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International Journal of Proteomics logoLink to International Journal of Proteomics
. 2012 Mar 20;2012:832569. doi: 10.1155/2012/832569

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, Zhuchu Chen 1,*
PMCID: PMC3317213  PMID: 22500222

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 (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 display  514 ± 83  protein 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).

Figure 1.

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.

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, X 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.

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).

Figure 2.

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/).

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

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 (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 (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.

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 (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 (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.

Figure 4.

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.

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 +

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 [1820]. Comparison of the current study with the literature data [1820] 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 [1820], 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 [1820].

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 [1820].

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 dehydrogenase
Alcohol dehydrogenase A
Short-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/reductase
Retinol dehydrogenase 10
Hydroxysteroid (17-Beta) dehydrogenase 8
Oxidoreductase HTATIP2 NADH-ubiquinone oxidoreductase 24 kDa subunit
NADH-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.

Supplementary Material

Supplemental figure 1: is detection and validation of the purity of isolated microsomes.

Supplemental figure 2: is distribution of subcellular locations of 2DE-derived proteins.

Supplemental figure 3: is distribution of 1DE-derived proteins over the GRAVY scores.

Supplemental figure 4: is distribution of 1DE-derived proteins over the pI values.

Supplemental figure 5: is significant enrichment of GO terms for mouse liver microsome proteins (n = 259) that were derived from 1DE and 2DE strategies.

Supplemental Table 1: MS/MS identification of 2DE-arrayed proteins from mouse liver microsomal preparations.

Supplemental Table 2: MS/MS identification of 1DE-separated proteins from Na2CO3-extracted mouse liver microsomal membrane preparations

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).

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.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental figure 1: is detection and validation of the purity of isolated microsomes.

Supplemental figure 2: is distribution of subcellular locations of 2DE-derived proteins.

Supplemental figure 3: is distribution of 1DE-derived proteins over the GRAVY scores.

Supplemental figure 4: is distribution of 1DE-derived proteins over the pI values.

Supplemental figure 5: is significant enrichment of GO terms for mouse liver microsome proteins (n = 259) that were derived from 1DE and 2DE strategies.

Supplemental Table 1: MS/MS identification of 2DE-arrayed proteins from mouse liver microsomal preparations.

Supplemental Table 2: MS/MS identification of 1DE-separated proteins from Na2CO3-extracted mouse liver microsomal membrane preparations


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