International Journal of ProteomicsVolume 2012 (2012), Article ID 832569, 24 pagesdoi:10.1155/2012/832569

Research Article

Proteomic and Bioinformatics Analyses of Mouse Liver Microsomes

Fang Peng,¹ Xianquan Zhan,¹ Mao-Yu Li,¹ Fan Fang,¹ Guoqing Li,1,2 Cui Li,¹ Peng-Fei Zhang,¹ and Zhuchu Chen¹

¹Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Hunan, Changsha 410008, China²Department of Biology, School of Pharmacy and Life Science, University of South China, Hengyang 421001, China

Received 28 July 2011; Revised 9 November 2011; Accepted 20 November 2011

Academic Editor: Visith Thongboonkerd

Copyright © 2012 Fang Peng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

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 (400 μg) 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 (75 μm 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 . 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 displayprotein spots ( 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 () from ER and Golgi, 11% of proteins () from mitochondria and other membranes, 50% of proteins () from cytosolic and other soluble proteins, 8% of secreted proteins (), and 9% of proteins without unambiguous location ().

Table 1: Proteins identified from mouse liver microsomal preparations with 2DE-based strategy.

Spot no.
IPIa
Protein name
Predicted TMD
Location

90,91
IPI00108939
glyceraldehyde-3-phosphate dehydrogenase, spermatogenic
0
ER

6
IPI00111936
UDP-glucuronosyltransferase 1-2 precursor, microsomal
1
ER

145
IPI00121833
Acetyl-coenzyme A acyltransferase 1
0
ER

102
IPI00622235
Transitional endoplasmic reticulum ATPase
0
ER

6,61,194
IPI00122815
Prolyl 4-hydroxylase, beta polypeptide
0
ER

17
IPI00123176
Similar to glyceraldehyde-3-phosphate dehydrogenase, 37 kDa protein
0
ER

134,135
IPI00123342
Hypoxia upregulated 1
1
ER

2
IPI00129526
Endoplasmin
0
ER

139
IPI00131459
Nucleoside diphosphate kinase A
0
ER

179
IPI00132874
Splice isoform 1 of monoglyceride lipase
0
ER

163
IPI00133522
Protein disulfide-isomerase precursor
0
ER

49
IPI00134058
Thioredoxin domain containing protein 4 precursor
0
ER

108,145,65
IPI00135284
Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ISOFORM 1
0
ER

147,148,149
IPI00135686
Mus musculus adult male kidney cDNA, RIKEN full-length enriched library, clone: 0610008
1
ER

174,178,179,183
IPI00135726
Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
0
ER

49,50
IPI00163011
Thioredoxin domain containing protein 5 precursor
0
ER

137
IPI00226993
Thioredoxin
0
ER

148
IPI00229551
ADAM4
1
ER

62,157,158,162
IPI00230108
Glucose-regulated protein, full insert sequence
0
ER

148
IPI00271869
Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
0
ER

146,147,149,150,153
IPI00273646
Glyceraldehyde-3-phosphate dehydrogenase
0
ER

187
IPI00555023
Glutathione S-transferase P 1
0
ER

144
IPI00319652
Glutathione peroxidase
0
ER

84
IPI00319992
78 kDa glucose-regulated protein precursor
0
ER

153
IPI00320208
Elongation factor 1-beta
0
ER

118
IPI00323357
Heat shock cognate 71 kDa protein
0
ER

173
IPI00323661
Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
0
ER

145,201
IPI00462605
Similar to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
0
ER

127
IPI00469307
Alpha-2-macroglobulin receptor-associated protein precursor
0
ER

143,152,203
IPI00480343
2700050F09Rik protein
0
ER

162
IPI00831714
Leucine-rich repeat-containing protein 7
0
ER (integral to membrane)

149,150
IPI00352124
Flavin containing monooxygenase 5
1
ER (integral to membrane)

131
IPI00132397
GTP-binding protein SAR1b
0
ER (peripheral membrane protein)

107
IPI00227657
Stromal cell-derived factor 2-like protein 1 precursor
0
ER lumen

145
IPI00123281
Expressed sequence AA959742
1
ER membrane

7
IPI00222496
Protein disulfide isomerase-associated 6
1
ER, membrane proteinb

7
IPI00112322
UDP-glucuronosyltransferase 2B5 precursor
1
ER, membrane proteinsb

156
IPI00331322
Microsomal glutathione S-transferase 1
3
ER, outer membrane

151
IPI00319973
Membrane-associated progesterone receptor component 1
1
ER, membrane-bound

152
IPI00170316
Multiple coagulation factor deficiency protein 2 homolog precursor
0
ER-Golgi intermediate compartment

170
IPI00408892
RAS-related protein RAB-7
0
Golgi, endosomes, lysosomes

153
IPI00123316
Splice isoform 1 of tropomyosin 1 alpha chain
0
Cytoplasm

116
IPI00133456
Senescence marker protein-30
0
Cytoplasm

89,129,130,151,152
IPI00135085
Heme-binding protein
0
Cytoplasm

165
IPI00109061
Tubulin beta-4 chain homolog
0
Cytoplasmic

88
IPI00109073
Tubulin beta-4 chain
0
Cytoplasmic

105,138
IPI00110753
Tubulin alpha-1 chain
0
Cytoplasmic

113,166,167,197,204
IPI00110827
Actin, alpha skeletal muscle
0
Cytoplasmic

9,129,130,151.153,166,167,197,198
IPI00110850
Actin, cytoplasmic 1
0
Cytoplasmic

90
IPI00114162
Fatty acid-binding protein, epidermal
0
Cytoplasmic

145
IPI00116277
T-complex protein 1, delta subunit
0
Cytoplasmic

144
IPI00117264
DJ-1 protein
0
Cytoplasmic

191,164,165
IPI00117348
Tubulin alpha-2 chain
0
Cytoplasmic

137,138,164
IPI00117350
Tubulin alpha-4 chain
0
Cytoplasmic

141,153,165,132
IPI00117352
Tubulin beta-5 chain
0
Cytoplasmic

126
IPI00117914
Arginase 1
0
Cytoplasmic

152
IPI00120532
21 kDa protein
0
Cytoplasmic

107,108,139,143,182
IPI00125489
44 KD protein (Argininosuccinate synthase)
0
Cytoplasmic

191
IPI00626790
Glutamine synthetase
0
Cytoplasmic

176,182,194
IPI00130950
Betaine-homocysteine S-methyltransferase
0
Cytoplasmic

99
IPI00131204
UDP-glucose pyrophosphorylase 2
0
Cytoplasmic

204
IPI00136929
Gamma actin-like protein
0
Cytoplasmic

101,132
IPI00169463
Tubulin beta-2C Chain
0
Cytoplasmic

202,133
IPI00221400
Alcohol dehydrogenase A chain
0
Cytoplasmic

89
IPI00221528
Actin, cytoplasmic type 5 homolog
0
Cytoplasmic

168
IPI00221890
Carbonic anhydrase III
0
Cytoplasmic

202,133
IPI00317740
Guanine nucleotide-binding protein beta subunit 2-like 1
0
Cytoplasmic

159
IPI00331174
T-complex protein 1, eta subunit
0
Cytoplasmic

154
IPI00338039
Tubulin, beta 2
0
Cytoplasmic

141
IPI00348094
Predicted: similar to tubulin M beta 1
0
Cytoplasmic

136
IPI00404011
Microtubule-associated protein
0
Cytoplasmic

153
IPI00421223
Tropomyosin alpha 4 chain
0
Cytoplasmic

194,195
IPI00457825
Similar to argininosuccinate synthase (Citrulline-aspartate ligase)
0
Cytoplasmic

60
IPI00462072
Similar to alpha enolase (2-phospho-D-glycerate hydro-lyase)
0
Cytoplasmic

178
IPI00467066
Glycine N-methyltransferase
0
Cytoplasmic

63,109
IPI00467833
Triosephosphate isomerase
0
Cytoplasmic

153
IPI00605380
Similar to tubulin alpha-2 chain (Alpha-tubulin 2)
0
Cytoplasmic

162
IPI00123313
Ubiquitin-activating enzyme E1 1
0
Cytoplasmic and nuclear

64
IPI00420745
Proteasome subunit, alpha type 2, full insert sequence
0
Cytoplasmic and nuclear

145
IPI00320165
Oxidoreductase HTATIP2
0
Cytoplasmic and nuclear

153
IPI00117978
Cytochrome c oxidase subunit IV isoform 1, mitochondrial precursor
1
Mitochondrial inner membrane

19
IPI00109167
NADH-ubiquinone oxidoreductase 24 kDa subunit
0
Mitochondrial inner membrane

158
IPI00111885
Ubiquinol-cytochrome-c reductase complex core protein I, mitochondrial precursor
0
Mitchondrial inner membrane

175
IPI00121322
Electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial precursor
0
Mitchondrial inner membrane

196
IPI00128023
NADH-ubiquinone oxidoreductase 49 kDa subunit, mitochondrial precursor
0
Mitchondrial inner membrane

134
IPI00111908
Predicted: carbamoyl-phosphate synthetase 1
0
Mitochondrial

145
IPI00114840
Endonuclease G, mitochondrial precursor
0
Mitochondrial

70
IPI00331555
2-oxoisovalerate dehydrogenase alpha subunit, mitochondrial precursor
0
Mitochondrial

94,95
IPI00115607
Trifunctional enzyme beta subunit, mitochondrial precursor
0
Mitochondrial

145
IPI00115824
NipSnap1 protein
0
Mitochondrial

22
IPI00116154
Cytochrome c oxidase, subunit vb, full insert sequence
0
Mitochondrial

15,146,147,148,149,100
IPI00118986
ATP synthase oligomycin sensitivity conferral protein, mitochondrial precursor
0
Mitochondrial

127
IPI00119138
Ubiquinol-cytochrome-c reductase complex core protein 2, mitochondrial precursor
0
Mitochondrial

147,148
IPI00120984
NADH-ubiquinone oxidoreductase 19 kDa subunit
0
Mitochondrial

137
IPI00129516
Ubiquinol-cytochrome c reductase complex 11 kDa protein, mitochondrial precursor
0
Mitochondrial

93,99,100,192,203
IPI00130280
ATP synthase alpha chain, mitochondrial precursor
0
Mitochondrial

149,150
IPI00132217
Tetratricopeptide repeat protein 11
1
Mitochondrial

150,151
IPI00132390
NADH-ubiquinone oxidoreductase B15 subunit
1
Mitochondrial

101,132,137,141,153
IPI00170093
NADH-ubiquinone oxidoreductase 23 kDa subunit, mitochondrial precursor
0
Mitochondrial

92,93,94,95,96
IPI00223092
Hydroxyacyl-coenzyme A dehydrogenase/3-ketoacyl-coenzyme A
0
Mitochondrial

142,143,152
IPI00230507
ATP synthase D chain, mitochondrial
0
Mitochondrial

162
IPI00308882
NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial precursor
0
Mitochondrial

149
IPI00344004
13 KDa differentiation-associated protein
0
Mitochondrial

145,146
IPI00420718
Hydroxymethylglutaryl-CoA synthase, mitochondrial precursor
0
Mitochondrial

51
IPI00308885
60 kDa heat shock protein, mitochondrial
0
Mitochondrial

153
IPI00462250
Similar to adenine nucleotide translocase
3
Mitochondrial

85,165,167,203
IPI00468481
ATP synthase beta chain, mitochondrial precursor
0
Mitochondrial

147
IPI00117281
Phospholipid hydroperoxide glutathione peroxidase, mitochondrial precursor
0
Mitochondrial and cytoplasmic

169
IPI00133240
Ubiquinol-cytochrome c reductase iron-sulfur subunit, mitochondrial precursor
0
Mitochondrial inner membrane

200,201
IPI00230351
Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial precursor
0
Mitochondrial inner membrane

174
IPI00132042
Pyruvate dehydrogenase E1 component beta subunit, mitochondrial precursor
0
Mitochondrial matrix

156
IPI00315794
Cytochrome b5 outer mitochondrial membrane isoform precursor
1
Mitochondrial outer membrane

65,109,144,170,176,100
IPI00134746
Argininosuccinate synthase
0
mitochondrion

145,146
IPI00338536
Succinate dehydrogenase [ubiquinone] iron-sulfur protein, mitochondrial precursor
0
Mitochondrion

112
IPI00122547
Voltage-dependent anion-selective channel protein 2
0
Mitochondrion outer membrane

147
IPI00131186
Splice isoform 2 of transcription factor BTF3
0
Nuclear

149
IPI00317794
Nucleolin
0
Nuclear

53
IPI00331146
UMP-CMP kinase
0
Nuclear

150
IPI00458856
Similar to ZNF91L isoform 1
0
Nuclear

6
IPI00461822
E1A binding protein p300
0
Nuclear

55
IPI00126172
RIKEN cDNA 4931406C07, PTD012 homolog
0
Nuclear

150,151
IPI00113241
40S ribosomal protein S19
0
Ribosome

104
IPI00116908
Similar to 40 s ribosomal protein S12
0
Ribosome

147
IPI00849793
60S ribosomal protein L12
0
Ribosome

81,127,145
IPI00125971
26S protease regulatory subunit S10B
0
Ribosome

199
IPI00123604
40S ribosomal protein SA
0
Ribosome

125
IPI00135640
26S protease regulatory subunit 8
0
Ribosome

149,150,151
IPI00139780
60S ribosomal protein L23
0
Ribosome

149,150
IPI00222546
60S ribosomal protein L22
0
Ribosome

145
IPI00314950
60S acidic ribosomal protein P0
0
Ribosome

149
IPI00322562
40S ribosomal protein S14
0
Ribosome

146
IPI00331092
40S ribosomal protein S4, isoform
0
Ribosome

149,150
IPI00331461
60S ribosomal protein L11
0
Ribosome

188
IPI00351894
Similar to ribosomal protein
0
Ribosome

148
IPI00849793
60S ribosomal protein L12
0
Ribosome

149
IPI00465880
40S ribosomal protein S17
0
Ribosome

199
IPI00123604
40S ribosomal protein SA
0
Ribosome

4,11
IPI00121209
Apolipoprotein A-I precursor
0
Secreted

149
IPI00121837
Ribonuclease 4 precursor
1
Secreted

12
IPI00122429
Plasma retinol-binding protein precursor
0
Secreted

163
IPI00123920
Alpha-1-antitrypsin 1–3 precursor
0
Secreted

163
IPI00123924
Alpha-1-antitrypsin 1–4 precursor
0
Secreted

163
IPI00123927
Alpha-1-antitrypsin 1–5 precursor
0
Secreted

162
IPI00128484
Hemopexin precursor
1
Secreted

3,117,118
IPI00131695
Serum albumin precursor
0
Secreted

98
IPI00139788
Serotransferrin precursor
0
Secreted

199
IPI00323571
Apolipoprotein E precursor
0
Secreted

135,136
IPI00377351
Apolipoprotein A-IV precursor
0
Secreted

163
IPI00406302
Alpha-1-antitrypsin 1-1 precursor
0
Secreted

100,155,156
IPI00466399
21 kDa protein
0
Secreted

156
IPI00480401
Major urinary protein 1 precursor
0
Secreted

122
IPI00130661
Tripeptidyl-peptidase I precursor
0
Secreted (lysosomal)

101
IPI00115302
Branched chain ketoacid dehydrogenase E1, beta polypeptide
0
Membrane

197
IPI00120716
Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 1
0
Membrane

21,137
IPI00120719
Cytochrome c oxidase, subunit va, full insert sequence
0
Membrane

125
IPI00124790
Polyposis locus protein 1-like 1
3
Membrane

129
IPI00132076
Catechol O-methyltransferase
1
Membrane

130,142
IPI00138406
Ras-related protein Rap-1A
0
Membrane

174
IPI00162780
Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 2
0
Membrane

88,154
IPI00230113
Cytochrome b5
1
Membrane

199,200
IPI00353727
Annexin A4
0
Membrane

110
IPI00117416
Neighbor of COX4
0
Unknown

143
IPI00121271
Hypothetical S-adenosyl-L-methionine-dependent methyltransferases structure containing protein
0
Unknown

144,108
IPI00267667
RIKEN cDNA 6330409N04, CLLL6 protein homolog
0
Unknown

101
IPI00269613
Eukaryotic translation initiation factor 3 subunit 2
0
Unknown

149,150
IPI00307837
51 kDa protein
0
Unknown

203
IPI00318204
Sid6061p
0
Unknown

105
IPI00273646
Similar to glyceraldehyde-3-phosphate dehydrogenase
0
Unknown

189,190,194
IPI00626790
Glutamine synthetase
0
Unknown

50
IPI00345842
86 KDa PROTEIN
0
Unknown

51
IPI00350780
45 kDa protein
0
Unknown

133
IPI00381231
77 KDa protein
0
Unknown

144
IPI00923085
Probable ubiquitin-conjugating enzyme E2 FLJ25076 homolog
0
Unknown

146,147,150,173
IPI00460295
44 KDa protein
0
Unknown

156
IPI00330913
Major urinary protein 26
0
Unknown

59
IPI00467988
169 kDa protein
0
Unknown

100,155,156
IPI00469517
21 kDa protein
0
Unknown

149
IPI00130554
Splice isoform 1 of SNARE-associated protein Snapin
0
Vesicular membrane

101,127,134
IPI00131366
Keratin, type II cytoskeletal 6B
0
Sarcolemma

83,106,107
IPI00121788
Peroxiredoxin 1
0
Microbody

101,139
IPI00348328
Keratin Kb40
0
Intermediate filament

156
IPI00137414
Left-right dynein
0
Cilium

aESI-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 (400 μg) 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%; ), (b) putative membrane proteins (5%; ), and (c) other proteins (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 name
Predicted TMD
GRAVY score
PI value
Subcellular location

9
IPI00112322
UDP-glucuronosyltransferase 2B5 precursor
1
−0.031
7.94
ER

9
IPI00127223
UDP glucuronosyltransferase 2 family, polypeptide B36
1
−0.036
8.47
ER

9
IPI00222496
Protein disulfide-isomerase A6
1
−0.292
5.05
ER

8
IPI00417182
UDP-glycosyltransferase 1 family polypeptide A5
1
0.044
8.33
ER

9
IPI00116572
Cytochrome P450, family 2, subfamily d, polypeptide 9
2
−0.043
6.37
ER

15
IPI00113655
40S ribosomal protein S6
0
−0.918
10.68
ER

5
IPI00129526
Endoplasmin precursor (ER protein 99,94 kDa glucose-regulated protein)
0
−0.72
4.74
ER

13
IPI00130985
Short-chain dehydrogenase CRAD2
0
0.026
8.35
ER

6
IPI00222809
Similar to GDH/6PGL endoplasmic bifunctional protein
0
−0.18
6.61
ER

8
IPI00230108
Glucose-regulated protein, full insert sequence
0
−0.479
5.78
ER

10,11
IPI00317356
Paraoxonase 1
0
−0.01
5.02
ER

7
IPI00319992
78 kDa glucose-regulated protein precursor
0
−0.481
5.07
ER

13
IPI00121079
NADH-cytochrome b5 reductase 3
0
−0.203
8.56
ER, membrane bound

9
IPI00123964
Cytochrome P450 2A5
1
−0.203
9.23
ER, membrane bound

9
IPI00114779
Cytochrome P450 2C38
0
−0.147
8.69
ER, membrane bound

17
IPI00331322
Microsomal glutathione S-transferase 1
3
0.14
9.67
ER and mitochondrial outer membrane

17
IPI00119766
Cis-retinol androgen dehydrogenase 1
0
0.005
9.25
ER lumen

8
IPI00134691
UDP-glucuronosyltransferase 1-1 precursor, microsomal
2
0.087
8.87
ER, integral to plasma membrane

8
IPI00128287
Cytochrome P450 1A2
1
−0.203
8.92
ER, membrane bound

10
IPI00136910
Cytochrome P450 2D11
2
−0.009
6.15
ER, membrane bound

9
IPI00308328
Cytochrome P450 2F2
1
−0.135
7.74
ER, membrane bound

9,10
IPI00323908
Cytochrome P450 2D10
2
−0.073
6.16
ER, membrane bound

7
IPI00112549
Long-chain-fatty-acid-CoA ligase 1
1
−0.045
6.81
ER, type III membrane protein

8
IPI00133522
Protein disulfide-isomerase precursor
0
−0.386
4.79
ER

9
IPI00116572
Cytochrome P450 2D9
0
−0.063
5.93
ER, membrane bound

5,6
IPI00119618
Calnexin precursor
1
−0.875
4.5
ER, type I membrane protein

1,10,12,14,15
IPI00319973
Membrane-associated progesterone receptor component 1
1
−0.616
4.57
ER, membrane bound

8
IPI00132475
Protein ERGIC-53
1
−0.545
5.92
ER-Golgi intermediate compartment (ERGIC), type I membrane protein

8,17
IPI00109061
Tubulin beta-4 chain homolog
0
−0.406
4.78
Cytoplasmic

10
IPI00110827
Actin, alpha skeletal muscle
0
−0.232
5.23
Cytoplasmic

10,12,14
IPI00110850
Actin, cytoplasmic 1
0
−0.2
5.29
Cytoplasmic

1,2,3
IPI00111908
Carbamoyl-phosphate synthase
0
−0.12
6.42
Cytoplasmic

1,8,9,13
IPI00117348
Tubulin alpha-2 chain
0
−0.23
4.94
Cytoplasmic

9,10,11,12,13
IPI00117914
Arginase 1
0
−0.187
6.52
Cytoplasmic

17
IPI00120451
Fatty acid-binding protein, liver
0
−0.409
8.59
Cytoplasmic

9
IPI00129028
Similar to tubulin, alpha 3C isoform 1
0
−0.204
4.98
Cytoplasmic

1–11,13,17
IPI00130950
Betaine-homocysteine S-methyltransferase
0
−0.36
8.01
Cytoplasmic

1,4,6,10,11,14,15
IPI00134746
Argininosuccinate synthase
0
−0.361
8.36
Cytoplasmic

3,4
IPI00114710
Pyruvate carboxylase, mitochondrial precursor
0
−0.173
6.25
Mitochondrial

17
IPI00553333
Hemoglobin subunit beta-1
0
0.092
7.13
Mitochondrial

9
IPI00134809
Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex
0
−0.171
9.1
Mitochondrial

17
IPI00117978
Cytochrome c oxidase subunit IV isoform 1, mitochondrial precursor
1
−0.412
9.25
Mitochondrial inner membrane

15,16
IPI00315794
Cytochrome b5 outer mitochondrial membrane isoform precursor
1
−0.602
4.79
Mitochondrial outer membrane

13
IPI00321718
Prohibitin-2
0
−2.58
9.83
Mitochondrial, cytoplasmic, nuclear

13
IPI00122547
Voltage-dependent anion-selective channel protein 2
0
−0.223
7.44
Outer mitochondrial Membrane

17
IPI00114559
Histone H2A type 1
0
−0.572
11.22
Nuclear

16,17
IPI00114642
Histone H2B F
0
−0.762
10.32
Nuclear

8
IPI00387318
Cell cycle control protein 50A
2
−0.331
8.58
Membrane

15
IPI00113849
Splice isoform 2 of cell division control protein 42 homolog
0
−0.157
6.16
Membrane

13
IPI00122549
Splice isoform Pl-VDAC1 of voltage-dependent anion-selective channel protein 1
0
−0.334
8.55
Membrane

15
IPI00127408
Ras-related C3 botulinum substrate 1
0
−0.101
8.77
Membrane

15
IPI00138406
Ras-related protein Rap-1A
0
−0.375
6.39
Membrane

6
IPI00116921
Scavenger receptor class B member 1
2
0.073
8.29
Integral membrane protein

1
IPI00121985
Splice Isoform 1 of solute carrier organic anion transporter family, member 1B2
12
0.172
8.95
Integral membrane protein

9
IPI00124830
Integrin-associated protein precursor
5
0.563
8.58
Integral membrane protein

14,15
IPI00131176
Cytochrome c oxidase subunit 2
2
0.27
4.6
Integral membrane protein

1
IPI00132604
Secretedretory carrier-associated membrane protein 3
4
0.028
7.55
Integral membrane protein

1
IPI00135701
Solute carrier organic anion transporter family, member 1A1
11
0.244
8.58
Integral membrane protein

1
IPI00311682
Sodium/potassium-transporting ATPase alpha-1 chain precursor
10
0.002
5.3
Integral membrane protein

6
IPI00331214
Platelet glycoprotein IV
2
−0.053
8.61
Integral membrane protein

2
IPI00119063
Prolow-density lipoprotein receptor-related protein 1
1
−0.502
5.17
Integral to membrane

1,12,16,17
IPI00124790
Polyposis locus protein 1-like 1
3
0.375
6.82
Integral to membrane

10
IPI00129677
Asialoglycoprotein receptor major subunit
1
−0.66
5.99
Integral to membrane

17
IPI00467119
Camello-like protein 1
1
0.302
9.61
Integral to membrane

5,8
IPI00316329
Keratin, type II cytoskeletal 1
0
−0.588
8.2
Intermediate filament

10,11,12
IPI00108844
Cation-dependent mannose-6-phosphate receptor precursor
1
−0.168
5.24
Type I membrane protein

9
IPI00109998
H-2 class I histocompatibility antigen, D-B alpha chain precursor
1
−0.508
6.28
Type I membrane protein

4
IPI00120245
Integrin alpha-V precursor
1
−0.246
5.46
Type I membrane protein

3,4
IPI00121190
Epidermal growth factor receptor precursor
2
−0.316
6.46
Type I membrane protein

2
IPI00126186
Macrophage mannose receptor 1 precursor
1
−0.5
6.47
Type I membrane protein

5
IPI00134549
Splice isoform LAMP-2A of lysosome-associated membrane glycoprotein 2 precursor
1
−0.036
7.05
Type I membrane protein

13
IPI00312018
Malectin
1
−0.203
5.73
Type I membrane protein

3
IPI00312063
Low-density lipoprotein receptor precursor
1
−0.391
4.88
Type I membrane protein

16
IPI00466570
Transmembrane emp24 domain-containing protein 10
2
−0.169
6.25
Type I membrane protein

4
IPI00108535
Carcinoembryonic antigen-related cell adhesion molecule 1
1
−0.302
5.35
Type I membrane protein

5
IPI00310059
Polymeric-immunoglobulin receptor precursor
1
−0.499
5.26
Type I membrane protein also secreted

9
IPI00121550
Sodium/potassium-transporting ATPase beta-1 chain
1
−0.55
8.83
Type II membrane protein

2,3
IPI00134585
Glutamyl aminopeptidase
1
−0.344
5.28
Type II membrane protein

10
IPI00307966
ADP-ribosyl cyclase 1
1
−0.106
8.64
Type II membrane protein

3
IPI00319509
Aminopeptidase N
1
−0.277
5.62
Type II membrane protein

3
IPI00458003
Ectonucleotide pyrophosphatase/phosphodiesterase 3
1
−0.346
6.13
Unknown

9
IPI00409409
CD1D1 protein
1
−0.178
9.22
Unknown

7
IPI00621548
NADPH-cytochrome P450 reductase
1
−0.463
5.37
Unknown

9
IPI00321644
Cytochrome P450 2D26
2
−0.105
6.16
Unknown

1
IPI00127016
Hydroxysteroid 17-beta dehydrogenase 6
0
−0.075
8.63
Unknown

16
IPI00221721
Hypothetical krab box containing protein, full insert sequence
0
−0.142
9.84
Unknown

8
IPI00224073
Hypothetical peptidase family M20/M25/M40 containing protein, full insert sequence
0
−0.01
5.99
Unknown

16
IPI00228379
Ferritin light chain 2
0
−0.479
6.37
Unknown

17
IPI00266842
17 kDa protein
0
−0.668
10.48
Unknown

15
IPI00379258
Similar to ferritin light chain 2
0
−0.454
8.51
Unknown

3
IPI00405742
Plexin B2
0
−0.3
5.67
Unknown

2
IPI00408258
Structure-specific endonuclease subunit SLX4
0
−0.714
5.33
Unknown

11
IPI00462251
Hypothetical protein LOC72792 isoform 1
0
−0.429
5.82
Unknown

15
IPI00605814
Similar to Ferritin light chain 1
0
−0.358
6.42
Unknown

10
IPI00131366
Keratin, type II cytoskeletal 6B
0
−0.488
8.32
Sarcolemma

10
IPI00322209
Keratin, type II cytoskeletal 8
0
−0.602
5.7
Sarcolemma

8
IPI00853991
Similar to VH coding region
0
−0.102
5.31
Secreted

10
IPI00126458
MRNA
1
−0.55
5.66
MHC class I protein complex

15
IPI00121788
Peroxiredoxin 1
0
−0.221
8.26
Microbody

(a)

(b)

Figure 2: 1DE 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/).

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 (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 ( proteins; Table 1) and 1DE dataset ( 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 ( 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 (Table 1) and 1DE dataset of Na₂CO₃-extracted microsomal proteins (Table 2).

Accession number
Protein name
Predicted TMD
GRAVY scores
PI value
Location

IPI00108454
Similar to 40S ribosomal protein S6
0
−0.918
10.68
ER

IPI00112322a
UDP-glucuronosyltransferase 2B5 precursor
1
−0.031
7.94
ER

IPI00129526
Endoplasmin precursor (ER protein 99, 94 kDa glucose-regulated protein)
0
−0.72
4.74
ER

IPI00133522
Protein disulfide-isomerase precursor
0
−0.386
4.79
ER

IPI00222496a
Protein disulfide isomerase-associated 6
1
−0.292
5.05
ER

IPI00230108
Glucose-regulated protein, full insert sequence
0
−0.479
5.78
ER

IPI00319992
78 kDa glucose-regulated protein precursor
0
−0.481
5.07
ER

IPI00331322a
Microsomal glutathione S-transferase 1
3
0.14
9.67
ER and mitochondrial outer membrane

IPI00319973a
Membrane-associated progesterone receptor component 1
1
−0.616
4.57
ER, membrane bound

IPI00109061
Tubulin beta-4 chain homolog
0
−0.406
4.78
Cytoplasmic

IPI00110827
Actin, alpha skeletal muscle
0
−0.232
5.23
Cytoplasmic

IPI00110850
Actin, cytoplasmic 1
0
−0.2
5.29
Cytoplasmic

IPI00111908
Predicted: carbamoyl-phosphate synthetase 1
0
−0.12
6.42
Cytoplasmic

IPI00117348
Tubulin alpha-2 chain
0
−0.23
4.94
Cytoplasmic

IPI00117914
Arginase 1
0
−0.187
6.52
Cytoplasmic

IPI00134746
Argininosuccinate synthase
0
−0.361
8.36
Cytoplasmic

IPI00117978a
Cytochrome c oxidase subunit IV isoform 1, mitochondrial precursor
1
−0.412
9.25
Mitochondrial inner membrane

IPI00315794a
Cytochrome b5 outer mitochondrial membrane isoform precursor
1
−0.602
4.79
Mitochondrial outer membrane

IPI00122547a
Voltage-dependent anion-selective channel protein 2
0
−0.223
7.44
Outer mitochondrial membrane

IPI00124790a
Polyposis locus protein 1-like 1
3
0.375
6.82
Integral to membrane

IPI00138406a
Ras-related protein Rap-1A
0
−0.375
6.39
Membrane

IPI00121788
Peroxiredoxin 1
0
−0.221
8.26
Microbody

IPI00131366
Keratin, type II cytoskeletal 6B
0
−0.488
8.32
Sarcolemma

aMembrane 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 (, 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 () 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 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 () 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 () that were derived from 1DE and 2DE strategies. KEGG pathway enrichment analysis was performed using WebGestalt. The pathways having enrichment () 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 (Figure 4). 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 () 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 pathway
Protein ID
Protein name
MS-identified proteins

A. Metabolism of exnobiotics
EC:1.14.14.1
IPI00128287
Cytochrome P450 1A2
+

IPI00123964
Cytochrome P450 2A5
+

IPI00116572
Cytochrome P450 2D9
+

IPI00323908
Cytochrome P450 2D10
+

IPI00321644
Cytochrome P450 2D26
+

IPI00114779
Cytochrome P450 2C38
+

IPI00308328
Cytochrome P450 2F2
+

EC:2.5.1.18
IPI00331322
Microsomal glutathione S-transferase 1
+

EC:1.1.1.1
IPI00221400
Alcohol dehydrogenase A chain
+

B. Ribosome
Small subunit
IPI00135640
26S protease regulatory subunit 8
+

IPI00125971
26S protease regulatory subunit S10B
+

IPI00331092
40S ribosomal protein S4, X isoform
+

IPI00116908
Similar to 40s ribosomal protein S12
+

IPI00322562
40S ribosomal protein S14
+

IPI00465880
40S ribosomal protein S17
+

IPI00113241
40S ribosomal protein S19
+

IPI00123604
40S ribosomal protein SA
+

IPI00314950
60S acidic ribosomal protein P0
+

Large subunit
IPI00331461
60S ribosomal protein L11
+

IPI00849793
60S ribosomal protein L12
+

IPI00222546
60S ribosomal protein L22
+

IPI00139780
60S ribosomal protein L23
+

(a)

(b)

Figure 4: 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.

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 (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 () 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 LimitationMammalian 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 ( proteins) of mouse liver microsome proteome relative to the documented data ( 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 study
Ref. [19]
Ref. [18]
Ref. [20]

Species
Mouse
Mouse
Mouse
Rat

Sample
Liver microsome
Liver microsome
Liver microsome
ER, Golgi

Pretreatment
None
Phenobarbital
Phenobarbital or 3-methylcholanthrene
None

Sample preparation
Subfractionated by differential ultra-centrifugation + sucrose gradient centrifugation + Na₂CO₃
Subfractionated by differential ultra-centrifugation
Differential ultracentrifugation
Subfractionated by differential ultra-centrifugation + density gradient centrifugation

Protein separation
2DE, 1DE
1DE
2DE
1DE

1D/2D-Gel Stain
Coomassie brilliant blue (2DE; 1DE)
—
Silver stain
—

Protein identification
MS/MS
MS/MS
PMF
MS/MS

Protein spots on 2D-Gel
514
—
1100
—

Proteins identified in 2D-Gel
183
—
139
—

Proteins identified in 1DE
99
519
—
832 (ER)

Proteins identified in 2-D LC
—
1410
—
—

Proteins identified in 3-D LC
—
3703
—
—

Total identified proteins
259
4142
Unspecified
832 (ER)

Membrane proteins
2DE: 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%)
Unspecified
183 (183/832 = 22%)

Protein superfamily

    P450 family members
10
29
2
11

    Ribosomal proteins
13
16
Unspecified
45

    UDP glycosyltransferases, UGTs
6
8
Unspecified
3

    Tubulins
11
5
Unspecified
2

    Short-chain     dehydrogenase/reductase
32
9
Unspecified
56

    Protein disulfide isomerase
2
4
Unspecified
1

Table 6: Comparison of selected proteins between the current study and the literature data [18–20].

Protein
Current study
Ref. [19]
Ref. [18]
Ref. [20]

P450 family members
2D9, 2A5, 2C38, 1A2, 2D11, 2F2, 2D10, 2D26

2C37
17A1, 20A1, 2B2, 2J3, 4A1, 4A8, 4F1, 4F4, 4V3, 8B1, NA2

GRP-170
Hypoxia upregulated protein 1

170 kDa glucose regulated protein
—

Endoplasmin
Endoplasmin

Tumor rejection antigen gp96

Serotransferrin
Serotransferrin

Transferrin
—

78 kDa glucose-regulated protein
78 kDa glucose-regulated protein

78 kDa glucose-regulated protein
—

Stress-induced phosphoprotein 1
—

Stress-induced phosphoprotein 1
—

Calreticulin family
Calnexin

Calreticulin
—

Protein disulfide-isomerase
Protein disulfide-isomerase precursor (PDI)

Protein disulfide-isomerase precursor (PDI)
Similar to disulfide isomerase

Glucose-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
—

Tubulins
Tubulin beta-4, alpha-1, alpha-2, alpha-4, beta-5, beta-2C, beta 2

Tubulin alpha
Tubulin alpha 6

Fibrinogen
—

Fibrinogen, gamma polypeptide
—

Serine protease inhibitor
—

Similar to serine protease inhibitor 1–4
—

Argininosuccinate synthetase 1
Argininosuccinate synthetase 1

Argininosuccinate synthetase 1
—

Interferon-inducible GTPase
—

Interferon-inducible GTPase

Progesterone receptor membrane component
Progesterone receptor membrane component

Progesterone receptor membrane component
—

Major urinary protein 2
Major urinary protein 2

Major urinary protein 2
—

Superoxide dismutase I
—

Superoxide dismutase I
—

Ribosomal proteins
26S 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, P0

Unspecified
40S 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, L7A

UDP glycosyltransferases, UGTs
UDP-glucuronosyltransferase 2B5, 2B36, 1A5

Unspecified
UDP-Glucuronosyltransferase 1A7

UDP-glucuronosyltransferase 1-1 precursor

UDP-Glucuronosyltransferase GTNA2

UDP-glucuronosyltransferase 1-2 precursor

Short-chain dehydrogenase/reductase
Glyceraldehyde-3-phosphate dehydrogenaseAlcohol dehydrogenase AShort-chain dehydrogenase CRAD2 Cis-retinol androgen dehydrogenase 1 Hydroxysteroid 17-beta dehydrogenase 6

Unspecified
Glyceraldehyde 3-phosphate dehydrogenase Alcohol dehydrogenase [NADP+]Similar to retinal short-chain Dehydrogenase/reductaseRetinol dehydrogenase 10 Hydroxysteroid (17-Beta) dehydrogenase 8

Oxidoreductase HTATIP2 NADH-ubiquinone oxidoreductase 24 kDa subunitNADH-cytochrome b5 reductase 3 NADPH-cytochrome P450 reductase

Unspecified
Oxidoreductase ero1-L endoplasmic oxidoreductase 1 Beta

No 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.

Abbreviations

ACN: | Acetonitrile
BinGO: | Biological Networks Gene Ontology
DTT: | Dithiothreitol
1DE: | One-dimensional gel electrophoresis
2DE: | Two-dimensional gel electrophoresis
ER: | Endoplasmic reticulum
GO: | Gene ontology
GRAVY: | Grand average of hydropathy
HLPP: | Human Liver Proteome Project
IEF: | Isoelectric focusing
KEGG: | Kyoto Encyclopedia of Genes and Genomes
LC: | Liquid chromatography
MS: | Mass spectrometry
MS/MS: | Tandem mass spectrometry
Q-TOF: | Quadrupole-time of flight
RP: | Reverse phase
TMD: | Transmembrane domains.

Acknowledgments

This work was supported by China National Haman Liver Proteome Project (Grant no. 2004 BA711A18) and The National Basic Research Program of China (Grant No. 2011CB910704).

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