# Proteomic and Bioinformatics Analyses of Mouse Liver Microsomes

Authors: Fang Peng; Xianquan Zhan; Mao-Yu Li; Fan Fang; Guoqing Li; Cui Li; Peng-Fei Zhang; Zhuchu Chen
Journal: International Journal of Proteomics
 (2012)

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## Abstract

Microsomes are derived mostly from endoplasmic reticulum and are an ideal target to investigate compound metabolism, membrane-bound enzyme functions, lipid-protein interactions, and drug-drug interactions. To better understand the molecular mechanisms of the liver and its diseases, mouse liver microsomes were isolated and enriched with differential centrifugation and sucrose gradient centrifugation, and microsome membrane proteins were further extracted from isolated microsomal fractions by the carbonate method. The enriched microsome proteins were arrayed with two-dimensional gel electrophoresis (2DE) and carbonate-extracted microsome membrane proteins with one-dimensional gel electrophoresis (1DE). A total of 183 2DE-arrayed proteins and 99 1DE-separated proteins were identified with tandem mass spectrometry. A total of 259 nonredundant microsomal proteins were obtained and represent the proteomic profile of mouse liver microsomes, including 62 definite microsome membrane proteins. The comprehensive bioinformatics analyses revealed the functional categories of those microsome proteins and provided clues into biological functions of the liver. The systematic analyses of the proteomic profile of mouse liver microsomes not only reveal essential, valuable information about the biological function of the liver, but they also provide important reference data to analyze liver disease-related microsome proteins for biomarker discovery and mechanism clarification of liver disease.


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## Body

## 1. Introduction

The liver, a vital organ, has a wide range of physiological functions and plays a major role in metabolism, biosynthesis, and chemical neutralizing. Liver diseases, such as viral hepatitis and liver cancer, pose a worldwide public health challenge. The Human Liver Proteome Project (HLPP) was launched in 2002 to better understand molecular liver functions and diseases, and liver proteome expression profile is one of the major parts of HLPP [1]. Because of the complexity, no single proteomic analysis strategy can sufficiently address all components of a proteome. Analysis of the subcellular proteome would provide insight into the functions of a given tissue or cell line. Subcellular proteomics reduces the complexity of a proteome [2, 3], detects some low-abundance proteins, and offers more detailed information that would contribute to the understanding of the function of the entire proteome.Microsomes are composed primarily of closed sacs of membrane called vesicles that are derived mostly from endoplasmic reticulum (ER). As for liver, in addition to components of the protein secretary pathway, microsomes contain a multitude of proteins that are involved in lipid/lipoprotein biosynthesis and drug metabolism. The liver microsome is an ideal way to study the metabolism of compounds, the functional properties of membrane-bound enzymes, lipid-protein interactions, and drug-drug interactions [4, 5]. The proteomic profiling of the microsomes combined with bioinformatics analysis can reveal more essential information about the biological function of the liver. The main goal of this study was to systematically identify the protein components of the liver microsomes, to conduct the functional annotation with bioinformatics analysis, and to provide insight into the biological functions of the liver.Two-dimensional gel electrophoresis (2DE) is one of the most widespread techniques for the proteomic profiling of soluble proteins and visualizes isoforms and posttranslational modifications in a proteome [6, 7]. Membrane proteins, however, are less amenable to solubilization in protein extraction buffers and are also susceptible to precipitation during isoelectric focusing (IEF) because of their hydrophobicity and alkaline pH value. One study showed that the analytical performance of one-dimensional gel electrophoresis (1DE) that separates endoplasmic reticulum membrane proteins is incomparably greater than that of 2DE [8]. Other studies [7, 9] demonstrated that the proteomic analysis of subcellular organelles, such as microsomes that contain a considerable number of highly hydrophobic membrane proteins, should be performed by combining 1DE and 2DE.Although many of microsome proteins have been studied, many more remain to be isolated and characterized. With the improvement of current methodologies and experimental techniques, more proteomic data will be obtained. Also, biological interpretation of proteomic data and extracting biological knowledge are essential to further understanding liver function.In our study, 2DE was first used to array the isolated microsome proteins of the liver. Because of the low performance of 2DE in separating membrane proteins [10] and the high efficiency of the carbonate procedure in separating membrane proteins [11, 12], the membrane proteins from Na2CO3-treated microsomes were separated by 1DE. Moreover, bioinformatics analysis of microsome proteomic data was performed to discover biological roles of the proteins. The results showed that the combination of 1DE and 2DE was more efficient for analyzing microsomes. Bioinformatics analysis can provide a valuable molecular basis to interpret the mechanisms underlying microsome biological functions and give insight into the biological function of the liver at the level of microsomes.


## 2. Material and Methods

### 2.1. Animals

Male C57 mice (9 weeks old) were purchased from the Experimental Animal Center of Central South University (Changsha, China). The mice were starved overnight for liver subcellular fractionation. All experiments were performed with the approval of the institutional ethics committee on animal research.


### 2.2. Preparation, Validation, and 2DE Analysis of Microsomes

#### 2.2.1. Preparation of Microsomes

Microsome apparatus-rich fractions were prepared from mice livers with differential centrifugation and sucrose gradient centrifugation as described [13]. Mice livers (approximately 10 g each) were drained of blood, minced thoroughly with scalpels, and transferred to 50 mL of chilled homogenization medium (0.25 M sucrose, pH 7.4) for 5–10 min with occasional stirring. The liquid was decanted and replaced with 50 mL of fresh homogenization medium followed by homogenization (30–60 sec.) on a TAMATO homogenizer (1,000 rpm × 3 and 1,500 rpm × 3). The homogenate was squeezed through a single layer of microcloth and centrifuged (10 min, 1,000 g; HITACHI centrifuge). The supernatant was centrifuged (30 min, 3,000 g), and sequentially centrifuged (30 min, 8,000 g) after discarding the sediment. The remainder supernatant was centrifuged (30 min, 34,000 g), carefully decanted, and centrifuged again (130,000 g, 1 h; Beckman Instruments, Palo Alto, CA) to get the “light” microsomes. The pink sediment was gently resuspended with a glass homogenizer in ~7 mL of 52% sucrose-0.1 M H3PO4 buffer (pH 7.1), and the density of sucrose was adjusted to 43.7%. The fraction was placed in one type-70i rotor centrifuge tube; overlayered sequentially with 7 mL, 5 mL, 5 mL, and 6 mL of 38.7%, 36.0%, 33.0%, and 29.0% sucrose, respectively, and centrifuged (80,000 g, 1 h). The upper four layers of the sucrose gradient were discarded by aspiration, and the bottom layer (43.7%) was diluted with two volumes of cold distilled water and centrifuged (130,000 g, 1 h) in a type-70i rotor to get the “heavy” microsomes. The pellets, light and heavy microsomes, were suspended in 3 mL of 0.25 M sucrose (pH 7.0) and combined. The mixture was diluted to 14 mL with 0.25 M sucrose containing CsCl with its final concentration of 0.015 M. The suspension was layered into an equal volume of 1.3 M sucrose/0.015 M CsCl and then centrifuged (240,000 g, 1 h) in an SW 55Ti rotor. The rough microsomes were in the pink sediment, and the smooth microsomes were at the interface. The smooth microsomes were diluted with an equal volume of 0.25 M sucrose (pH 7.0) and centrifuged (140,000 g, 1 h) in an SW 55i rotor.


#### 2.2.2. Detection and Validation of the Purity of Microsomes

Electron microscopy and Western blotting were used to detect and validate the purity of prepared microsomes. For electron microscope analysis, the prepared microsomes were fixed with 2.5% glutaraldehyde for 24 h and 2% OsO4 for 2 h, dehydrated with alcohol (50%, 70%, 90%, and 100% in turn), and processed into epoxy resin. Thin sections (500 Å‌) were prepared and stained with uranyl acetate and lead citrate then examined with a transmission electron microscope (H-600-1, Hitachi, Japan). For Western blotting analysis, the microsome fractions were lysed (4°C; 30 min) in lysis buffer (50 mM Tris-Hcl, 150 mM NaCl, 1 mM EDTA, 1% Triton-X100, and 0.1% SDS). The protein samples (50 μg) were subjected to electrophoresis on SDS-PAGE with 12% gel and transferred to PVDF membrane (Millipore). The PVDF membranes with proteins were immunoblotted with antibodies to endoplasmin (ER marker), OxPhos complex IV subunit I (mitochondrial marker), catalase (peroxisomal marker), and cadherin (cytoplasmic marker), respectively.


#### 2.2.3. Separation of Microsome Proteins by 2DE

2DE was performed as described by the manufacturer (Amersham Biosciences). Protein samples (400μg) were diluted to 450 μL with rehydration solution (7 mol/L urea, 2 mol/L thiourea, 0.2% DTT, 0.5% (v/v) pH3–10 NL IPG buffer, and trace bromophenol blue) and applied to IPG strips (pH 3–10 NL; 24 cm) for rehydration (14 h; 30 V). Proteins were focused successively (1 h at 500 V, 1 h at 1,000 V, and 8.5 h at 8,000 V) to give a total of 68 kVh on an IPGphor. After equilibration, SDS-PAGE was performed with 12% gel on Ettan DALT II system. Then, the blue silver staining method was used to visualize the protein spots on the 2DE gels [14].


### 2.3. Na2CO3 Extraction and 1DE Analysis of Microsome Membrane Proteins

Microsome membrane proteins were further extracted by the carbonate method from isolated microsomal fractions [12]. Microsomal fractions were diluted 50- to 1,000-fold with 100 mM sodium carbonate (pH 11.5; final protein concentration to 0.02 to 1 mg/mL), and incubated (0°C; 30 min) with slow stirring and accompanying sonication for 15 sec at 3-4 W at 0 min, 15 min, and 30 min. The suspensions were centrifuged and decanted, and the membrane pellets were gently rinsed three times with ice-cold distilled water. These pellets were diluted with denaturing sample buffer (5% mercaptoethanol, 2% SDS, 0.06 M Tris-HCl, pH 6.8, and 10% glycerol), heated (95°C; 5 min), and then subjected to 1D SDS-PAGE with a 12% gel. Electrophoresis was performed at 80 V for 20 min, followed by 100 V for 2 h. Gels were visualized with Coomassie Brilliant Blue G [14].


### 2.4. Tandem Mass Spectrometry (MS/MS) Identification of Proteins

#### 2.4.1. In-Gel Digestion

The proteins contained in the 2D gel spots and 1D gel bands were subjected to in-gel digestion with trypsin. Gel spots or bands were excised and destained with 100 mM NH4HCO3 in 50% acetonitrile (ACN) at room temperature. The proteins were reduced with 10 mM dithiothreitol (DDT) (56°C; 30 min) and alkylated with 50 mM iodoacetamide in 100 mM NH4HCO3 (dark, room temperature, 30 min). The gel pieces that contained proteins were dried and then incubated in the digestion solution (40 mM NH4HCO3, 9% ACN, and 20 μg/mL trypsin; 18 h, 37°C). The tryptic peptides were extracted with 50% ACN/2.5% TFA and then dried using a Speed-Vac.


#### 2.4.2. Nanoliquid Chromatography (LC) MS/MS and Protein Identification

The tryptic peptide mixture was fractionated with reverse-phase (RP) high-performance liquid chromatography (HPLC) by using an Ultimate nano-HPLC system (Dionex). Peptide samples were purified and concentrated with a C18-PepMap precolumn and then separated on an analytical C18-PepMap column (75μm ID × 150 mm, 100 Å pore size, 3 mm particle size) at a column flow rate of 300 nL/min. The ACN gradient (solution A: 0.1% formic acid, 2% ACN; solution B: 0.1% formic acid, 80% ACN) started at 5% B and ended at 70% B in 45 min. Mass spectrometry (MS) and MS/MS data were acquired using a Micromass quadrupole time of flight Micromass spectrometer (Waters). Database searches were carried out with the MASCOT server by using a decoy database (concatenated forward-reverse mouse IPI database, version 3.07; release date June 20, 2005). A mass tolerance of 0.3 Da for both parent (MS) and fragmented (MS/MS) ions, allowance for up to one trypsin miscleavage, variable amino acid modifications consisting of methionine oxidation and cysteine carbamidomethylation were used. MS/MS ion score threshold was determined to produce a false-positive rate less than 5% for a significant hit (P<0.05). The false-positive rate was calculated with 2* reverse/(reverse + forward)/100. In the current study, the MS/MS ion score threshold was 23 and a false-positive rate was approximately 3.1%. For all the proteins that were identified with only one peptide, each MS/MS spectrum was checked manually.


### 2.5. Bioinformatics Analysis of Identified Proteins

Protein annotations were obtained primarily from UniProt 7.0 including accession, entry name, comments such as function, catalytic activity, subcellular location, and similarity. The Cytoscape plugin, Biological Networks Gene Ontology (BinGO), was used to find statistically overrepresented GO categories of the protein dataset. An online tool, WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/), was used to map target proteins to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The pathway visualization was based on the pathway mapping service provided in KEGG.


## 2.1. Animals

Male C57 mice (9 weeks old) were purchased from the Experimental Animal Center of Central South University (Changsha, China). The mice were starved overnight for liver subcellular fractionation. All experiments were performed with the approval of the institutional ethics committee on animal research.


## 2.2. Preparation, Validation, and 2DE Analysis of Microsomes

### 2.2.1. Preparation of Microsomes

Microsome apparatus-rich fractions were prepared from mice livers with differential centrifugation and sucrose gradient centrifugation as described [13]. Mice livers (approximately 10 g each) were drained of blood, minced thoroughly with scalpels, and transferred to 50 mL of chilled homogenization medium (0.25 M sucrose, pH 7.4) for 5–10 min with occasional stirring. The liquid was decanted and replaced with 50 mL of fresh homogenization medium followed by homogenization (30–60 sec.) on a TAMATO homogenizer (1,000 rpm × 3 and 1,500 rpm × 3). The homogenate was squeezed through a single layer of microcloth and centrifuged (10 min, 1,000 g; HITACHI centrifuge). The supernatant was centrifuged (30 min, 3,000 g), and sequentially centrifuged (30 min, 8,000 g) after discarding the sediment. The remainder supernatant was centrifuged (30 min, 34,000 g), carefully decanted, and centrifuged again (130,000 g, 1 h; Beckman Instruments, Palo Alto, CA) to get the “light” microsomes. The pink sediment was gently resuspended with a glass homogenizer in ~7 mL of 52% sucrose-0.1 M H3PO4 buffer (pH 7.1), and the density of sucrose was adjusted to 43.7%. The fraction was placed in one type-70i rotor centrifuge tube; overlayered sequentially with 7 mL, 5 mL, 5 mL, and 6 mL of 38.7%, 36.0%, 33.0%, and 29.0% sucrose, respectively, and centrifuged (80,000 g, 1 h). The upper four layers of the sucrose gradient were discarded by aspiration, and the bottom layer (43.7%) was diluted with two volumes of cold distilled water and centrifuged (130,000 g, 1 h) in a type-70i rotor to get the “heavy” microsomes. The pellets, light and heavy microsomes, were suspended in 3 mL of 0.25 M sucrose (pH 7.0) and combined. The mixture was diluted to 14 mL with 0.25 M sucrose containing CsCl with its final concentration of 0.015 M. The suspension was layered into an equal volume of 1.3 M sucrose/0.015 M CsCl and then centrifuged (240,000 g, 1 h) in an SW 55Ti rotor. The rough microsomes were in the pink sediment, and the smooth microsomes were at the interface. The smooth microsomes were diluted with an equal volume of 0.25 M sucrose (pH 7.0) and centrifuged (140,000 g, 1 h) in an SW 55i rotor.


### 2.2.2. Detection and Validation of the Purity of Microsomes

Electron microscopy and Western blotting were used to detect and validate the purity of prepared microsomes. For electron microscope analysis, the prepared microsomes were fixed with 2.5% glutaraldehyde for 24 h and 2% OsO4 for 2 h, dehydrated with alcohol (50%, 70%, 90%, and 100% in turn), and processed into epoxy resin. Thin sections (500 Å‌) were prepared and stained with uranyl acetate and lead citrate then examined with a transmission electron microscope (H-600-1, Hitachi, Japan). For Western blotting analysis, the microsome fractions were lysed (4°C; 30 min) in lysis buffer (50 mM Tris-Hcl, 150 mM NaCl, 1 mM EDTA, 1% Triton-X100, and 0.1% SDS). The protein samples (50 μg) were subjected to electrophoresis on SDS-PAGE with 12% gel and transferred to PVDF membrane (Millipore). The PVDF membranes with proteins were immunoblotted with antibodies to endoplasmin (ER marker), OxPhos complex IV subunit I (mitochondrial marker), catalase (peroxisomal marker), and cadherin (cytoplasmic marker), respectively.


### 2.2.3. Separation of Microsome Proteins by 2DE

2DE was performed as described by the manufacturer (Amersham Biosciences). Protein samples (400μg) were diluted to 450 μL with rehydration solution (7 mol/L urea, 2 mol/L thiourea, 0.2% DTT, 0.5% (v/v) pH3–10 NL IPG buffer, and trace bromophenol blue) and applied to IPG strips (pH 3–10 NL; 24 cm) for rehydration (14 h; 30 V). Proteins were focused successively (1 h at 500 V, 1 h at 1,000 V, and 8.5 h at 8,000 V) to give a total of 68 kVh on an IPGphor. After equilibration, SDS-PAGE was performed with 12% gel on Ettan DALT II system. Then, the blue silver staining method was used to visualize the protein spots on the 2DE gels [14].


## 2.2.1. Preparation of Microsomes

Microsome apparatus-rich fractions were prepared from mice livers with differential centrifugation and sucrose gradient centrifugation as described [13]. Mice livers (approximately 10 g each) were drained of blood, minced thoroughly with scalpels, and transferred to 50 mL of chilled homogenization medium (0.25 M sucrose, pH 7.4) for 5–10 min with occasional stirring. The liquid was decanted and replaced with 50 mL of fresh homogenization medium followed by homogenization (30–60 sec.) on a TAMATO homogenizer (1,000 rpm × 3 and 1,500 rpm × 3). The homogenate was squeezed through a single layer of microcloth and centrifuged (10 min, 1,000 g; HITACHI centrifuge). The supernatant was centrifuged (30 min, 3,000 g), and sequentially centrifuged (30 min, 8,000 g) after discarding the sediment. The remainder supernatant was centrifuged (30 min, 34,000 g), carefully decanted, and centrifuged again (130,000 g, 1 h; Beckman Instruments, Palo Alto, CA) to get the “light” microsomes. The pink sediment was gently resuspended with a glass homogenizer in ~7 mL of 52% sucrose-0.1 M H3PO4 buffer (pH 7.1), and the density of sucrose was adjusted to 43.7%. The fraction was placed in one type-70i rotor centrifuge tube; overlayered sequentially with 7 mL, 5 mL, 5 mL, and 6 mL of 38.7%, 36.0%, 33.0%, and 29.0% sucrose, respectively, and centrifuged (80,000 g, 1 h). The upper four layers of the sucrose gradient were discarded by aspiration, and the bottom layer (43.7%) was diluted with two volumes of cold distilled water and centrifuged (130,000 g, 1 h) in a type-70i rotor to get the “heavy” microsomes. The pellets, light and heavy microsomes, were suspended in 3 mL of 0.25 M sucrose (pH 7.0) and combined. The mixture was diluted to 14 mL with 0.25 M sucrose containing CsCl with its final concentration of 0.015 M. The suspension was layered into an equal volume of 1.3 M sucrose/0.015 M CsCl and then centrifuged (240,000 g, 1 h) in an SW 55Ti rotor. The rough microsomes were in the pink sediment, and the smooth microsomes were at the interface. The smooth microsomes were diluted with an equal volume of 0.25 M sucrose (pH 7.0) and centrifuged (140,000 g, 1 h) in an SW 55i rotor.


## 2.2.2. Detection and Validation of the Purity of Microsomes

Electron microscopy and Western blotting were used to detect and validate the purity of prepared microsomes. For electron microscope analysis, the prepared microsomes were fixed with 2.5% glutaraldehyde for 24 h and 2% OsO4 for 2 h, dehydrated with alcohol (50%, 70%, 90%, and 100% in turn), and processed into epoxy resin. Thin sections (500 Å‌) were prepared and stained with uranyl acetate and lead citrate then examined with a transmission electron microscope (H-600-1, Hitachi, Japan). For Western blotting analysis, the microsome fractions were lysed (4°C; 30 min) in lysis buffer (50 mM Tris-Hcl, 150 mM NaCl, 1 mM EDTA, 1% Triton-X100, and 0.1% SDS). The protein samples (50 μg) were subjected to electrophoresis on SDS-PAGE with 12% gel and transferred to PVDF membrane (Millipore). The PVDF membranes with proteins were immunoblotted with antibodies to endoplasmin (ER marker), OxPhos complex IV subunit I (mitochondrial marker), catalase (peroxisomal marker), and cadherin (cytoplasmic marker), respectively.


## 2.2.3. Separation of Microsome Proteins by 2DE

2DE was performed as described by the manufacturer (Amersham Biosciences). Protein samples (400μg) were diluted to 450 μL with rehydration solution (7 mol/L urea, 2 mol/L thiourea, 0.2% DTT, 0.5% (v/v) pH3–10 NL IPG buffer, and trace bromophenol blue) and applied to IPG strips (pH 3–10 NL; 24 cm) for rehydration (14 h; 30 V). Proteins were focused successively (1 h at 500 V, 1 h at 1,000 V, and 8.5 h at 8,000 V) to give a total of 68 kVh on an IPGphor. After equilibration, SDS-PAGE was performed with 12% gel on Ettan DALT II system. Then, the blue silver staining method was used to visualize the protein spots on the 2DE gels [14].


## 2.3. Na2CO3 Extraction and 1DE Analysis of Microsome Membrane Proteins

Microsome membrane proteins were further extracted by the carbonate method from isolated microsomal fractions [12]. Microsomal fractions were diluted 50- to 1,000-fold with 100 mM sodium carbonate (pH 11.5; final protein concentration to 0.02 to 1 mg/mL), and incubated (0°C; 30 min) with slow stirring and accompanying sonication for 15 sec at 3-4 W at 0 min, 15 min, and 30 min. The suspensions were centrifuged and decanted, and the membrane pellets were gently rinsed three times with ice-cold distilled water. These pellets were diluted with denaturing sample buffer (5% mercaptoethanol, 2% SDS, 0.06 M Tris-HCl, pH 6.8, and 10% glycerol), heated (95°C; 5 min), and then subjected to 1D SDS-PAGE with a 12% gel. Electrophoresis was performed at 80 V for 20 min, followed by 100 V for 2 h. Gels were visualized with Coomassie Brilliant Blue G [14].


## 2.4. Tandem Mass Spectrometry (MS/MS) Identification of Proteins

### 2.4.1. In-Gel Digestion

The proteins contained in the 2D gel spots and 1D gel bands were subjected to in-gel digestion with trypsin. Gel spots or bands were excised and destained with 100 mM NH4HCO3 in 50% acetonitrile (ACN) at room temperature. The proteins were reduced with 10 mM dithiothreitol (DDT) (56°C; 30 min) and alkylated with 50 mM iodoacetamide in 100 mM NH4HCO3 (dark, room temperature, 30 min). The gel pieces that contained proteins were dried and then incubated in the digestion solution (40 mM NH4HCO3, 9% ACN, and 20 μg/mL trypsin; 18 h, 37°C). The tryptic peptides were extracted with 50% ACN/2.5% TFA and then dried using a Speed-Vac.


### 2.4.2. Nanoliquid Chromatography (LC) MS/MS and Protein Identification

The tryptic peptide mixture was fractionated with reverse-phase (RP) high-performance liquid chromatography (HPLC) by using an Ultimate nano-HPLC system (Dionex). Peptide samples were purified and concentrated with a C18-PepMap precolumn and then separated on an analytical C18-PepMap column (75μm ID × 150 mm, 100 Å pore size, 3 mm particle size) at a column flow rate of 300 nL/min. The ACN gradient (solution A: 0.1% formic acid, 2% ACN; solution B: 0.1% formic acid, 80% ACN) started at 5% B and ended at 70% B in 45 min. Mass spectrometry (MS) and MS/MS data were acquired using a Micromass quadrupole time of flight Micromass spectrometer (Waters). Database searches were carried out with the MASCOT server by using a decoy database (concatenated forward-reverse mouse IPI database, version 3.07; release date June 20, 2005). A mass tolerance of 0.3 Da for both parent (MS) and fragmented (MS/MS) ions, allowance for up to one trypsin miscleavage, variable amino acid modifications consisting of methionine oxidation and cysteine carbamidomethylation were used. MS/MS ion score threshold was determined to produce a false-positive rate less than 5% for a significant hit (P<0.05). The false-positive rate was calculated with 2* reverse/(reverse + forward)/100. In the current study, the MS/MS ion score threshold was 23 and a false-positive rate was approximately 3.1%. For all the proteins that were identified with only one peptide, each MS/MS spectrum was checked manually.


## 2.4.1. In-Gel Digestion

The proteins contained in the 2D gel spots and 1D gel bands were subjected to in-gel digestion with trypsin. Gel spots or bands were excised and destained with 100 mM NH4HCO3 in 50% acetonitrile (ACN) at room temperature. The proteins were reduced with 10 mM dithiothreitol (DDT) (56°C; 30 min) and alkylated with 50 mM iodoacetamide in 100 mM NH4HCO3 (dark, room temperature, 30 min). The gel pieces that contained proteins were dried and then incubated in the digestion solution (40 mM NH4HCO3, 9% ACN, and 20 μg/mL trypsin; 18 h, 37°C). The tryptic peptides were extracted with 50% ACN/2.5% TFA and then dried using a Speed-Vac.


## 2.4.2. Nanoliquid Chromatography (LC) MS/MS and Protein Identification

The tryptic peptide mixture was fractionated with reverse-phase (RP) high-performance liquid chromatography (HPLC) by using an Ultimate nano-HPLC system (Dionex). Peptide samples were purified and concentrated with a C18-PepMap precolumn and then separated on an analytical C18-PepMap column (75μm ID × 150 mm, 100 Å pore size, 3 mm particle size) at a column flow rate of 300 nL/min. The ACN gradient (solution A: 0.1% formic acid, 2% ACN; solution B: 0.1% formic acid, 80% ACN) started at 5% B and ended at 70% B in 45 min. Mass spectrometry (MS) and MS/MS data were acquired using a Micromass quadrupole time of flight Micromass spectrometer (Waters). Database searches were carried out with the MASCOT server by using a decoy database (concatenated forward-reverse mouse IPI database, version 3.07; release date June 20, 2005). A mass tolerance of 0.3 Da for both parent (MS) and fragmented (MS/MS) ions, allowance for up to one trypsin miscleavage, variable amino acid modifications consisting of methionine oxidation and cysteine carbamidomethylation were used. MS/MS ion score threshold was determined to produce a false-positive rate less than 5% for a significant hit (P<0.05). The false-positive rate was calculated with 2* reverse/(reverse + forward)/100. In the current study, the MS/MS ion score threshold was 23 and a false-positive rate was approximately 3.1%. For all the proteins that were identified with only one peptide, each MS/MS spectrum was checked manually.


## 2.5. Bioinformatics Analysis of Identified Proteins

Protein annotations were obtained primarily from UniProt 7.0 including accession, entry name, comments such as function, catalytic activity, subcellular location, and similarity. The Cytoscape plugin, Biological Networks Gene Ontology (BinGO), was used to find statistically overrepresented GO categories of the protein dataset. An online tool, WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/), was used to map target proteins to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The pathway visualization was based on the pathway mapping service provided in KEGG.


## 3. Results

### 3.1. Characterization and Detection of Liver Microsomes

It was essential to obtain a highly pure fraction to conduct proteomic characterization of microsomes. The purity of prepared microsomes was monitored with electron microscope and Western blotting analysis. A large number of nearly spherical membrane vesicles were visualized with electron microscope without other contaminated organelle compositions (see Supplemental Figure  1(a) in Supplementary Material available online at doi:10.1155/2012/832569). Western blotting analyses showed that, with the standard immunoblotting protocol, the ER marker endoplasmin was enriched in the isolated microsome fractions without the contamination marker (mitochondrial marker OxPhos Complex IV subunit I, peroxisomal marker catalase, and cytoplasmic marker cadherin) being detected (Supplemental Figure  1(b)). The results demonstrated an optimized preparation of microsomes.


### 3.2. Fractionation and Identification of Microsome Proteins Identified by 2DE and MS/MS

The 2DE reference maps display514±83protein spots (n=10 gels). A representative 2DE map of microsome proteins was shown (Figure 1). A total of 183 proteins were identified with ESI-Q-TOF MS/MS from 204 excised gel spots. Those proteins are summarized (Table 1 and Supplemental Table  1), including 2D gel-spot number, IPI number, protein name, predicted TMD, and subcellular location. The microsomal marker proteins such as endoplasmin (Spot 2) and UDP glucuronosyltransferase (Spots 6 and 7) were identified. Those proteins were located in different subcellular locations (Table 1) including ER, mitochondrial membrane, cytoplasmic, ribosome, microbody, microsome membrane, nuclear, vesicular membrane, sarcolemma, extracellular space, cilium, ER-Golgi intermediate compartment, and secreted proteins. Supplemental Figure  2 shows the percentage of each group of proteins, according to their subcellular locations, derived from the annotations in the Swiss-Prot database and Gene Ontology: 22% of proteins (n=41) from ER and Golgi, 11% of proteins (n=20) from mitochondria and other membranes, 50% of proteins (n=91) from cytosolic and other soluble proteins, 8% of secreted proteins (n=15), and 9% of proteins without unambiguous location (n=16).Table 1
Proteins identified from mouse liver microsomal preparations with 2DE-based strategy.
Spot no.IPIaProtein namePredicted TMDLocation90,91IPI00108939glyceraldehyde-3-phosphate dehydrogenase, spermatogenic0ER6IPI00111936UDP-glucuronosyltransferase 1-2 precursor, microsomal1ER145IPI00121833Acetyl-coenzyme A acyltransferase 10ER102IPI00622235Transitional endoplasmic reticulum ATPase0ER6,61,194IPI00122815Prolyl 4-hydroxylase, beta polypeptide0ER17IPI00123176Similar to glyceraldehyde-3-phosphate dehydrogenase, 37 kDa protein0ER134,135IPI00123342Hypoxia upregulated 11ER2IPI00129526Endoplasmin0ER139IPI00131459Nucleoside diphosphate kinase A0ER179IPI00132874Splice isoform 1 of monoglyceride lipase0ER163IPI00133522Protein disulfide-isomerase precursor0ER49IPI00134058Thioredoxin domain containing protein 4 precursor0ER108,145,65IPI00135284Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ISOFORM 10ER147,148,149IPI00135686Mus musculus adult male kidney cDNA, RIKEN full-length enriched library, clone: 06100081ER174,178,179,183IPI00135726Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)0ER49,50IPI00163011Thioredoxin domain containing protein 5 precursor0ER137IPI00226993Thioredoxin0ER148IPI00229551ADAM41ER62,157,158,162IPI00230108Glucose-regulated protein, full insert sequence0ER148IPI00271869Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)0ER146,147,149,150,153IPI00273646Glyceraldehyde-3-phosphate dehydrogenase0ER187IPI00555023Glutathione S-transferase P 10ER144IPI00319652Glutathione peroxidase0ER84IPI0031999278 kDa glucose-regulated protein precursor0ER153IPI00320208Elongation factor 1-beta0ER118IPI00323357Heat shock cognate 71 kDa protein0ER173IPI00323661Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)0ER145,201IPI00462605Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)0ER127IPI00469307Alpha-2-macroglobulin receptor-associated protein precursor0ER143,152,203IPI004803432700050F09Rik protein0ER162IPI00831714Leucine-rich repeat-containing protein 70ER (integral to membrane)149,150IPI00352124Flavin containing monooxygenase 51ER (integral to membrane)131IPI00132397GTP-binding protein SAR1b0ER (peripheral membrane protein)107IPI00227657Stromal cell-derived factor 2-like protein 1 precursor0ER lumen145IPI00123281Expressed sequence AA9597421ER membrane7IPI00222496Protein disulfide isomerase-associated 61ER, membrane proteinb7IPI00112322UDP-glucuronosyltransferase 2B5 precursor1ER, membrane proteinsb156IPI00331322Microsomal glutathione S-transferase 13ER, outer membrane151IPI00319973Membrane-associated progesterone receptor component 11ER, membrane-bound152IPI00170316Multiple coagulation factor deficiency protein 2 homolog precursor0ER-Golgi intermediate compartment170IPI00408892RAS-related protein RAB-70Golgi, endosomes, lysosomes153IPI00123316Splice isoform 1 of tropomyosin 1 alpha chain0Cytoplasm116IPI00133456Senescence marker protein-300Cytoplasm89,129,130,151,152IPI00135085Heme-binding protein0Cytoplasm165IPI00109061Tubulin beta-4 chain homolog0Cytoplasmic88IPI00109073Tubulin beta-4 chain0Cytoplasmic105,138IPI00110753Tubulin alpha-1 chain0Cytoplasmic113,166,167,197,204IPI00110827Actin, alpha skeletal muscle0Cytoplasmic9,129,130,151.153,166,167,197,198IPI00110850Actin, cytoplasmic 10Cytoplasmic90IPI00114162Fatty acid-binding protein, epidermal0Cytoplasmic145IPI00116277T-complex protein 1, delta subunit0Cytoplasmic144IPI00117264DJ-1 protein0Cytoplasmic191,164,165IPI00117348Tubulin alpha-2 chain0Cytoplasmic137,138,164IPI00117350Tubulin alpha-4 chain0Cytoplasmic141,153,165,132IPI00117352Tubulin beta-5 chain0Cytoplasmic126IPI00117914Arginase 10Cytoplasmic152IPI0012053221 kDa protein0Cytoplasmic107,108,139,143,182IPI0012548944 KD protein (Argininosuccinate synthase)0Cytoplasmic191IPI00626790Glutamine synthetase0Cytoplasmic176,182,194IPI00130950Betaine-homocysteine S-methyltransferase0Cytoplasmic99IPI00131204UDP-glucose pyrophosphorylase 20Cytoplasmic204IPI00136929Gamma actin-like protein0Cytoplasmic101,132IPI00169463Tubulin beta-2C Chain0Cytoplasmic202,133IPI00221400Alcohol dehydrogenase A chain0Cytoplasmic89IPI00221528Actin, cytoplasmic type 5 homolog0Cytoplasmic168IPI00221890Carbonic anhydrase III0Cytoplasmic202,133IPI00317740Guanine nucleotide-binding protein beta subunit 2-like 10Cytoplasmic159IPI00331174T-complex protein 1, eta subunit0Cytoplasmic154IPI00338039Tubulin, beta 20Cytoplasmic141IPI00348094Predicted: similar to tubulin M beta 10Cytoplasmic136IPI00404011Microtubule-associated protein0Cytoplasmic153IPI00421223Tropomyosin alpha 4 chain0Cytoplasmic194,195IPI00457825Similar to argininosuccinate synthase (Citrulline-aspartate ligase)0Cytoplasmic60IPI00462072Similar to alpha enolase (2-phospho-D-glycerate hydro-lyase)0Cytoplasmic178IPI00467066Glycine N-methyltransferase0Cytoplasmic63,109IPI00467833Triosephosphate isomerase0Cytoplasmic153IPI00605380Similar to tubulin alpha-2 chain (Alpha-tubulin 2)0Cytoplasmic162IPI00123313Ubiquitin-activating enzyme E1 10Cytoplasmic and nuclear64IPI00420745Proteasome subunit, alpha type 2, full insert sequence0Cytoplasmic and nuclear145IPI00320165Oxidoreductase HTATIP20Cytoplasmic and nuclear153IPI00117978Cytochrome c oxidase subunit IV isoform 1, mitochondrial precursor1Mitochondrial inner membrane19IPI00109167NADH-ubiquinone oxidoreductase 24 kDa subunit0Mitochondrial inner membrane158IPI00111885Ubiquinol-cytochrome-c reductase complex core protein I, mitochondrial precursor0Mitchondrial inner membrane175IPI00121322Electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial precursor0Mitchondrial inner membrane196IPI00128023NADH-ubiquinone oxidoreductase 49 kDa subunit, mitochondrial precursor0Mitchondrial inner membrane134IPI00111908Predicted: carbamoyl-phosphate synthetase 10Mitochondrial145IPI00114840Endonuclease G, mitochondrial precursor0Mitochondrial70IPI003315552-oxoisovalerate dehydrogenase alpha subunit, mitochondrial precursor0Mitochondrial94,95IPI00115607Trifunctional enzyme beta subunit, mitochondrial precursor0Mitochondrial145IPI00115824NipSnap1 protein0Mitochondrial22IPI00116154Cytochrome c oxidase, subunit vb, full insert sequence0Mitochondrial15,146,147,148,149,100IPI00118986ATP synthase oligomycin sensitivity conferral protein, mitochondrial precursor0Mitochondrial127IPI00119138Ubiquinol-cytochrome-c reductase complex core protein 2, mitochondrial precursor0Mitochondrial147,148IPI00120984NADH-ubiquinone oxidoreductase 19 kDa subunit0Mitochondrial137IPI00129516Ubiquinol-cytochrome c reductase complex 11 kDa protein, mitochondrial precursor0Mitochondrial93,99,100,192,203IPI00130280ATP synthase alpha chain, mitochondrial precursor0Mitochondrial149,150IPI00132217Tetratricopeptide repeat protein 111Mitochondrial150,151IPI00132390NADH-ubiquinone oxidoreductase B15 subunit1Mitochondrial101,132,137,141,153IPI00170093NADH-ubiquinone oxidoreductase 23 kDa subunit, mitochondrial precursor0Mitochondrial92,93,94,95,96IPI00223092Hydroxyacyl-coenzyme A dehydrogenase/3-ketoacyl-coenzyme A0Mitochondrial142,143,152IPI00230507ATP synthase D chain, mitochondrial0Mitochondrial162IPI00308882NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial precursor0Mitochondrial149IPI0034400413 KDa differentiation-associated protein0Mitochondrial145,146IPI00420718Hydroxymethylglutaryl-CoA synthase, mitochondrial precursor0Mitochondrial51IPI0030888560 kDa heat shock protein, mitochondrial0Mitochondrial153IPI00462250Similar to adenine nucleotide translocase3Mitochondrial85,165,167,203IPI00468481ATP synthase beta chain, mitochondrial precursor0Mitochondrial147IPI00117281Phospholipid hydroperoxide glutathione peroxidase, mitochondrial precursor0Mitochondrial and cytoplasmic169IPI00133240Ubiquinol-cytochrome c reductase iron-sulfur subunit, mitochondrial precursor0Mitochondrial inner membrane200,201IPI00230351Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial precursor0Mitochondrial inner membrane174IPI00132042Pyruvate dehydrogenase E1 component beta subunit, mitochondrial precursor0Mitochondrial matrix156IPI00315794Cytochrome b5 outer mitochondrial membrane isoform precursor1Mitochondrial outer membrane65,109,144,170,176,100IPI00134746Argininosuccinate synthase0mitochondrion145,146IPI00338536Succinate dehydrogenase [ubiquinone] iron-sulfur protein, mitochondrial precursor0Mitochondrion112IPI00122547Voltage-dependent anion-selective channel protein 20Mitochondrion outer membrane147IPI00131186Splice isoform 2 of transcription factor BTF30Nuclear149IPI00317794Nucleolin0Nuclear53IPI00331146UMP-CMP kinase0Nuclear150IPI00458856Similar to ZNF91L isoform 10Nuclear6IPI00461822E1A binding protein p3000Nuclear55IPI00126172RIKEN cDNA 4931406C07, PTD012 homolog0Nuclear150,151IPI0011324140S ribosomal protein S190Ribosome104IPI00116908Similar to 40 s ribosomal protein S120Ribosome147IPI0084979360S ribosomal protein L120Ribosome81,127,145IPI0012597126S protease regulatory subunit S10B0Ribosome199IPI0012360440S ribosomal protein SA0Ribosome125IPI0013564026S protease regulatory subunit 80Ribosome149,150,151IPI0013978060S ribosomal protein L230Ribosome149,150IPI0022254660S ribosomal protein L220Ribosome145IPI0031495060S acidic ribosomal protein P00Ribosome149IPI0032256240S ribosomal protein S140Ribosome146IPI0033109240S ribosomal protein S4,X isoform0Ribosome149,150IPI0033146160S ribosomal protein L110Ribosome188IPI00351894Similar to ribosomal protein0Ribosome148IPI0084979360S ribosomal protein L120Ribosome149IPI0046588040S ribosomal protein S170Ribosome199IPI0012360440S ribosomal protein SA0Ribosome4,11IPI00121209Apolipoprotein A-I precursor0Secreted149IPI00121837Ribonuclease 4 precursor1Secreted12IPI00122429Plasma retinol-binding protein precursor0Secreted163IPI00123920Alpha-1-antitrypsin 1–3 precursor0Secreted163IPI00123924Alpha-1-antitrypsin 1–4 precursor0Secreted163IPI00123927Alpha-1-antitrypsin 1–5 precursor0Secreted162IPI00128484Hemopexin precursor1Secreted3,117,118IPI00131695Serum albumin precursor0Secreted98IPI00139788Serotransferrin precursor0Secreted199IPI00323571Apolipoprotein E precursor0Secreted135,136IPI00377351Apolipoprotein A-IV precursor0Secreted163IPI00406302Alpha-1-antitrypsin 1-1 precursor0Secreted100,155,156IPI0046639921 kDa protein0Secreted156IPI00480401Major urinary protein 1 precursor0Secreted122IPI00130661Tripeptidyl-peptidase I precursor0Secreted (lysosomal)101IPI00115302Branched chain ketoacid dehydrogenase E1, beta polypeptide0Membrane197IPI00120716Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 10Membrane21,137IPI00120719Cytochrome c oxidase, subunit va, full insert sequence0Membrane125IPI00124790Polyposis locus protein 1-like 13Membrane129IPI00132076Catechol O-methyltransferase1Membrane130,142IPI00138406Ras-related protein Rap-1A0Membrane174IPI00162780Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 20Membrane88,154IPI00230113Cytochrome b51Membrane199,200IPI00353727Annexin A40Membrane110IPI00117416Neighbor of COX40Unknown143IPI00121271Hypothetical S-adenosyl-L-methionine-dependent methyltransferases structure containing protein0Unknown144,108IPI00267667RIKEN cDNA 6330409N04, CLLL6 protein homolog0Unknown101IPI00269613Eukaryotic translation initiation factor 3 subunit 20Unknown149,150IPI0030783751 kDa protein0Unknown203IPI00318204Sid6061p0Unknown105IPI00273646Similar to glyceraldehyde-3-phosphate dehydrogenase0Unknown189,190,194IPI00626790Glutamine synthetase0Unknown50IPI0034584286 KDa PROTEIN0Unknown51IPI0035078045 kDa protein0Unknown133IPI0038123177 KDa protein0Unknown144IPI00923085Probable ubiquitin-conjugating enzyme E2 FLJ25076 homolog0Unknown146,147,150,173IPI0046029544 KDa protein0Unknown156IPI00330913Major urinary protein 260Unknown59IPI00467988169 kDa protein0Unknown100,155,156IPI0046951721 kDa protein0Unknown149IPI00130554Splice isoform 1 of SNARE-associated protein Snapin0Vesicular membrane101,127,134IPI00131366Keratin, type II cytoskeletal 6B0Sarcolemma83,106,107IPI00121788Peroxiredoxin 10Microbody101,139IPI00348328Keratin Kb400Intermediate filament156IPI00137414Left-right dynein0CiliumaESI-Q-TOF identification, subcellular location are given for each ID number.bThis protein is nonmembrane associated according to the annotation in the Swiss-Prot database but has one predicted TMD.Figure 1
2DE pattern of mouse liver microsome. Microsomal proteins (400μg) were arrayed by 2DE with IPG strip (pH 3–10 NL; 24 cm) and SDS-PAGE with 12% gel and visualized with blue silver staining method. A total of 204 spots denoted by circles were MS-analyzed.


### 3.3. Fractionation and Identification of Microsomal Membrane Proteins Identified by 1DE and MS/MS

The Na2CO3-treated microsome membrane proteins were separated on SDS-PAGE gels and visualized with Coomassie brilliant blue staining (Figure 2(a)). A total of 99 proteins (Table 2 and Supplemental Table  2) was identified with electrospray ionization- (ESI-) Q-TOF MS/MS from 17 gel bands (Figure 2(a)). Those proteins were derived from the ER, type I/II membrane proteins, integral membrane proteins, major histocompatibility complex class I protein, ER-Golgi intermediate compartment, mitochondrial membrane, nuclear, cytoplasm, microbody, sarcolemma, and secreted and unknown proteins (Table 2). Those membrane proteins were classified into three categories (Figure 2(b)): (a) proteins with known membrane associations (55%; n=54), (b) putative membrane proteins (5%; n=5), and (c) other proteins (40%; n=40). Those identified proteins were categorized according to the reported annotation in the UniProt database (http://www.uniprot.org/) and predictions for transmembrane regions (http://www.cbs.dtu.dk/services/TMHMM/). Of the 99 proteins, 59 (60%) were described as “membrane-associated” proteins (category (a) and (b)), including ER-characteristic proteins (cytochromes P-450 and b5, calnexin, integral membrane enzymes such as NADPH-cytochrome c reductase, and microsomal glutathione S-transferase 1).Table 2
Proteins identified from Na2CO3-extracted mouse liver microsomal membrane preparations with 1DE-based strategy.
Bands no.Accession no.Protein namePredicted TMDGRAVY scorePI valueSubcellular location9IPI00112322UDP-glucuronosyltransferase 2B5 precursor1−0.0317.94ER9IPI00127223UDP glucuronosyltransferase 2 family, polypeptide B361−0.0368.47ER9IPI00222496Protein disulfide-isomerase A61−0.2925.05ER8IPI00417182UDP-glycosyltransferase 1 family polypeptide A510.0448.33ER9IPI00116572Cytochrome P450, family 2, subfamily d, polypeptide 92−0.0436.37ER15IPI0011365540S ribosomal protein S60−0.91810.68ER5IPI00129526Endoplasmin precursor (ER protein 99,94 kDa glucose-regulated protein)0−0.724.74ER13IPI00130985Short-chain dehydrogenase CRAD200.0268.35ER6IPI00222809Similar to GDH/6PGL endoplasmic bifunctional protein0−0.186.61ER8IPI00230108Glucose-regulated protein, full insert sequence0−0.4795.78ER10,11IPI00317356Paraoxonase 10−0.015.02ER7IPI0031999278 kDa glucose-regulated protein precursor0−0.4815.07ER13IPI00121079NADH-cytochrome b5 reductase 30−0.2038.56ER, membrane bound9IPI00123964Cytochrome P450 2A51−0.2039.23ER, membrane bound9IPI00114779Cytochrome P450 2C380−0.1478.69ER, membrane bound17IPI00331322Microsomal glutathione S-transferase 130.149.67ER and mitochondrial outer membrane17IPI00119766Cis-retinol androgen dehydrogenase 100.0059.25ER lumen8IPI00134691UDP-glucuronosyltransferase 1-1 precursor, microsomal20.0878.87ER, integral to plasma membrane8IPI00128287Cytochrome P450 1A21−0.2038.92ER, membrane bound10IPI00136910Cytochrome P450 2D112−0.0096.15ER, membrane bound9IPI00308328Cytochrome P450 2F21−0.1357.74ER, membrane bound9,10IPI00323908Cytochrome P450 2D102−0.0736.16ER, membrane bound7IPI00112549Long-chain-fatty-acid-CoA ligase 11−0.0456.81ER, type III membrane protein8IPI00133522Protein disulfide-isomerase precursor0−0.3864.79ER9IPI00116572Cytochrome P450 2D90−0.0635.93ER, membrane bound5,6IPI00119618Calnexin precursor1−0.8754.5ER, type I membrane protein1,10,12,14,15IPI00319973Membrane-associated progesterone receptor component 11−0.6164.57ER, membrane bound8IPI00132475Protein ERGIC-531−0.5455.92ER-Golgi intermediate compartment (ERGIC), type I membrane protein8,17IPI00109061Tubulin beta-4 chain homolog0−0.4064.78Cytoplasmic10IPI00110827Actin, alpha skeletal muscle0−0.2325.23Cytoplasmic10,12,14IPI00110850Actin, cytoplasmic 10−0.25.29Cytoplasmic1,2,3IPI00111908Carbamoyl-phosphate synthase0−0.126.42Cytoplasmic1,8,9,13IPI00117348Tubulin alpha-2 chain0−0.234.94Cytoplasmic9,10,11,12,13IPI00117914Arginase 10−0.1876.52Cytoplasmic17IPI00120451Fatty acid-binding protein, liver0−0.4098.59Cytoplasmic9IPI00129028Similar to tubulin, alpha 3C isoform 10−0.2044.98Cytoplasmic1–11,13,17IPI00130950Betaine-homocysteine S-methyltransferase0−0.368.01Cytoplasmic1,4,6,10,11,14,15IPI00134746Argininosuccinate synthase0−0.3618.36Cytoplasmic3,4IPI00114710Pyruvate carboxylase, mitochondrial precursor0−0.1736.25Mitochondrial17IPI00553333Hemoglobin subunit beta-100.0927.13Mitochondrial9IPI00134809Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex0−0.1719.1Mitochondrial17IPI00117978Cytochrome c oxidase subunit IV isoform 1, mitochondrial precursor1−0.4129.25Mitochondrial inner membrane15,16IPI00315794Cytochrome b5 outer mitochondrial membrane isoform precursor1−0.6024.79Mitochondrial outer membrane13IPI00321718Prohibitin-20−2.589.83Mitochondrial, cytoplasmic, nuclear13IPI00122547Voltage-dependent anion-selective channel protein 20−0.2237.44Outer mitochondrial Membrane17IPI00114559Histone H2A type 10−0.57211.22Nuclear16,17IPI00114642Histone H2B F0−0.76210.32Nuclear8IPI00387318Cell cycle control protein 50A2−0.3318.58Membrane15IPI00113849Splice isoform 2 of cell division control protein 42 homolog0−0.1576.16Membrane13IPI00122549Splice isoform Pl-VDAC1 of voltage-dependent anion-selective channel protein 10−0.3348.55Membrane15IPI00127408Ras-related C3 botulinum substrate 10−0.1018.77Membrane15IPI00138406Ras-related protein Rap-1A0−0.3756.39Membrane6IPI00116921Scavenger receptor class B member 120.0738.29Integral membrane protein1IPI00121985Splice Isoform 1 of solute carrier organic anion transporter family, member 1B2120.1728.95Integral membrane protein9IPI00124830Integrin-associated protein precursor50.5638.58Integral membrane protein14,15IPI00131176Cytochrome c oxidase subunit 220.274.6Integral membrane protein1IPI00132604Secretedretory carrier-associated membrane protein 340.0287.55Integral membrane protein1IPI00135701Solute carrier organic anion transporter family, member 1A1110.2448.58Integral membrane protein1IPI00311682Sodium/potassium-transporting ATPase alpha-1 chain precursor100.0025.3Integral membrane protein6IPI00331214Platelet glycoprotein IV2−0.0538.61Integral membrane protein2IPI00119063Prolow-density lipoprotein receptor-related protein 11−0.5025.17Integral to membrane1,12,16,17IPI00124790Polyposis locus protein 1-like 130.3756.82Integral to membrane10IPI00129677Asialoglycoprotein receptor major subunit1−0.665.99Integral to membrane17IPI00467119Camello-like protein 110.3029.61Integral to membrane5,8IPI00316329Keratin, type II cytoskeletal 10−0.5888.2Intermediate filament10,11,12IPI00108844Cation-dependent mannose-6-phosphate receptor precursor1−0.1685.24Type I membrane protein9IPI00109998H-2 class I histocompatibility antigen, D-B alpha chain precursor1−0.5086.28Type I membrane protein4IPI00120245Integrin alpha-V precursor1−0.2465.46Type I membrane protein3,4IPI00121190Epidermal growth factor receptor precursor2−0.3166.46Type I membrane protein2IPI00126186Macrophage mannose receptor 1 precursor1−0.56.47Type I membrane protein5IPI00134549Splice isoform LAMP-2A of lysosome-associated membrane glycoprotein 2 precursor1−0.0367.05Type I membrane protein13IPI00312018Malectin1−0.2035.73Type I membrane protein3IPI00312063Low-density lipoprotein receptor precursor1−0.3914.88Type I membrane protein16IPI00466570Transmembrane emp24 domain-containing protein 102−0.1696.25Type I membrane protein4IPI00108535Carcinoembryonic antigen-related cell adhesion molecule 11−0.3025.35Type I membrane protein5IPI00310059Polymeric-immunoglobulin receptor precursor1−0.4995.26Type I membrane protein also secreted9IPI00121550Sodium/potassium-transporting ATPase beta-1 chain1−0.558.83Type II membrane protein2,3IPI00134585Glutamyl aminopeptidase1−0.3445.28Type II membrane protein10IPI00307966ADP-ribosyl cyclase 11−0.1068.64Type II membrane protein3IPI00319509Aminopeptidase N1−0.2775.62Type II membrane protein3IPI00458003Ectonucleotide pyrophosphatase/phosphodiesterase 31−0.3466.13Unknown9IPI00409409CD1D1 protein1−0.1789.22Unknown7IPI00621548NADPH-cytochrome P450 reductase1−0.4635.37Unknown9IPI00321644Cytochrome P450 2D262−0.1056.16Unknown1IPI00127016Hydroxysteroid 17-beta dehydrogenase 60−0.0758.63Unknown16IPI00221721Hypothetical krab box containing protein, full insert sequence0−0.1429.84Unknown8IPI00224073Hypothetical peptidase family M20/M25/M40 containing protein, full insert sequence0−0.015.99Unknown16IPI00228379Ferritin light chain 20−0.4796.37Unknown17IPI0026684217 kDa protein0−0.66810.48Unknown15IPI00379258Similar to ferritin light chain 20−0.4548.51Unknown3IPI00405742Plexin B20−0.35.67Unknown2IPI00408258Structure-specific endonuclease subunit SLX40−0.7145.33Unknown11IPI00462251Hypothetical protein LOC72792 isoform 10−0.4295.82Unknown15IPI00605814Similar to Ferritin light chain 10−0.3586.42Unknown10IPI00131366Keratin, type II cytoskeletal 6B0−0.4888.32Sarcolemma10IPI00322209Keratin, type II cytoskeletal 80−0.6025.7Sarcolemma8IPI00853991Similar to VH coding region0−0.1025.31Secreted10IPI00126458MRNA1−0.555.66MHC class I protein complex15IPI00121788Peroxiredoxin 10−0.2218.26Microbody1DE pattern and membrane-associated characteristic classification of Na2CO3-extracted microsomal membrane proteins. (a) 1DE pattern. Molecular weight markers are shown on the left and bands excised for MS analysis are indicated on the right. Lanes S1 and S2 were loaded with the same protein samples (50 μg per lane). (b) Classification via membrane-associated characteristic. The criteria used for this classification were published reports, annotations in the genome database (http://www.uniprot.org/), and predictions for transmembrane regions (http://www.cbs.dtu.dk/services/TMHMM/).
(a)(b)Hydrophobicity is an important characteristic of a membrane protein. The grand average of hydropathy (GRAVY) scores (>−0.4) (http://us.expasy.org/tools/protparam.html) is an index to evaluate the hydrophobic status of a protein, indicates a hydrophobic protein, and suggests a membrane association. In the current study, 69 (70%) of the 99 proteins identified from 1DE had a GRAVY>-0.4 (Supplemental Figure  3), a score indicating the probability for membrane association. Moreover, some alkaline proteins with PI values close to or greater than 10 were separated by 1DE (Supplemental Figure  4), but they could not be detected in a conventional 2DE map.


### 3.4. Comparison of 2DE and 1DE Datasets

Among the 2DE dataset (n=183 proteins; Table 1) and 1DE dataset (n=99 proteins; Table 2), only 23 proteins (Table 3) were consistent between 2DE and 1DE datasets (23% of 1DE dataset, and 13% of 2DE dataset). A total of 259 nonredundant proteins (n=183+99-23) were identified in the microsome fraction through the strategy of combining 2DE with 1DE protein-separation technologies followed by ESI-Q-TOF MS/MS. The microsome consisted of a complex network of continuous membranes including ER, ER-Golgi intermediate complex—also referred to as the vesiculotubular clusters or pre-Golgi intermediates—and the Golgi apparatus [5]. Among those identified proteins, 62 located in ER and Golgi were definitely classified as microsome proteins by annotation in the Swiss-Prot database and the Gene Ontology (GO).Table 3
Proteins that are consistently present in both 2DE dataset of microsomal proteins (Table1) and 1DE dataset of Na2CO3-extracted microsomal proteins (Table 2).
Accession numberProtein namePredicted TMDGRAVY scoresPI valueLocationIPI00108454Similar to 40S ribosomal protein S60−0.91810.68ERIPI00112322aUDP-glucuronosyltransferase 2B5 precursor1−0.0317.94ERIPI00129526Endoplasmin precursor (ER protein 99, 94 kDa glucose-regulated protein)0−0.724.74ERIPI00133522Protein disulfide-isomerase precursor0−0.3864.79ERIPI00222496aProtein disulfide isomerase-associated 61−0.2925.05ERIPI00230108Glucose-regulated protein, full insert sequence0−0.4795.78ERIPI0031999278 kDa glucose-regulated protein precursor0−0.4815.07ERIPI00331322aMicrosomal glutathione S-transferase 130.149.67ER and mitochondrial outer membraneIPI00319973aMembrane-associated progesterone receptor component 11−0.6164.57ER, membrane boundIPI00109061Tubulin beta-4 chain homolog0−0.4064.78CytoplasmicIPI00110827Actin, alpha skeletal muscle0−0.2325.23CytoplasmicIPI00110850Actin, cytoplasmic 10−0.25.29CytoplasmicIPI00111908Predicted: carbamoyl-phosphate synthetase 10−0.126.42CytoplasmicIPI00117348Tubulin alpha-2 chain0−0.234.94CytoplasmicIPI00117914Arginase 10−0.1876.52CytoplasmicIPI00134746Argininosuccinate synthase0−0.3618.36CytoplasmicIPI00117978aCytochrome c oxidase subunit IV isoform 1, mitochondrial precursor1−0.4129.25Mitochondrial inner membraneIPI00315794aCytochrome b5 outer mitochondrial membrane isoform precursor1−0.6024.79Mitochondrial outer membraneIPI00122547aVoltage-dependent anion-selective channel protein 20−0.2237.44Outer mitochondrial membraneIPI00124790aPolyposis locus protein 1-like 130.3756.82Integral to membraneIPI00138406aRas-related protein Rap-1A0−0.3756.39MembraneIPI00121788Peroxiredoxin 10−0.2218.26MicrobodyIPI00131366Keratin, type II cytoskeletal 6B0−0.4888.32SarcolemmaaMembrane proteins with one or more predicted trans-membrane origins or validated by references.


### 3.5. Significantly Enriched GO Terms for Mouse Liver Microsome Proteins

Biological Networks Gene Ontology [15] and Cytoscape [16] plugins to find statistically overrepresented GO categories were used for the enrichment analysis of our protein dataset. The microsome protein dataset (n=259, from 1DE and 2DE datasets) was compared to a reference set of complete mouse proteome (IPI mouse) that was provided by Biological Networks Gene Ontology. The analysis was done with a hypergeometric test, and all significant (P<0.01) GO terms were selected after correcting for a multiple term testing with a Benjamini and Hochberg false discovery rate. The analysis was performed separately for molecular function, cellular component, and biological process categories, and x-fold enrichment for every overrepresented term in three GO categories was calculated (Supplemental Figure  5). The results showed that the terms were related to mostly catalytic activity in terms of molecular function, including metabolism-related oxidoreductase, hydrolase, and dehydrogenase. Similarly, terms belonging to the cellular component namespace include mitochondrion, ER, and ribosome. Finally, terms from the biological process namespace included metabolic process, localization, transport, and translation. All of the information suggested the main functions and compositions of microsome.


### 3.6. Significant Enrichment of KEGG Pathway for Mouse Liver Microsome Proteins

Biological pathways analysis based on KEGG pathway database was performed with an analysis toolkit—WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/) [17]. This toolkit allowed the functional annotation of gene/protein sets into well-characterized functional signaling pathways (KEGG: http://www.genome.jp/kegg/). In addition, an enrichment score was obtained of the frequency of occurrence of a specific protein (or gene) within any given experimental subset with respect to a species-specific background set. Thus, an enrichment factor (the observed frequency in input set/the expected frequency in background set) was created with a statistical value that indicated that the protein (or gene) was specifically overrepresented in the input dataset. In this current study, all the proteins except 81 (n=259-81=178) were linked to a total of 99 biological pathways in the KEGG, including metabolic pathway, glycolysis/gluconeogenesis, metabolism of xenobiotics by cytochrome P450, and PPAR signaling pathway. Among those pathways, 34 significantly (P<0.01) enriched biological processes analyzed by WebGestalt were obtained (Figure 3). Those biological processes were involved in cell metabolism, benzoate degradation, metabolism of xenobiotics, ribosome, biosynthesis, signaling pathway, and oxidative stress. Those results are known to be related to microsome.Figure 3
Significantly enriched KEGG pathways for mouse liver microsome proteins (n=259) that were derived from 1DE and 2DE strategies. KEGG pathway enrichment analysis was performed using WebGestalt. The pathways having enrichment (P<0.01) are presented. For each KEGG pathway, the bar shows the x-fold enrichment of the pathway in our dataset.To ascertain the coverage of our dataset with the enriched pathways or biological processes, the KEGG search service was used to map our dataset on KEGG pathways. Two of the aforementioned enriched KEGG pathways (metabolism of xenobiotics and ribosome) were related to the well-known function and composition of the microsome (Figure4). Enzyme Commission numbers (EC no., e.g, 1.14.14.1) are used to represent enzymes in metabolism. Highlighted in green background are known mouse enzymes annotated in the KEGG database and the red boxed are enzymes in our dataset (Figure 4(a)). All enzymes (n=9) that played a key role in every pathway of metabolism of xenobiotics were included in our dataset (Table 4). Thirteen proteins from large and small subunits of ribosome were also found in our dataset (Table 4) and are indicated with a red box (Figure 4(b)). These proteins interact physically with each other and form a large protein complex—the ribosome. All the identified proteins that are involved in those two pathways are summarized in Table 4, including their KEGG pathway, protein ID, and protein name.Table 4
Proteins involved in KEGG pathways. (a) Metabolism of xenobiotics. (b) Ribosome.
KEGG pathwayProtein IDProtein nameMS-identified proteinsA. Metabolism of exnobioticsEC:1.14.14.1IPI00128287Cytochrome P450 1A2+IPI00123964Cytochrome P450 2A5+IPI00116572Cytochrome P450 2D9+IPI00323908Cytochrome P450 2D10+IPI00321644Cytochrome P450 2D26+IPI00114779Cytochrome P450 2C38+IPI00308328Cytochrome P450 2F2+EC:2.5.1.18IPI00331322Microsomal glutathione S-transferase 1+EC:1.1.1.1IPI00221400Alcohol dehydrogenase A chain+B. RibosomeSmall subunitIPI0013564026S protease regulatory subunit 8+IPI0012597126S protease regulatory subunit S10B+IPI0033109240S ribosomal protein S4, X isoform+IPI00116908Similar to 40s ribosomal protein S12+IPI0032256240S ribosomal protein S14+IPI0046588040S ribosomal protein S17+IPI0011324140S ribosomal protein S19+IPI0012360440S ribosomal protein SA+IPI0031495060S acidic ribosomal protein P0+Large subunitIPI0033146160S ribosomal protein L11+IPI0084979360S ribosomal protein L12+IPI0022254660S ribosomal protein L22+IPI0013978060S ribosomal protein L23+Metabolism of xenobiotics by cytochrome P450 pathway, and ribosome map views of identified proteins. The two enriched metabolic pathway maps were generated by KEGG, which incorporated the proteomic data into the KEGG pathway maps. All of the genes in mouse are colored; the genes contained in the protein dataset are red.
(a)(b)


## 3.1. Characterization and Detection of Liver Microsomes

It was essential to obtain a highly pure fraction to conduct proteomic characterization of microsomes. The purity of prepared microsomes was monitored with electron microscope and Western blotting analysis. A large number of nearly spherical membrane vesicles were visualized with electron microscope without other contaminated organelle compositions (see Supplemental Figure  1(a) in Supplementary Material available online at doi:10.1155/2012/832569). Western blotting analyses showed that, with the standard immunoblotting protocol, the ER marker endoplasmin was enriched in the isolated microsome fractions without the contamination marker (mitochondrial marker OxPhos Complex IV subunit I, peroxisomal marker catalase, and cytoplasmic marker cadherin) being detected (Supplemental Figure  1(b)). The results demonstrated an optimized preparation of microsomes.


## 3.2. Fractionation and Identification of Microsome Proteins Identified by 2DE and MS/MS

The 2DE reference maps display514±83protein spots (n=10 gels). A representative 2DE map of microsome proteins was shown (Figure 1). A total of 183 proteins were identified with ESI-Q-TOF MS/MS from 204 excised gel spots. Those proteins are summarized (Table 1 and Supplemental Table  1), including 2D gel-spot number, IPI number, protein name, predicted TMD, and subcellular location. The microsomal marker proteins such as endoplasmin (Spot 2) and UDP glucuronosyltransferase (Spots 6 and 7) were identified. Those proteins were located in different subcellular locations (Table 1) including ER, mitochondrial membrane, cytoplasmic, ribosome, microbody, microsome membrane, nuclear, vesicular membrane, sarcolemma, extracellular space, cilium, ER-Golgi intermediate compartment, and secreted proteins. Supplemental Figure  2 shows the percentage of each group of proteins, according to their subcellular locations, derived from the annotations in the Swiss-Prot database and Gene Ontology: 22% of proteins (n=41) from ER and Golgi, 11% of proteins (n=20) from mitochondria and other membranes, 50% of proteins (n=91) from cytosolic and other soluble proteins, 8% of secreted proteins (n=15), and 9% of proteins without unambiguous location (n=16).Table 1
Proteins identified from mouse liver microsomal preparations with 2DE-based strategy.
Spot no.IPIaProtein namePredicted TMDLocation90,91IPI00108939glyceraldehyde-3-phosphate dehydrogenase, spermatogenic0ER6IPI00111936UDP-glucuronosyltransferase 1-2 precursor, microsomal1ER145IPI00121833Acetyl-coenzyme A acyltransferase 10ER102IPI00622235Transitional endoplasmic reticulum ATPase0ER6,61,194IPI00122815Prolyl 4-hydroxylase, beta polypeptide0ER17IPI00123176Similar to glyceraldehyde-3-phosphate dehydrogenase, 37 kDa protein0ER134,135IPI00123342Hypoxia upregulated 11ER2IPI00129526Endoplasmin0ER139IPI00131459Nucleoside diphosphate kinase A0ER179IPI00132874Splice isoform 1 of monoglyceride lipase0ER163IPI00133522Protein disulfide-isomerase precursor0ER49IPI00134058Thioredoxin domain containing protein 4 precursor0ER108,145,65IPI00135284Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ISOFORM 10ER147,148,149IPI00135686Mus musculus adult male kidney cDNA, RIKEN full-length enriched library, clone: 06100081ER174,178,179,183IPI00135726Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)0ER49,50IPI00163011Thioredoxin domain containing protein 5 precursor0ER137IPI00226993Thioredoxin0ER148IPI00229551ADAM41ER62,157,158,162IPI00230108Glucose-regulated protein, full insert sequence0ER148IPI00271869Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)0ER146,147,149,150,153IPI00273646Glyceraldehyde-3-phosphate dehydrogenase0ER187IPI00555023Glutathione S-transferase P 10ER144IPI00319652Glutathione peroxidase0ER84IPI0031999278 kDa glucose-regulated protein precursor0ER153IPI00320208Elongation factor 1-beta0ER118IPI00323357Heat shock cognate 71 kDa protein0ER173IPI00323661Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)0ER145,201IPI00462605Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)0ER127IPI00469307Alpha-2-macroglobulin receptor-associated protein precursor0ER143,152,203IPI004803432700050F09Rik protein0ER162IPI00831714Leucine-rich repeat-containing protein 70ER (integral to membrane)149,150IPI00352124Flavin containing monooxygenase 51ER (integral to membrane)131IPI00132397GTP-binding protein SAR1b0ER (peripheral membrane protein)107IPI00227657Stromal cell-derived factor 2-like protein 1 precursor0ER lumen145IPI00123281Expressed sequence AA9597421ER membrane7IPI00222496Protein disulfide isomerase-associated 61ER, membrane proteinb7IPI00112322UDP-glucuronosyltransferase 2B5 precursor1ER, membrane proteinsb156IPI00331322Microsomal glutathione S-transferase 13ER, outer membrane151IPI00319973Membrane-associated progesterone receptor component 11ER, membrane-bound152IPI00170316Multiple coagulation factor deficiency protein 2 homolog precursor0ER-Golgi intermediate compartment170IPI00408892RAS-related protein RAB-70Golgi, endosomes, lysosomes153IPI00123316Splice isoform 1 of tropomyosin 1 alpha chain0Cytoplasm116IPI00133456Senescence marker protein-300Cytoplasm89,129,130,151,152IPI00135085Heme-binding protein0Cytoplasm165IPI00109061Tubulin beta-4 chain homolog0Cytoplasmic88IPI00109073Tubulin beta-4 chain0Cytoplasmic105,138IPI00110753Tubulin alpha-1 chain0Cytoplasmic113,166,167,197,204IPI00110827Actin, alpha skeletal muscle0Cytoplasmic9,129,130,151.153,166,167,197,198IPI00110850Actin, cytoplasmic 10Cytoplasmic90IPI00114162Fatty acid-binding protein, epidermal0Cytoplasmic145IPI00116277T-complex protein 1, delta subunit0Cytoplasmic144IPI00117264DJ-1 protein0Cytoplasmic191,164,165IPI00117348Tubulin alpha-2 chain0Cytoplasmic137,138,164IPI00117350Tubulin alpha-4 chain0Cytoplasmic141,153,165,132IPI00117352Tubulin beta-5 chain0Cytoplasmic126IPI00117914Arginase 10Cytoplasmic152IPI0012053221 kDa protein0Cytoplasmic107,108,139,143,182IPI0012548944 KD protein (Argininosuccinate synthase)0Cytoplasmic191IPI00626790Glutamine synthetase0Cytoplasmic176,182,194IPI00130950Betaine-homocysteine S-methyltransferase0Cytoplasmic99IPI00131204UDP-glucose pyrophosphorylase 20Cytoplasmic204IPI00136929Gamma actin-like protein0Cytoplasmic101,132IPI00169463Tubulin beta-2C Chain0Cytoplasmic202,133IPI00221400Alcohol dehydrogenase A chain0Cytoplasmic89IPI00221528Actin, cytoplasmic type 5 homolog0Cytoplasmic168IPI00221890Carbonic anhydrase III0Cytoplasmic202,133IPI00317740Guanine nucleotide-binding protein beta subunit 2-like 10Cytoplasmic159IPI00331174T-complex protein 1, eta subunit0Cytoplasmic154IPI00338039Tubulin, beta 20Cytoplasmic141IPI00348094Predicted: similar to tubulin M beta 10Cytoplasmic136IPI00404011Microtubule-associated protein0Cytoplasmic153IPI00421223Tropomyosin alpha 4 chain0Cytoplasmic194,195IPI00457825Similar to argininosuccinate synthase (Citrulline-aspartate ligase)0Cytoplasmic60IPI00462072Similar to alpha enolase (2-phospho-D-glycerate hydro-lyase)0Cytoplasmic178IPI00467066Glycine N-methyltransferase0Cytoplasmic63,109IPI00467833Triosephosphate isomerase0Cytoplasmic153IPI00605380Similar to tubulin alpha-2 chain (Alpha-tubulin 2)0Cytoplasmic162IPI00123313Ubiquitin-activating enzyme E1 10Cytoplasmic and nuclear64IPI00420745Proteasome subunit, alpha type 2, full insert sequence0Cytoplasmic and nuclear145IPI00320165Oxidoreductase HTATIP20Cytoplasmic and nuclear153IPI00117978Cytochrome c oxidase subunit IV isoform 1, mitochondrial precursor1Mitochondrial inner membrane19IPI00109167NADH-ubiquinone oxidoreductase 24 kDa subunit0Mitochondrial inner membrane158IPI00111885Ubiquinol-cytochrome-c reductase complex core protein I, mitochondrial precursor0Mitchondrial inner membrane175IPI00121322Electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial precursor0Mitchondrial inner membrane196IPI00128023NADH-ubiquinone oxidoreductase 49 kDa subunit, mitochondrial precursor0Mitchondrial inner membrane134IPI00111908Predicted: carbamoyl-phosphate synthetase 10Mitochondrial145IPI00114840Endonuclease G, mitochondrial precursor0Mitochondrial70IPI003315552-oxoisovalerate dehydrogenase alpha subunit, mitochondrial precursor0Mitochondrial94,95IPI00115607Trifunctional enzyme beta subunit, mitochondrial precursor0Mitochondrial145IPI00115824NipSnap1 protein0Mitochondrial22IPI00116154Cytochrome c oxidase, subunit vb, full insert sequence0Mitochondrial15,146,147,148,149,100IPI00118986ATP synthase oligomycin sensitivity conferral protein, mitochondrial precursor0Mitochondrial127IPI00119138Ubiquinol-cytochrome-c reductase complex core protein 2, mitochondrial precursor0Mitochondrial147,148IPI00120984NADH-ubiquinone oxidoreductase 19 kDa subunit0Mitochondrial137IPI00129516Ubiquinol-cytochrome c reductase complex 11 kDa protein, mitochondrial precursor0Mitochondrial93,99,100,192,203IPI00130280ATP synthase alpha chain, mitochondrial precursor0Mitochondrial149,150IPI00132217Tetratricopeptide repeat protein 111Mitochondrial150,151IPI00132390NADH-ubiquinone oxidoreductase B15 subunit1Mitochondrial101,132,137,141,153IPI00170093NADH-ubiquinone oxidoreductase 23 kDa subunit, mitochondrial precursor0Mitochondrial92,93,94,95,96IPI00223092Hydroxyacyl-coenzyme A dehydrogenase/3-ketoacyl-coenzyme A0Mitochondrial142,143,152IPI00230507ATP synthase D chain, mitochondrial0Mitochondrial162IPI00308882NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial precursor0Mitochondrial149IPI0034400413 KDa differentiation-associated protein0Mitochondrial145,146IPI00420718Hydroxymethylglutaryl-CoA synthase, mitochondrial precursor0Mitochondrial51IPI0030888560 kDa heat shock protein, mitochondrial0Mitochondrial153IPI00462250Similar to adenine nucleotide translocase3Mitochondrial85,165,167,203IPI00468481ATP synthase beta chain, mitochondrial precursor0Mitochondrial147IPI00117281Phospholipid hydroperoxide glutathione peroxidase, mitochondrial precursor0Mitochondrial and cytoplasmic169IPI00133240Ubiquinol-cytochrome c reductase iron-sulfur subunit, mitochondrial precursor0Mitochondrial inner membrane200,201IPI00230351Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial precursor0Mitochondrial inner membrane174IPI00132042Pyruvate dehydrogenase E1 component beta subunit, mitochondrial precursor0Mitochondrial matrix156IPI00315794Cytochrome b5 outer mitochondrial membrane isoform precursor1Mitochondrial outer membrane65,109,144,170,176,100IPI00134746Argininosuccinate synthase0mitochondrion145,146IPI00338536Succinate dehydrogenase [ubiquinone] iron-sulfur protein, mitochondrial precursor0Mitochondrion112IPI00122547Voltage-dependent anion-selective channel protein 20Mitochondrion outer membrane147IPI00131186Splice isoform 2 of transcription factor BTF30Nuclear149IPI00317794Nucleolin0Nuclear53IPI00331146UMP-CMP kinase0Nuclear150IPI00458856Similar to ZNF91L isoform 10Nuclear6IPI00461822E1A binding protein p3000Nuclear55IPI00126172RIKEN cDNA 4931406C07, PTD012 homolog0Nuclear150,151IPI0011324140S ribosomal protein S190Ribosome104IPI00116908Similar to 40 s ribosomal protein S120Ribosome147IPI0084979360S ribosomal protein L120Ribosome81,127,145IPI0012597126S protease regulatory subunit S10B0Ribosome199IPI0012360440S ribosomal protein SA0Ribosome125IPI0013564026S protease regulatory subunit 80Ribosome149,150,151IPI0013978060S ribosomal protein L230Ribosome149,150IPI0022254660S ribosomal protein L220Ribosome145IPI0031495060S acidic ribosomal protein P00Ribosome149IPI0032256240S ribosomal protein S140Ribosome146IPI0033109240S ribosomal protein S4,X isoform0Ribosome149,150IPI0033146160S ribosomal protein L110Ribosome188IPI00351894Similar to ribosomal protein0Ribosome148IPI0084979360S ribosomal protein L120Ribosome149IPI0046588040S ribosomal protein S170Ribosome199IPI0012360440S ribosomal protein SA0Ribosome4,11IPI00121209Apolipoprotein A-I precursor0Secreted149IPI00121837Ribonuclease 4 precursor1Secreted12IPI00122429Plasma retinol-binding protein precursor0Secreted163IPI00123920Alpha-1-antitrypsin 1–3 precursor0Secreted163IPI00123924Alpha-1-antitrypsin 1–4 precursor0Secreted163IPI00123927Alpha-1-antitrypsin 1–5 precursor0Secreted162IPI00128484Hemopexin precursor1Secreted3,117,118IPI00131695Serum albumin precursor0Secreted98IPI00139788Serotransferrin precursor0Secreted199IPI00323571Apolipoprotein E precursor0Secreted135,136IPI00377351Apolipoprotein A-IV precursor0Secreted163IPI00406302Alpha-1-antitrypsin 1-1 precursor0Secreted100,155,156IPI0046639921 kDa protein0Secreted156IPI00480401Major urinary protein 1 precursor0Secreted122IPI00130661Tripeptidyl-peptidase I precursor0Secreted (lysosomal)101IPI00115302Branched chain ketoacid dehydrogenase E1, beta polypeptide0Membrane197IPI00120716Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 10Membrane21,137IPI00120719Cytochrome c oxidase, subunit va, full insert sequence0Membrane125IPI00124790Polyposis locus protein 1-like 13Membrane129IPI00132076Catechol O-methyltransferase1Membrane130,142IPI00138406Ras-related protein Rap-1A0Membrane174IPI00162780Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 20Membrane88,154IPI00230113Cytochrome b51Membrane199,200IPI00353727Annexin A40Membrane110IPI00117416Neighbor of COX40Unknown143IPI00121271Hypothetical S-adenosyl-L-methionine-dependent methyltransferases structure containing protein0Unknown144,108IPI00267667RIKEN cDNA 6330409N04, CLLL6 protein homolog0Unknown101IPI00269613Eukaryotic translation initiation factor 3 subunit 20Unknown149,150IPI0030783751 kDa protein0Unknown203IPI00318204Sid6061p0Unknown105IPI00273646Similar to glyceraldehyde-3-phosphate dehydrogenase0Unknown189,190,194IPI00626790Glutamine synthetase0Unknown50IPI0034584286 KDa PROTEIN0Unknown51IPI0035078045 kDa protein0Unknown133IPI0038123177 KDa protein0Unknown144IPI00923085Probable ubiquitin-conjugating enzyme E2 FLJ25076 homolog0Unknown146,147,150,173IPI0046029544 KDa protein0Unknown156IPI00330913Major urinary protein 260Unknown59IPI00467988169 kDa protein0Unknown100,155,156IPI0046951721 kDa protein0Unknown149IPI00130554Splice isoform 1 of SNARE-associated protein Snapin0Vesicular membrane101,127,134IPI00131366Keratin, type II cytoskeletal 6B0Sarcolemma83,106,107IPI00121788Peroxiredoxin 10Microbody101,139IPI00348328Keratin Kb400Intermediate filament156IPI00137414Left-right dynein0CiliumaESI-Q-TOF identification, subcellular location are given for each ID number.bThis protein is nonmembrane associated according to the annotation in the Swiss-Prot database but has one predicted TMD.Figure 1
2DE pattern of mouse liver microsome. Microsomal proteins (400μg) were arrayed by 2DE with IPG strip (pH 3–10 NL; 24 cm) and SDS-PAGE with 12% gel and visualized with blue silver staining method. A total of 204 spots denoted by circles were MS-analyzed.


## 3.3. Fractionation and Identification of Microsomal Membrane Proteins Identified by 1DE and MS/MS

The Na2CO3-treated microsome membrane proteins were separated on SDS-PAGE gels and visualized with Coomassie brilliant blue staining (Figure 2(a)). A total of 99 proteins (Table 2 and Supplemental Table  2) was identified with electrospray ionization- (ESI-) Q-TOF MS/MS from 17 gel bands (Figure 2(a)). Those proteins were derived from the ER, type I/II membrane proteins, integral membrane proteins, major histocompatibility complex class I protein, ER-Golgi intermediate compartment, mitochondrial membrane, nuclear, cytoplasm, microbody, sarcolemma, and secreted and unknown proteins (Table 2). Those membrane proteins were classified into three categories (Figure 2(b)): (a) proteins with known membrane associations (55%; n=54), (b) putative membrane proteins (5%; n=5), and (c) other proteins (40%; n=40). Those identified proteins were categorized according to the reported annotation in the UniProt database (http://www.uniprot.org/) and predictions for transmembrane regions (http://www.cbs.dtu.dk/services/TMHMM/). Of the 99 proteins, 59 (60%) were described as “membrane-associated” proteins (category (a) and (b)), including ER-characteristic proteins (cytochromes P-450 and b5, calnexin, integral membrane enzymes such as NADPH-cytochrome c reductase, and microsomal glutathione S-transferase 1).Table 2
Proteins identified from Na2CO3-extracted mouse liver microsomal membrane preparations with 1DE-based strategy.
Bands no.Accession no.Protein namePredicted TMDGRAVY scorePI valueSubcellular location9IPI00112322UDP-glucuronosyltransferase 2B5 precursor1−0.0317.94ER9IPI00127223UDP glucuronosyltransferase 2 family, polypeptide B361−0.0368.47ER9IPI00222496Protein disulfide-isomerase A61−0.2925.05ER8IPI00417182UDP-glycosyltransferase 1 family polypeptide A510.0448.33ER9IPI00116572Cytochrome P450, family 2, subfamily d, polypeptide 92−0.0436.37ER15IPI0011365540S ribosomal protein S60−0.91810.68ER5IPI00129526Endoplasmin precursor (ER protein 99,94 kDa glucose-regulated protein)0−0.724.74ER13IPI00130985Short-chain dehydrogenase CRAD200.0268.35ER6IPI00222809Similar to GDH/6PGL endoplasmic bifunctional protein0−0.186.61ER8IPI00230108Glucose-regulated protein, full insert sequence0−0.4795.78ER10,11IPI00317356Paraoxonase 10−0.015.02ER7IPI0031999278 kDa glucose-regulated protein precursor0−0.4815.07ER13IPI00121079NADH-cytochrome b5 reductase 30−0.2038.56ER, membrane bound9IPI00123964Cytochrome P450 2A51−0.2039.23ER, membrane bound9IPI00114779Cytochrome P450 2C380−0.1478.69ER, membrane bound17IPI00331322Microsomal glutathione S-transferase 130.149.67ER and mitochondrial outer membrane17IPI00119766Cis-retinol androgen dehydrogenase 100.0059.25ER lumen8IPI00134691UDP-glucuronosyltransferase 1-1 precursor, microsomal20.0878.87ER, integral to plasma membrane8IPI00128287Cytochrome P450 1A21−0.2038.92ER, membrane bound10IPI00136910Cytochrome P450 2D112−0.0096.15ER, membrane bound9IPI00308328Cytochrome P450 2F21−0.1357.74ER, membrane bound9,10IPI00323908Cytochrome P450 2D102−0.0736.16ER, membrane bound7IPI00112549Long-chain-fatty-acid-CoA ligase 11−0.0456.81ER, type III membrane protein8IPI00133522Protein disulfide-isomerase precursor0−0.3864.79ER9IPI00116572Cytochrome P450 2D90−0.0635.93ER, membrane bound5,6IPI00119618Calnexin precursor1−0.8754.5ER, type I membrane protein1,10,12,14,15IPI00319973Membrane-associated progesterone receptor component 11−0.6164.57ER, membrane bound8IPI00132475Protein ERGIC-531−0.5455.92ER-Golgi intermediate compartment (ERGIC), type I membrane protein8,17IPI00109061Tubulin beta-4 chain homolog0−0.4064.78Cytoplasmic10IPI00110827Actin, alpha skeletal muscle0−0.2325.23Cytoplasmic10,12,14IPI00110850Actin, cytoplasmic 10−0.25.29Cytoplasmic1,2,3IPI00111908Carbamoyl-phosphate synthase0−0.126.42Cytoplasmic1,8,9,13IPI00117348Tubulin alpha-2 chain0−0.234.94Cytoplasmic9,10,11,12,13IPI00117914Arginase 10−0.1876.52Cytoplasmic17IPI00120451Fatty acid-binding protein, liver0−0.4098.59Cytoplasmic9IPI00129028Similar to tubulin, alpha 3C isoform 10−0.2044.98Cytoplasmic1–11,13,17IPI00130950Betaine-homocysteine S-methyltransferase0−0.368.01Cytoplasmic1,4,6,10,11,14,15IPI00134746Argininosuccinate synthase0−0.3618.36Cytoplasmic3,4IPI00114710Pyruvate carboxylase, mitochondrial precursor0−0.1736.25Mitochondrial17IPI00553333Hemoglobin subunit beta-100.0927.13Mitochondrial9IPI00134809Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex0−0.1719.1Mitochondrial17IPI00117978Cytochrome c oxidase subunit IV isoform 1, mitochondrial precursor1−0.4129.25Mitochondrial inner membrane15,16IPI00315794Cytochrome b5 outer mitochondrial membrane isoform precursor1−0.6024.79Mitochondrial outer membrane13IPI00321718Prohibitin-20−2.589.83Mitochondrial, cytoplasmic, nuclear13IPI00122547Voltage-dependent anion-selective channel protein 20−0.2237.44Outer mitochondrial Membrane17IPI00114559Histone H2A type 10−0.57211.22Nuclear16,17IPI00114642Histone H2B F0−0.76210.32Nuclear8IPI00387318Cell cycle control protein 50A2−0.3318.58Membrane15IPI00113849Splice isoform 2 of cell division control protein 42 homolog0−0.1576.16Membrane13IPI00122549Splice isoform Pl-VDAC1 of voltage-dependent anion-selective channel protein 10−0.3348.55Membrane15IPI00127408Ras-related C3 botulinum substrate 10−0.1018.77Membrane15IPI00138406Ras-related protein Rap-1A0−0.3756.39Membrane6IPI00116921Scavenger receptor class B member 120.0738.29Integral membrane protein1IPI00121985Splice Isoform 1 of solute carrier organic anion transporter family, member 1B2120.1728.95Integral membrane protein9IPI00124830Integrin-associated protein precursor50.5638.58Integral membrane protein14,15IPI00131176Cytochrome c oxidase subunit 220.274.6Integral membrane protein1IPI00132604Secretedretory carrier-associated membrane protein 340.0287.55Integral membrane protein1IPI00135701Solute carrier organic anion transporter family, member 1A1110.2448.58Integral membrane protein1IPI00311682Sodium/potassium-transporting ATPase alpha-1 chain precursor100.0025.3Integral membrane protein6IPI00331214Platelet glycoprotein IV2−0.0538.61Integral membrane protein2IPI00119063Prolow-density lipoprotein receptor-related protein 11−0.5025.17Integral to membrane1,12,16,17IPI00124790Polyposis locus protein 1-like 130.3756.82Integral to membrane10IPI00129677Asialoglycoprotein receptor major subunit1−0.665.99Integral to membrane17IPI00467119Camello-like protein 110.3029.61Integral to membrane5,8IPI00316329Keratin, type II cytoskeletal 10−0.5888.2Intermediate filament10,11,12IPI00108844Cation-dependent mannose-6-phosphate receptor precursor1−0.1685.24Type I membrane protein9IPI00109998H-2 class I histocompatibility antigen, D-B alpha chain precursor1−0.5086.28Type I membrane protein4IPI00120245Integrin alpha-V precursor1−0.2465.46Type I membrane protein3,4IPI00121190Epidermal growth factor receptor precursor2−0.3166.46Type I membrane protein2IPI00126186Macrophage mannose receptor 1 precursor1−0.56.47Type I membrane protein5IPI00134549Splice isoform LAMP-2A of lysosome-associated membrane glycoprotein 2 precursor1−0.0367.05Type I membrane protein13IPI00312018Malectin1−0.2035.73Type I membrane protein3IPI00312063Low-density lipoprotein receptor precursor1−0.3914.88Type I membrane protein16IPI00466570Transmembrane emp24 domain-containing protein 102−0.1696.25Type I membrane protein4IPI00108535Carcinoembryonic antigen-related cell adhesion molecule 11−0.3025.35Type I membrane protein5IPI00310059Polymeric-immunoglobulin receptor precursor1−0.4995.26Type I membrane protein also secreted9IPI00121550Sodium/potassium-transporting ATPase beta-1 chain1−0.558.83Type II membrane protein2,3IPI00134585Glutamyl aminopeptidase1−0.3445.28Type II membrane protein10IPI00307966ADP-ribosyl cyclase 11−0.1068.64Type II membrane protein3IPI00319509Aminopeptidase N1−0.2775.62Type II membrane protein3IPI00458003Ectonucleotide pyrophosphatase/phosphodiesterase 31−0.3466.13Unknown9IPI00409409CD1D1 protein1−0.1789.22Unknown7IPI00621548NADPH-cytochrome P450 reductase1−0.4635.37Unknown9IPI00321644Cytochrome P450 2D262−0.1056.16Unknown1IPI00127016Hydroxysteroid 17-beta dehydrogenase 60−0.0758.63Unknown16IPI00221721Hypothetical krab box containing protein, full insert sequence0−0.1429.84Unknown8IPI00224073Hypothetical peptidase family M20/M25/M40 containing protein, full insert sequence0−0.015.99Unknown16IPI00228379Ferritin light chain 20−0.4796.37Unknown17IPI0026684217 kDa protein0−0.66810.48Unknown15IPI00379258Similar to ferritin light chain 20−0.4548.51Unknown3IPI00405742Plexin B20−0.35.67Unknown2IPI00408258Structure-specific endonuclease subunit SLX40−0.7145.33Unknown11IPI00462251Hypothetical protein LOC72792 isoform 10−0.4295.82Unknown15IPI00605814Similar to Ferritin light chain 10−0.3586.42Unknown10IPI00131366Keratin, type II cytoskeletal 6B0−0.4888.32Sarcolemma10IPI00322209Keratin, type II cytoskeletal 80−0.6025.7Sarcolemma8IPI00853991Similar to VH coding region0−0.1025.31Secreted10IPI00126458MRNA1−0.555.66MHC class I protein complex15IPI00121788Peroxiredoxin 10−0.2218.26Microbody1DE pattern and membrane-associated characteristic classification of Na2CO3-extracted microsomal membrane proteins. (a) 1DE pattern. Molecular weight markers are shown on the left and bands excised for MS analysis are indicated on the right. Lanes S1 and S2 were loaded with the same protein samples (50 μg per lane). (b) Classification via membrane-associated characteristic. The criteria used for this classification were published reports, annotations in the genome database (http://www.uniprot.org/), and predictions for transmembrane regions (http://www.cbs.dtu.dk/services/TMHMM/).
(a)(b)Hydrophobicity is an important characteristic of a membrane protein. The grand average of hydropathy (GRAVY) scores (>−0.4) (http://us.expasy.org/tools/protparam.html) is an index to evaluate the hydrophobic status of a protein, indicates a hydrophobic protein, and suggests a membrane association. In the current study, 69 (70%) of the 99 proteins identified from 1DE had a GRAVY>-0.4 (Supplemental Figure  3), a score indicating the probability for membrane association. Moreover, some alkaline proteins with PI values close to or greater than 10 were separated by 1DE (Supplemental Figure  4), but they could not be detected in a conventional 2DE map.


## 3.4. Comparison of 2DE and 1DE Datasets

Among the 2DE dataset (n=183 proteins; Table 1) and 1DE dataset (n=99 proteins; Table 2), only 23 proteins (Table 3) were consistent between 2DE and 1DE datasets (23% of 1DE dataset, and 13% of 2DE dataset). A total of 259 nonredundant proteins (n=183+99-23) were identified in the microsome fraction through the strategy of combining 2DE with 1DE protein-separation technologies followed by ESI-Q-TOF MS/MS. The microsome consisted of a complex network of continuous membranes including ER, ER-Golgi intermediate complex—also referred to as the vesiculotubular clusters or pre-Golgi intermediates—and the Golgi apparatus [5]. Among those identified proteins, 62 located in ER and Golgi were definitely classified as microsome proteins by annotation in the Swiss-Prot database and the Gene Ontology (GO).Table 3
Proteins that are consistently present in both 2DE dataset of microsomal proteins (Table1) and 1DE dataset of Na2CO3-extracted microsomal proteins (Table 2).
Accession numberProtein namePredicted TMDGRAVY scoresPI valueLocationIPI00108454Similar to 40S ribosomal protein S60−0.91810.68ERIPI00112322aUDP-glucuronosyltransferase 2B5 precursor1−0.0317.94ERIPI00129526Endoplasmin precursor (ER protein 99, 94 kDa glucose-regulated protein)0−0.724.74ERIPI00133522Protein disulfide-isomerase precursor0−0.3864.79ERIPI00222496aProtein disulfide isomerase-associated 61−0.2925.05ERIPI00230108Glucose-regulated protein, full insert sequence0−0.4795.78ERIPI0031999278 kDa glucose-regulated protein precursor0−0.4815.07ERIPI00331322aMicrosomal glutathione S-transferase 130.149.67ER and mitochondrial outer membraneIPI00319973aMembrane-associated progesterone receptor component 11−0.6164.57ER, membrane boundIPI00109061Tubulin beta-4 chain homolog0−0.4064.78CytoplasmicIPI00110827Actin, alpha skeletal muscle0−0.2325.23CytoplasmicIPI00110850Actin, cytoplasmic 10−0.25.29CytoplasmicIPI00111908Predicted: carbamoyl-phosphate synthetase 10−0.126.42CytoplasmicIPI00117348Tubulin alpha-2 chain0−0.234.94CytoplasmicIPI00117914Arginase 10−0.1876.52CytoplasmicIPI00134746Argininosuccinate synthase0−0.3618.36CytoplasmicIPI00117978aCytochrome c oxidase subunit IV isoform 1, mitochondrial precursor1−0.4129.25Mitochondrial inner membraneIPI00315794aCytochrome b5 outer mitochondrial membrane isoform precursor1−0.6024.79Mitochondrial outer membraneIPI00122547aVoltage-dependent anion-selective channel protein 20−0.2237.44Outer mitochondrial membraneIPI00124790aPolyposis locus protein 1-like 130.3756.82Integral to membraneIPI00138406aRas-related protein Rap-1A0−0.3756.39MembraneIPI00121788Peroxiredoxin 10−0.2218.26MicrobodyIPI00131366Keratin, type II cytoskeletal 6B0−0.4888.32SarcolemmaaMembrane proteins with one or more predicted trans-membrane origins or validated by references.


## 3.5. Significantly Enriched GO Terms for Mouse Liver Microsome Proteins

Biological Networks Gene Ontology [15] and Cytoscape [16] plugins to find statistically overrepresented GO categories were used for the enrichment analysis of our protein dataset. The microsome protein dataset (n=259, from 1DE and 2DE datasets) was compared to a reference set of complete mouse proteome (IPI mouse) that was provided by Biological Networks Gene Ontology. The analysis was done with a hypergeometric test, and all significant (P<0.01) GO terms were selected after correcting for a multiple term testing with a Benjamini and Hochberg false discovery rate. The analysis was performed separately for molecular function, cellular component, and biological process categories, and x-fold enrichment for every overrepresented term in three GO categories was calculated (Supplemental Figure  5). The results showed that the terms were related to mostly catalytic activity in terms of molecular function, including metabolism-related oxidoreductase, hydrolase, and dehydrogenase. Similarly, terms belonging to the cellular component namespace include mitochondrion, ER, and ribosome. Finally, terms from the biological process namespace included metabolic process, localization, transport, and translation. All of the information suggested the main functions and compositions of microsome.


## 3.6. Significant Enrichment of KEGG Pathway for Mouse Liver Microsome Proteins

Biological pathways analysis based on KEGG pathway database was performed with an analysis toolkit—WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/) [17]. This toolkit allowed the functional annotation of gene/protein sets into well-characterized functional signaling pathways (KEGG: http://www.genome.jp/kegg/). In addition, an enrichment score was obtained of the frequency of occurrence of a specific protein (or gene) within any given experimental subset with respect to a species-specific background set. Thus, an enrichment factor (the observed frequency in input set/the expected frequency in background set) was created with a statistical value that indicated that the protein (or gene) was specifically overrepresented in the input dataset. In this current study, all the proteins except 81 (n=259-81=178) were linked to a total of 99 biological pathways in the KEGG, including metabolic pathway, glycolysis/gluconeogenesis, metabolism of xenobiotics by cytochrome P450, and PPAR signaling pathway. Among those pathways, 34 significantly (P<0.01) enriched biological processes analyzed by WebGestalt were obtained (Figure 3). Those biological processes were involved in cell metabolism, benzoate degradation, metabolism of xenobiotics, ribosome, biosynthesis, signaling pathway, and oxidative stress. Those results are known to be related to microsome.Figure 3
Significantly enriched KEGG pathways for mouse liver microsome proteins (n=259) that were derived from 1DE and 2DE strategies. KEGG pathway enrichment analysis was performed using WebGestalt. The pathways having enrichment (P<0.01) are presented. For each KEGG pathway, the bar shows the x-fold enrichment of the pathway in our dataset.To ascertain the coverage of our dataset with the enriched pathways or biological processes, the KEGG search service was used to map our dataset on KEGG pathways. Two of the aforementioned enriched KEGG pathways (metabolism of xenobiotics and ribosome) were related to the well-known function and composition of the microsome (Figure4). Enzyme Commission numbers (EC no., e.g, 1.14.14.1) are used to represent enzymes in metabolism. Highlighted in green background are known mouse enzymes annotated in the KEGG database and the red boxed are enzymes in our dataset (Figure 4(a)). All enzymes (n=9) that played a key role in every pathway of metabolism of xenobiotics were included in our dataset (Table 4). Thirteen proteins from large and small subunits of ribosome were also found in our dataset (Table 4) and are indicated with a red box (Figure 4(b)). These proteins interact physically with each other and form a large protein complex—the ribosome. All the identified proteins that are involved in those two pathways are summarized in Table 4, including their KEGG pathway, protein ID, and protein name.Table 4
Proteins involved in KEGG pathways. (a) Metabolism of xenobiotics. (b) Ribosome.
KEGG pathwayProtein IDProtein nameMS-identified proteinsA. Metabolism of exnobioticsEC:1.14.14.1IPI00128287Cytochrome P450 1A2+IPI00123964Cytochrome P450 2A5+IPI00116572Cytochrome P450 2D9+IPI00323908Cytochrome P450 2D10+IPI00321644Cytochrome P450 2D26+IPI00114779Cytochrome P450 2C38+IPI00308328Cytochrome P450 2F2+EC:2.5.1.18IPI00331322Microsomal glutathione S-transferase 1+EC:1.1.1.1IPI00221400Alcohol dehydrogenase A chain+B. RibosomeSmall subunitIPI0013564026S protease regulatory subunit 8+IPI0012597126S protease regulatory subunit S10B+IPI0033109240S ribosomal protein S4, X isoform+IPI00116908Similar to 40s ribosomal protein S12+IPI0032256240S ribosomal protein S14+IPI0046588040S ribosomal protein S17+IPI0011324140S ribosomal protein S19+IPI0012360440S ribosomal protein SA+IPI0031495060S acidic ribosomal protein P0+Large subunitIPI0033146160S ribosomal protein L11+IPI0084979360S ribosomal protein L12+IPI0022254660S ribosomal protein L22+IPI0013978060S ribosomal protein L23+Metabolism of xenobiotics by cytochrome P450 pathway, and ribosome map views of identified proteins. The two enriched metabolic pathway maps were generated by KEGG, which incorporated the proteomic data into the KEGG pathway maps. All of the genes in mouse are colored; the genes contained in the protein dataset are red.
(a)(b)


## 4. Discussion

Proteome analysis of the cell membrane-bound organelles is a daunting task mainly because of (a) isolation of membrane that is free from nonconstituents and (b) solubilization of membrane proteins in a manner amenable to isoelectric focusing [10]. 2DE is an effective tool to survey biological complexity at the molecular level and provides a systematic and comprehensive study of the proteins. However, because of the PI value range limited by the IPG strip and the high dependence on sample preparation, some problems exist for the available 2DE protocols to resolve membrane-associated proteins [10, 22]. Therefore, in the current study, the whole microsome lysate was arrayed with 2DE, and the membrane fraction of microsomes purified by the carbonate procedure was separated with 1DE. The complementary 2DE and 1DE approaches provided a much wider coverage of microsome proteome.Hydrophobicity and relatively low abundance causes a challenge for proteomic technology to separate and identify membrane proteins. The hydrophobicity of proteins is frequently expressed as GRAVY scores (http://us.expasy.org/tools/protparam.html). A calculated GRAVY score of up to –0.4 indicates a hydrophobic protein, suggesting a membrane association [21]. In the current study, 69 (70%) of the 99 proteins identified from 1DE had a GRAVY>-0.4 (Supplemental Figure  3), indicating the probability for membrane association [21]. As shown in Supplemental Figure  4, some alkaline proteins with PI values close to or greater than 10 were separated by 1DE; they could not be detected in conventional 2DE map. Only 23 proteins were found to be consistent between 2DE and 1DE datasets with 6 proteins classified as membrane proteins (Table 3). All these results indicate that 1DE is a potent supplement to 2DE, and the combination of the two approaches is necessary in protein profiling of microsomes.Microsome-sealed vesicles could be converted into flat membrane sheets with cisternal contents that were released effectively with the treatment solution (100 mM Na2CO3; 0°C). It appears to be as effective as the low detergent procedure in selectively releasing microsomal content. In the current study, some proteins that were identified from Na2CO3-extracted fraction were classified as membrane associated mainly based on published reports, even though their predicted transmembrane domains (TMDs) did not suggest a membrane origin. The observations point out the fact that structure alone may not be the deciding factor, as far as the association of proteins with cell membrane is concerned. First, the proteins may be bound to the membrane simply to perform their functional obligations. Consequently, they could become part of complexes involving membrane proteins and may not depart from them easily under the conditions of sample preparation. For example, many enzymes were identified in the extracted membrane fraction, such as Cis-retinol androgen dehydrogenase 1 (short-chain dehydrogenase family). It is anchored to the ER membrane facing the cytoplasm by an N-terminal signaling sequence of 22 residues and takes part in the membrane-associated retinoid metabolism [23], so is fatty acid-binding protein, which participates in the palmitic acid or retinylester metabolism that is incorporated in microsomal membranes [24] and the free fatty acid transferation to the membrane. Second, some truly cytosolic proteins may simply integrate with membrane vesicles during the sonication process and become difficult to remove by the extraction methods [25]. Studies [5] have demonstrated that hepatic microsomes are derived from the ER and other cell organelles. The ER represents a membrane tubular network that crosses the cytoplasm from the nucleus membrane to the plasma membrane. Moreover, some proteins perform their functions between cytoplasm and ER, such as fatty-acid-binding proteins [26]. From this point of view, taking all of the portions into account, 60%–70% of the proteins identified can be regarded as microsome proteins in this research. A part (~15%) of identified proteins did not have unambiguous locations in published reports or annotations in the genome database. This current study provides information relevant to subcellular locations of these proteins for subsequent studies.Two datasets from 1DE and 2DE are part of the complete protein composition of microsomes. A bioinformatics analysis of the two datasets combined offers more information. For an overview of the proteomic data and comprehending their biological importance, biological networks GO (BinGO) (http://www.psb.ugent.be/cbd/papers/BiNGO/index.html) was used to identify GO-category significant enrichment with all the identified proteins. BiNGO is a plugin for Cytoscape, which is an open source bioinformatics software platform to visualize and integrate molecular interaction networks. BinGO maps the predominant functional themes of a given gene set on the GO hierarchy. Of the 259 target proteins and direct partners analyzed, 182 target proteins linked to one or more GO terms. GO-term enrichment analysis revealed that the most highly represented GO terms in the cellular GO category component were organelles such as ER, mitochondrial, and organelle membrane. An analysis of the proteins that were identified according to their potential roles in biological processes indicated that the proteins were mainly involved in metabolic process, localization, transport, and translation. All the results were highly statistically significant.The KEGG pathway database integrates current knowledge on molecular interaction networks in biological processes. To gain a broad understanding of our dataset, WebGestalt (a web-based gene set analysis toolkit) was used to map the identified proteins to KEGG pathways. The results showed that 112 of the total proteins were associated with one or more KEGG pathways. Meanwhile, 97 of 112 target proteins (87%) fell into 34 KEGG pathways; they were specifically enriched (P<0.01) compared to statistical expectations. Pathways that are involved in benzoate degradation, metabolism of xenobiotic, glutamate metabolism, and cysteine metabolism were among the most enriched biologically. This finding was consistent with the fact that microsomes were used to investigate the metabolism of compounds and to examine drug-drug interaction by in vitro studies.Collectively, the bioinformatics analysis via enrichment analysis of GO annotation and KEGG pathways derived meaning from the proteomic data and assisted in the understanding of the function of liver at the subcellular level.Novelty and Limitation
Mammalian liver microsome proteomes have been studied by several groups [18–20]. Comparison of the current study with the literature data [18–20] was shown in Tables 5 and 6. Zgoda et al. [18] studied differential ultracentrifugation-separated mouse liver microsome proteome; 2DE and silver stain yielded 1,100 protein spots, and 138 proteins contained in 2D gel spots were identified with peptide mass fingerprint (PMF). Zgoda et al. [19] also studied differential ultracentrifugation-separated mouse liver microsome proteome with 1DE and MS/MS; 519 proteins were identified including 138 (138/519 = 27%) predicted membrane proteins. Gilchrist et al. [20] used 1DE and MS/MS to analyze rat ER and Golgi that were separated with differential ultracentrifugation and density gradient centrifugation; 832 ER proteins were identified including 183 (183/832 = 22%) membrane proteins. This current study combined differential ultracentrifugation and sucrose gradient centrifugation to prepare mouse liver microsomes; 2DE and Coomassie brilliant blue stain yielded 514 protein spots, and 183 proteins were identified with MS/MS from 204 excised gel spots, including 41 (41/183 = 22%) membrane proteins. Na2CO3 was used to further extract membrane proteins from isolated microsomes; 1DE and Coomassie brilliant blue stain yield 17 protein bands, and 99 proteins were identified with MS/MS from those 17 protein bands, including 54 (54/99 = 55%) membrane proteins. A total of 259 nonredundant proteins were identified including 62 (62/259 = 24%) membrane proteins. Compared to the documented data [18–20], the novelty of this current study is that the carbonate method significantly increased the identification rate of microsomal membrane proteins, that some proteins and functional annotations from this current study have not been identified in other literature, which expanded and enriched the documented data, and that the established analysis system and data will benefit the discovery of liver disease-related microsomal membrane proteins. Meanwhile, we also noted that the current study had a relatively low coverage (n=259 proteins) of mouse liver microsome proteome relative to the documented data (n=519 proteins [19] and 832 proteins [20]), which might be derived from several factors: (i) inconsistent protein-extracted procedures and protein-stained methods were used, (ii) only part of 2D gel spots were excised to identify proteins, (iii) only visualized 1D gel bands (not the entire 1D gel lane) were used for protein identification, (iv) MS/MS (not PMF) was used to identify 2D gel proteins, (v) different sensitivity mass spectrometers were used, (vi) different parameters were used to search protein database. The use of 2D/3D LC-MS/MS [19] and carbonate extraction of isolated microsomes would significantly improve the coverage of microsomal membrane proteome.Table 5
Comparison of the current study with the literature data [18–20].
Current studyRef. [19]Ref. [18]Ref. [20]SpeciesMouseMouseMouseRatSampleLiver microsomeLiver microsomeLiver microsomeER, GolgiPretreatmentNonePhenobarbitalPhenobarbital or 3-methylcholanthreneNoneSample preparationSubfractionated by differential ultra-centrifugation + sucrose gradient centrifugation + Na2CO3Subfractionated by differential ultra-centrifugationDifferential ultracentrifugationSubfractionated by differential ultra-centrifugation + density gradient centrifugationProtein separation2DE, 1DE1DE2DE1DE1D/2D-Gel StainCoomassie brilliant blue (2DE; 1DE)—Silver stain—Protein identificationMS/MSMS/MSPMFMS/MSProtein spots on 2D-Gel514—1100—Proteins identified in 2D-Gel183—139—Proteins identified in 1DE99519—832 (ER)Proteins identified in 2-D LC—1410——Proteins identified in 3-D LC—3703——Total identified proteins2594142Unspecified832 (ER)Membrane proteins2DE: 41 (41/183 = 22%)1DE: 54 (54/99 = 55%)Total: 62 (62/259 = 24%)1DE: 138 (138/519 = 27%)2-D LC: 259 (259/1410 = 21%)3-D LC: 659 (659/3703 = 18%)Unspecified183 (183/832 = 22%)Protein superfamilyP450 family members1029211Ribosomal proteins1316Unspecified45UDP glycosyltransferases, UGTs68Unspecified3Tubulins115Unspecified2Short-chain  dehydrogenase/reductase329Unspecified56Protein disulfide isomerase24Unspecified1Table 6
Comparison of selected proteins between the current study and the literature data [18–20].
ProteinCurrent studyRef. [19]Ref. [18]Ref. [20]P450 family members2D9, 2A5, 2C38, 1A2, 2D11, 2F2, 2D10, 2D262C3717A1, 20A1, 2B2, 2J3, 4A1, 4A8, 4F1, 4F4, 4V3, 8B1, NA2GRP-170Hypoxia upregulated protein 1170 kDa glucose regulated protein—EndoplasminEndoplasminTumor rejection antigen gp96SerotransferrinSerotransferrinTransferrin—78 kDa glucose-regulated protein78 kDa glucose-regulated protein78 kDa glucose-regulated protein—Stress-induced phosphoprotein 1—Stress-induced phosphoprotein 1—Calreticulin familyCalnexinCalreticulin—Protein disulfide-isomeraseProtein disulfide-isomerase precursor (PDI)Protein disulfide-isomerase precursor (PDI)Similar to disulfide isomeraseGlucose-regulated protein similar to ER-60 protease—Glucose-regulated protein similar to ER-60 protease—Erp58—Erp58—Vitamin D-binding protein—Vitamin D-binding protein—TubulinsTubulin beta-4, alpha-1, alpha-2, alpha-4, beta-5, beta-2C, beta 2Tubulin alphaTubulin alpha 6Fibrinogen—Fibrinogen, gamma polypeptide—Serine protease inhibitor—Similar to serine protease inhibitor 1–4—Argininosuccinate synthetase 1Argininosuccinate synthetase 1Argininosuccinate synthetase 1—Interferon-inducible GTPase—Interferon-inducible GTPaseProgesterone receptor membrane componentProgesterone receptor membrane componentProgesterone receptor membrane component—Major urinary protein 2Major urinary protein 2Major urinary protein 2—Superoxide dismutase I—Superoxide dismutase I—Ribosomal proteins26S protease regulatory subunit 8, S10B; 40S ribosomal protein S17, SA, S6, S19, S12, SA, S14, S4 X isoform; 60S ribosomal protein L11, L12, L23, L22, P0Unspecified40S Ribosomal Protein S10, S12, S18, S20, S21, S23, S24, S25, S26, S27, S29, S30, S6, S9    60S Ribosomal Protein L12, L15, L18A, L19, L21, L22, L23, L23A, L24, L26, L27, L27A, L28, L3, L32, L34, L35, L35A, L36, L37, L37A, L39, L4, L40, L44, L6, L7AUDP glycosyltransferases, UGTsUDP-glucuronosyltransferase 2B5, 2B36, 1A5UnspecifiedUDP-Glucuronosyltransferase 1A7UDP-glucuronosyltransferase 1-1 precursorUDP-Glucuronosyltransferase GTNA2UDP-glucuronosyltransferase 1-2 precursorShort-chain dehydrogenase/reductaseGlyceraldehyde-3-phosphate dehydrogenaseAlcohol dehydrogenase AShort-chain dehydrogenase CRAD2 Cis-retinol androgen dehydrogenase 1 Hydroxysteroid 17-beta dehydrogenase 6UnspecifiedGlyceraldehyde 3-phosphate dehydrogenase Alcohol dehydrogenase [NADP+]Similar to retinal short-chain Dehydrogenase/reductaseRetinol dehydrogenase 10 Hydroxysteroid (17-Beta) dehydrogenase 8Oxidoreductase HTATIP2 NADH-ubiquinone oxidoreductase 24 kDa subunitNADH-cytochrome b5 reductase 3 NADPH-cytochrome P450 reductaseUnspecifiedOxidoreductase ero1-L endoplasmic oxidoreductase 1 BetaNo protein list was obtained from [19].—means “not included.”


## 5. Conclusions

The preparation of liver microsomes was optimized. The data presented here demonstrated that 1DE and 2DE are complementary approaches to analyze the intracellular microsomes that contain considerable numbers of highly hydrophobic membrane proteins. An integrated bioinformatics analysis of all of the microsome proteins identified with 1DE and 2DE can provide a relatively complete understanding of the protein composition and cellular function of the target microsome organelles. The information presented here will be useful for successful analysis of other membranous organelles. Our data will assist in understanding the function of liver and are an important reference for subsequent analysis of liver disease-related microsome proteins for biomarker discovery and mechanism clarification of a liver disease.



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