**1. Introduction**

62 Chromatography – The Most Versatile Method of Chemical Analysis

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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

© 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. © 2012 Van Chi and Dung, licensee InTech. This is a paper 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.

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 difcult 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.

2D-NanoLC-ESI-MS/MS for Separation and Identification of Mouse Brain Membrane Proteins 65

**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

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

(2D-nanoLC) coupled online with tandem mass spectrometry.

**2. Membrane protein enrichment and extraction** 

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 (UltiMateTM/FAMOS/SwitchosTM, 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.

## **2. Membrane protein enrichment and extraction**

64 Chromatography – The Most Versatile Method of Chemical Analysis

moved this field forward.

final detection method.

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 difcult 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

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

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 (UltiMateTM/FAMOS/SwitchosTM, LC Parking, Dionex, The Netherlands) coupled online with QSTAR®XL MS/MS mass

spectrometer (Appllied Biosystems/MDS SCIEX, Ontario, Canada) is presented.

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 4oC to sediment large organelles. The obtained supernatant was used for recentrifugation again at 10,000 rpm for 15 min at 4oC. The supernatant was collected and centrifuged at 40,000 rpm at 4oC 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 4oC. The sample was divided and stored at −80oC until use. The protein concentration of the extracted membrane fractions was assessed using a Quick StartTM Bradford Protein Assay Kit (Bio-Rad, Hercules, CA 94547 USA).

2D-NanoLC-ESI-MS/MS for Separation and Identification of Mouse Brain Membrane Proteins 67

The membrane fraction was solubilized in lysis buffer containing 3% SDS. Equal volumes containing approximately 25 μg/lane of MP were separated by 12% SDS-PAGE and were

**Figure 2.** The separation of membrane proteins (MPs) by SDS-PAGE. The gel was cut into 10 slices that covered known apparent mass ranges. Lane M, protein standard markers; lane 1 & lane 2: membrane protein fractions isolated from mouse brain; 1-10: slices to be cut for trypsin in-gel digestion, separation

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)

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

visualized by staining with Coomassie Brilliant Blue G-250.

and analyses by nanoLC-MS/MS.

prepared using deionized filter-sterilised MilliQ water.

**4.2. In-gel digestion** 
