteomics [6,9,10,21,22], particularly in the analysis of membrane proteomes [18,19]. With the development of new quantitative strategies and bioinformatics tools to cope with the analysis of the large amounts of data generated in proteomics experiments, the resolution and sensitivity state-of-the art LC-MS/MS systems has reached dimensions allowing not only the analysis of individual proteins but also investigations on the level of complete proteomes [8]. This approach is usually based on the injection of the digested protein sample onto a strong cation-exchange (SCX) column as a first-dimension separation. Peptides bound in SCX column are eluted and separated from the column as fractions by an injecting salt plugs/salt step gradient of increasing salt concentration. Each fraction is subsequently separated on a reversed-phase (RP) column as the second orthogonal separation dimension before being presented to mass spectrometry analysis. Different stationary phases in chromatography columns provide variable levels of resolution. Reversed-phase chromatography is highly compatible with subsequent mass spectrometric analysis due to the lack of salts in the buffers and provides relatively high-resolution separation. Most reversed-phase stationary phases for LC-MS analysis consist of silica beads of 3-5 μm in diameter with alkyl chains of either eight or eighteen carbons in length (C8 or C18) attached. Using column switching, the entire procedure is on-line and fully automated. In order to improve sensitivity the reversed phase separation is usually performed in the nanoflow scale and mass spectrometry is used as the final detection method.
In this chapter a strategy for enrichment, isolation, separation, identification and characterization of mouse brain membrane proteins with the basic setup of two-dimensional nano liquid chromatography (2D-nanoLC) system (UltiMate TM /FAMOS/Switchos TM , LC Parking, Dionex, The Netherlands) coupled online with QSTAR ® XL MS/MS mass spectrometer (Appllied Biosystems/MDS SCIEX, Ontario, Canada) is presented. Figure 1. A scheme illustrating the necessary steps, including enrichment and extraction, separation, identification and characterization for proteomic analyses of mouse brain membrane proteins using gelbased approach in combination with comprehensive two-dimensional nano liquid chromatography (2D-nanoLC) coupled online with tandem mass spectrometry.

Membrane protein enrichment and extraction
Swiss mouse brains were collected as soon as possible after the animals were killed. The samples (3-5 g) were excised into approximately 5 mm wide pieces using scissors and washed with 10 ml of ice cold PBS buffer (0.2 g KCl, 8 g NaCl, 1.44 Na2HPO4, 0.24 g KH2PO4) and then resuspended in 3 volumes of the homogenization medium (0.25 M sucrose in 5 mM Tris-HCl pH 7.4 with 1 mM tetrasodium EGTA, 1 mM sodium orthovanadate (Na3VO4) and 2 mM sodium fluoride in deionized filter-sterilised MilliQ water) containing protease inhibitors (Calbiochem Protease Inhibitor Cocktail Set 111, catalog number 39134, contains AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A). After the medium has been drained off, new medium was replaced and drained off again. 10 ml of homogenisation medium (containing inhibitors) was added and the sample was homogenised using a Polytron in a Potter homogeniser with motor driven teflon pestle at approximately 1,000 rpm. Completely homogenized samples were centrifuged at 10,000 rpm for 15 min at 4 o C to sediment large organelles. The obtained supernatant was used for recentrifugation again at 10,000 rpm for 15 min at 4 o C. The supernatant was collected and centrifuged at 40,000 rpm at 4 o C for 1 hr. After discarding the clear supernatant, the membrane pellets were retained and washed by resuspending in ice-cold 0.1 M Na2CO3 containing protease inhibitors for 1 hr. The mouse brain membrane protein fractions were obtained by centrifugation again at 40,000 rpm for 1 h at 4 o C. The sample was divided and stored at −80 o C until use. The protein concentration of the extracted membrane fractions was assessed using a Quick Start TM Bradford Protein Assay Kit (Bio-Rad, Hercules, CA 94547 USA).

Protein quantification
Protein concentration of the extracted membrane fractions was determined using Bio-Rad's Quick Start TM Bradford Protein Assay [5]. The assay is based on the observation that the maximum absorbance for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible colour change. For the standard curve, bovine serum albumin over a wide range of concentrations (0.1 -20 μg/μl) was used. The low concentration range assay was used in the test tube format. 2 μl of standard or sample was added to 798 μl of MilliQ water. 200 μL of Bio-Rad reagent was added, mixed, and incubated for 10 min at room temperature. The absorbance at the wavelength of 595 nm was measured in a spectrophotometer. Glass or polystyrene (cheap) cuvettes have been used, however the color reagent stained both. Disposable cuvettes were recommended.

In-gel digestion
In-gel digestion of proteins isolated by gel electrophoresis was carried out according to the protocol published by Shevchenko et al [25] with some modifications described in our previous study [3,28,29]. All chemicals including DTT, iodoacetamide (IAA), ammonium bicarbonate, ammonium acetate, trypsin (proteomics sequencing grade), sodium bicarbonate and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA) prepared using deionized filter-sterilised MilliQ water.
Upon electrophoresis, proteins were fixed within a polyacrylamide matrix by incubating the entire gel in 5% (vol/vol) acetic acid in 1:1 (vol/vol) water:methanol. Coomassie blue-stained protein bands were excised from gels and placed into 1.5 ml eppendorf tubes, destained with 50% ACN in 25 mM NH4HCO pH 8.0 at room temperature with occasional vortexing, until gel pieces became white and shrank, and then acetonitrile was removed. The gel pieces were then reduced by incubating with 5 mM DTT solution at 56 o C for 45 min and alkylated for 1 hr with 20 mM IAA solution in darkness at room temperature. The membrane proteins were digested by adding trypsin buffer (0.03 μg/μl in 10 mM ammonium bicarbonate containing 10% (vol/vol) acetonitrile) and incubating overnight at 37 o C. Check if all solution was absorbed and add more trypsin buffer, if necessary. Gel pieces should be completely covered with trypsin buffer (typically, 50 μl or more).

Sample cleanup with C-18 ZipTips
The resulting peptide digestion products were extracted by adding 100 μl of extraction buffer (1:2 (vol/vol) 5% formic acid/acetonitrile) to each tube and incubated for 15 min at 37 °C in a shaker. All extracts were saved and dried and re-dissolved in 10-20 μl of 0.1% FA, incubated for 2-5 min in the sonication bath and centrifuged for 15 min at 10,000 rpm at the bench-top centrifuge. The obtained supernatant was applied for binding the samples onto micro pipette tips (µC18), catalog number ZTC18S096 (ZipTip ® , Millipore Co., Billerica, MA 01821 USA), equilibrated by being aspirated and dispensed with 100% acetonitrile, 40% acetonitrile/0.1% FA, 0.1% FA solutions. The samples were washed (4 times by aspirating and dispensing) with 15 µl of 0.1% FA), then eluted with 10 µl of 40% acetonitrile/0.1% FA. Appropriate aliquots were withdrawn for LC-MS/MS analysis or store at −20°C as contingency.

Two-dimensional nano liquid chromatography (2D-nanoLC)
The basic setup of an online two-dimensional nano liquid chromatography (2D-nanoLC) system (LC Parking, Dionex, The Netherlands) was developed for improved separation and hydrophobic peptide recovery, especially for complex peptides made from enzymatic digests of selected proteomes. The system works with the principle of elution of the digested peptides from the first dimension SCX column with injected salt solution plugs of increasing concentration. The eluted peptides are again trapped and introduced into the nanoflow path for separation and analysis by second dimension RP column and tandem mass spectrometry. The great advantage of the system is a robust and fully automated separation. The methods are easy to set up and composed of identical runs differing only in the concentration of injected salt plugs.     After washing (~12 min), peptides were eluted from a reversed phase C18 column using the solvent B (0.1% FA in 85% LC-MS grade ACN) gradients: from 5 to 20% of solvent B in 25 min, 20 to 70% in 28 min, 70 to 100% in 10 min and maintaining 100% B in 10 min, and back to 5% B in 5 min.
According to the workflow, after 2D-nanoLC separation, peptides were independently analyzed by a QSTAR ® XL MS/MS mass spectrometer (Appllied Biosystems/MDS SCIEX, Ontario, Canada) equipped with a nanoESI source. MS and MS/MS spectra were recorded and processed in IDA mode (Information Dependent Acquisition) controlled by Analyst QS software. Typical settings are chosen to select multiply charged ions for MS/MS that produce at least 45-50 ion counts/s in a 0.5 s survey scan. The range of the MS full scan was from 400 to 1200 amu followed by MS/MS fragmentation of the three most intense precursor peptide ions for 1 s each.

Protein identification and validation
There are a number of different methods for identifying the proteins in the sample, and the most frequently used is the searching of the uninterpreted MS/MS data. The FASTA formatted protein sequences from National Center for BioTechnology Information (NCBI) and UniProtKB/Swiss-Prot databases are collected for proteins identified or identification by each MS experiment. Searching uninterpreted MS/MS data from a single peptide or from a complete nanoLC-MS/MS run was automatically analyzed with a non-redundant protein database by the program SEQUEST, which allows the correlation of experimental data with theoretical spectra generated from known protein sequences [7].
The precursor mass is used as a filter to find a list of candidate peptide sequences from the theoretical digest of the database. A variety of different systems are used to score the experimental MS/MS spectrum against spectra predicted from the candidate peptide sequences. For protein identification, experimental data were searched against the NCBInr and Swiss-Prot mouse protein database using Mascot v1.8 software in which the criteria were based on the manufacturer's definitions (Matrix Science Ltd, London, UK) [20]. The parameters were set as follows: enzymatic cleavage with trypsin; 1 potential missed cleavage; a peptide and fragment mass tolerance of ± 0.25 Da, and fixed modification of carbamidomethyl (cysteine); variable modification of oxidation (methionine); 1 + , 2 + , and 3 + peptide charge. Protein identifications were performed using a Mowse scoring algorithm with a confidence level of 95% and at least two matched peptides, showing a high score [12].
For further verification, proteins might be validated by MSQuant software [1,4,24] available at http://msquant.sourceforge.net. The MSQuant software is used as a validation and quantitation tool that produces the Mascot peptide identifications (HTLM files) and allows manual verification against the raw MS data (QSTAR XL raw files). The MSQuant software will pick up significant and verified hits from the Mascot output file and export information of identified proteins into an .xls file, including the GI (genInfo identifier) number and molecular-mass values.
The average hydrophobic values and transmembrane domains of the identified proteins were calculated using the SOSUI system that is available at http://bp.nuap.nagoya-u.ac.jp/sosui/ [11]. The proteins exhibiting positive GRAVY values were recognized as hydrophobic and those with negative values were hydrophilic [13].   . An example of hydropathy profile and transmembrane regions/domains of an identified mouse brain membrane protein calculated using the SOSUI system that is available at http://bp.nuap.nagoya-u.ac.jp/sosui/ [11].

Conclusion
Identification and characterization of membrane proteins is a crucial challenge in proteomics research. Thus, we have designed a strategy of gel-based approach in combination with comprehensive two-dimensional nano liquid chromatography (2D-nanoLC) that is robust and offers high separation capacity and high analysis throughput for mouse brain membrane proteins. By using this system, mixtures of in-gel trypsin-digested mouse brain membrane proteins were injected, desalted, separated and analyzed in complete automatization. The workflow started by the extraction and purification of the membrane fractions, then the SDS-PAGE was carried out as a useful preparative separation step. After staining, the gel slides with protein bands were cut, reduced, alkylated and trypsin-digested. The peptide mixtures extracted from each gel slice were fractionated by 2D-nanoLC coupled online with tandem mass spectrometry analysis (nanoESI-Q-TOF-MS/MS). The proteins were identified by MASCOT search against mouse protein database using a peptide and fragment mass tolerance of ±0.25 Da. Protein identification was carried out using a MOWSE scoring algorithm with a confidence level of 95% and processed by MSQuant software for further validation. In total, 298 identified membrane proteins from mouse brain tissues were verified by UniProt database, SOSUI and TMHMM prediction algorithms. Of these, 129 (43.3%) proteins have at least one transmembrane domain according to SOSUI and TMHMM. Furthermore, the function, subcellular location, molecular weight, post-translational modifications, transmembrane domains (TMD) and average of hydrophobicity of the identified membrane proteins might be categorized and analysed.