An Alternative Strategy for the Membrane Proteome Analysis of the Green Sulfur Bacterium Chlorobium tepidum Using Blue Native PAGE and 2-D PAGE on Purified Membranes Michalis Aivaliotis,†,# Michael Karas,‡ and Georgios Tsiotis*,† Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box 2208, GR-71003 Voutes, Heraklion, Greece, and Institut fu ¨ r Pharmazeutische Chemie, Johann Wolfgang Goethe-Universita¨t, Marie Curie Strasse 9, 60439 Frankfurt am Main, Germany Received October 19, 2006
To avoid the specific problems concerning intrinsic membrane proteins in proteome analysis, an alternative strategy is described that is complementary to previous investigations using 2-D polyacrylamide gel electrophoresis (PAGE) techniques. The strategy involves (a) obtaining purified preparations of the membranes from Chlorobium tepidum by washing with 2 M NaBr, which removed membraneassociated soluble proteins and membrane-associated organelles; (b) separation of membrane protein complexes using 1-D Blue-native polyacrylamide gel electrophoresis (BN-PAGE) after solubilization with n-dodecyl-β-D-maltoside (DDM); (c) combination of the BN with Tricine-SDS-PAGE; (d) highthroughput mass spectrometric analysis after gel band excision, in-gel digestion, and MALDI target spotting; and (e) protein identification from mixtures of tryptic peptides by peptide mass fingerprinting. Using this approach, we identified 143 different proteins, 70 of which have not been previously reported using 2-D PAGE techniques. Membrane proteins with up to 14 transmembrane helices were found, and this procedure proved to be efficient with proteins within a wide pI range (4.4-11.6). About 54% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, and protein translocation, while for the others, a function is not yet known, indicating the potential of the method for the elucidation of the membrane proteomes in general. Keywords: Mass spectrometric identification • Chlorobium tepidum • Membrane proteins • Two-dimensional polyacryamide gel electrophoresis • Blue native
1. Introduction Two-dimensional electrophoresis based on isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension is the most commonly used method for protein separation in proteomic approaches. However, despite recent developments1,2 and systematic evaluation of detergents,3,4 isoelectric focusing is still not the method of choice for separation of hydrophobic membrane proteins. Membrane proteins represent at least 30% of all proteins coded in the genome.5 Because of their interfacial location, membrane proteins play key roles in cellular processes such as energy conversion, signal transduction, and metabolite transportation. For differential display, hardly detectable intrinsic membrane proteins are even of higher significance than high-abundance soluble proteins. * Corresponding author: Georgios Tsiotis, Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box 2208, GR-71003 Voutes, Heraklion, Greece, E-mail,
[email protected]; tel, +30 2810 545006; fax, +30 2810 545001. † University of Crete. # Present address: Department of Membrane Biochemistry, Max-PlanckInstitute of Biochemistry, D-82152 Martinsried, Munich, Germany. ‡ Johann Wolfgang Goethe-Universita¨t.
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Journal of Proteome Research 2007, 6, 1048-1058
Published on Web 01/30/2007
Integral membrane proteins identified in recent studies often result from prepurified preparations6 or are found via specialized detection methods.7 Because of their hydrophobic nature, membrane proteins show poor solubility in the solvents used for IEF. They tend to precipitate either at their application position or during the IEF run close to their isoelectric point, where their solubility is even more decreased. Even the initial extraction step to solubilize membrane proteins from the membrane fraction may be critical and is highly dependable on the detergent selection.3 In addition, membrane proteins tend to have more basic isoelectric points8 and therefore could be missed in standard 2-DE gels.2 Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is an electrophoresis technique capable of separating native, catalytically active membrane protein complexes.9,10 BN-PAGE utilizes the anionic dye Coomassie Brilliant Blue G-250, which binds noncovalently to proteins and transfers a negative charge to the membrane protein complexes. Since the complexes are retained in a structural intact form, the hydrophilic subunits of membrane protein complexes remain connected to hydrophobic membrane integral parts during the migration through the gel, thereby shielding hydrophobic areas and reducing the overall hydrophobicity of the complex. During the initial 10.1021/pr060553u CCC: $37.00
2007 American Chemical Society
Membrane Proteome Analysis of C. tepidum
solubilization step of membrane proteins, as well as throughout electrophoresis, no chaotropes are present in order to avoid protein denaturation and to prevent exposure of additional hydrophobic protein sites to the solvent and thereby avoid protein aggregation. The proteome of Chlorobium tepidum comprises 2288 protein coding sequences, more than 973 of which are hypothetical or conserved hypothetical proteins .11 A search in the Chlorobium genome indicates the presence of at least 350 membrane proteins. In our previous studies on the membrane proteome of C. tepidum using standard 2-D electrophoresis technique, it was possible to identify about 88 cytoplasmatic, 25 outer membrane, 37 inner membrane, and 49 unknown proteins.4,12 In the present work, we report for the first time the analysis of proteins from membranes of C. tepidum using BN-PAGE. This method, in combination with Tricine-SDS-PAGE in the second dimension, enables the identification of integral membrane proteins from the cytoplasmatic membrane of the green sulfur anaerobic bacterium C. tepidum by peptide mass fingerprinting using MALDI TOF-MS. Optimized solubilization conditions, as well as improvements in the membrane isolation procedure allowed the identification of membrane proteins which contain up to 14 transmembrane R-helixes (TMH).
2. Materials and Methods 2.1. Isolation and Washing of Membranes. C. tepidum cells were grown for 24-30 h at 48 °C, and cells were harvested by centrifugation at 7000g for 10 min and stored at -80 °C. The isolation of the membrane fraction of C. tepidum was performed according to Aivaliotis et al.13 In brief, 10 g of cell paste were respundend in 30 mL of 20 mM Tricine-Tris, pH 7.5, buffer and disrupted by sonication (20 times, 15 s with 45 s intervals at 150 W) on ice. Unbroken cells and cell debris were removed from the resulting suspension by centrifugation at 10 000g for 15 min at 4 °C. The supernatant was then centrifuged at 200 000g for 1 h at 4 °C to pellet the membrane fraction. The membranes were suspended in 20 mM Tricine-Tris, to an OD750 nm of 200 cm-1 (about 2 mg of BChlc/mL) and stored at -80 °C until use. To optimize the removal of membrane-associated proteins, membranes were washed with salt or chaotropic compounds. Membranes were incubated in 20 mM Tricine-Tris, pH 7.5, 5 mM EDTA, and 2 M NaBr at room temperature and sonicated three times (15 s and 45 intervals) to minimize the size of vesicles and avoid the trapping of soluble proteins in them. Integral membrane proteins and membrane-associated proteins were separated by centrifugation at 100 000g for 30 min. The protein concentration in the membrane fraction and in the supernatant was determined according to Bradford.14 2.2. Solubilization of Membranes. Membranes were incubated for 30 min at 8 °C in solubilization buffer (20 mM Tris/ HCl, pH 8.0, 150 mM NaCl, and detergent at a ratio 1:1, 3:1, and 5:1) under agitation by a rotary shaker at 15 rpm. Nonsolubilized proteins and membrane fragments were sediment at 136 000g for 1 h at 8 °C. Triton X-100 was obtained from Sigma (Germany), and N-dodecyl β-D-maltoside (DDM) was obtained from Biomol (Hamburg, Germany). The optical spectra were recorded on an Aminco dual wavelength DW 2000 UV-vis spectrophotometer (SLM Aminco). 2.3. Gel Electrophoresis. 2.3.1. First-Dimensional Electrophoresis (BN-PAGE). The BN-PAGE was carried out in a 8% polyacrylamide gel (2.5% bis-acrylamide), as described in
research articles Schaegger and von Jagow.10 In brief, prior to electrophoresis, glycerol was added to the samples at a final concentration of 20% (v/v). Electrophoresis was applied at 10 °C, initially at 80 V until the entry of the protein sample into the stacking gel, and subsequently at 150 V. The run was terminated as soon as the dye front ran off from the gel. The high molecular weight calibration kit for native electrophoresis, containing thyroglobulin 669 kDa, ferritin 440 kDa, catalase 232 kDa, lactate dehydrogenase 140 kDa, and albumin 66 kDa which was obtained from Amersham Biosciences, was used for the molecular weight estimation of the separated complexes. 2.3.2. Second-Dimensional Electrophoresis (Tricine-SDSPAGE). Tricine-SDS-PAGE was performed according to Schaegger and von Jagow15 using a self-built system with 12% acrylamide gels (26 × 24 × 0.1 cm). The excised lane was transferred to a 26 × 24 cm glass plate at the position of the sample gel and soaked with 1% SDS and 1% mercaptoethanol for 2 h. One millimeter spacers were positioned, a glass plate was placed above the gel, and clamps were fixed. While still in a horizontal position, mercaptoethanol was removed with a paper towel. The spacers and glass plates were aligned and vertically placed in the casting stand. The acrylamide solution was then injected between the glass plates up to 3 cm from the first-dimensional gel strip. The freshly poured gel was overlaid with a small amount of water. After polymerization, the water was decanted, and a 2 cm layer of 3% T TricineSDS-gel mixture was added. Electrophoresis was performed at room temperature, starting for 3 h at 75 V, and the voltage was then raised to 150 V overnight (max 50 mA). Staining was carried out with 0.02% Coomassie Brilliant Blue G-250 in 10% acetic acid or colloidal Coomassie solution.16 SDSPAGE standards (Fluka Laboratories) which contained R-lactalbumin 14.2 kDa, trypsin inhibitor 20 kDa, trypsinogen 24 kDa, carbonic anhydrase 29 kDa, glyceraldeyde-3-phosphate dehydrogenase 36 kDa, albumin from chicken eggs 45 kDa, and albumin from bovine serum 67 kDa were used for molecular weight estimation. 2.4. Protein Identification Using Peptide Mass Fingerprinting by MALDI-TOF MS. Spots were excised from the Coomassie blue-stained gels and subjected to in-gel digestion protocol described in Aivaliotis et al.12 After 12 h, the supernatant was removed, and the remaining peptides were extracted three times with 50 µL of 50% acetonitrile/5% formic acid. All fractions were pooled and dried in a vacuum centrifuge prior to analysis. The digestion mixture was dissolved in 5 µL of 50% acetonitrile/1% TFA. The sample (0.5 µL) was mixed with 0.5 µL of matrix (2 mg/mL 1-cyano-4- hydroxycinnamic acid (Fluka) in 50% acetonitrile/0.5% TFA) directly on a stainless steel target (Applied Biosystems) and dried in ambient air. Delayed extraction (DE) MALDI-TOF mass spectra were recorded on a Voyager-DE STR instrument (Applied Biosystems) using the parameter as in Aivaliotis et al.12 Spectra were externally calibrated with sequazyme peptide mass standards kit (Applied Biosystems) with the following peptides: des-Arg1bradykinin (904.47 Da), angiotensin I (1296.69 Da), glu1fibrinopeptide B (1570.68 Da), neurotensin, ACTH (1-17 clips) (2093.0 Da), ACTH (18-39 clip) (2465.20 Da), and ACTH (738 clip) (3657.93 Da). Between 1000 and 2000 single scans were accumulated for each mass spectrum. All spectra were smoothed, noise-filtered, and deisotoped using the data explorer V4.0 software (Applied Biosystems). Deisotoped peaks were automatically labeled from the software, and the peaks were used for database search. Autolytic tryptic peptides or peptides Journal of Proteome Research • Vol. 6, No. 3, 2007 1049
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Figure 1. Characteristic absorption spectra of the membrane fractions (II) and the supernatant (I) after washing with 5 mM EDTA (A) and 2 M NaBr (B). (C) Protein composition of the isolated membrane fractions after washing with 5 mM EDTA (lane 1), 2 M NaBr (lane 2), and the respective supernatants (lanes 3 and 4). Numbers indicate the proteins bands which were analyzed by MALDI-TOF MS.
resulting from the identified protein were used for internal calibration. The PMF spectra were analyzed against the genome database of C. tepidum using spectrum mill V3.0 (Agilent technologies) installed on a local server. The parameters which were used for the database search were minimum number of matched peptides, 4; peptide mass tolerance, 50 ppm; maximum number of missed cleavages, 1; possible amino acid modifications, carbamidomethylation of cysteines and oxidation of methionines. All identified proteins were analyzed using TMtrep17 and PRED-TMBB18 programs for the prediction of transmembrane R-helices and β-strands, respectively. The localization of the identified proteins was predicted using SignalP19 and PSORTB20 programs. The grand average of hydrophobicity (GRAVY) value of the identified proteins was calculated using ProtParam program.21
3. Results 3.1. Washing of the Membranes. The aim of the present work was to develop an efficient method for the mapping of membrane proteins from C. tepidum using BN in the first dimension and Tricine-SDS-PAGE in the second dimension. For this purpose, the membrane fraction was separated from the cytosolic proteins. A preliminary MALDI-TOF MS analysis revealed that even after solubilization of the membranes in 3% Triton X-100 or 2% SDS many soluble and membrane-associated proteins remain in the membrane fraction (for example, the ribosomal proteins L1/L2/L3/L4/L5/L17, S1/S2/S3/S4, or the elongation factors Ts, Tu).12 These proteins are often highly abundant and may therefore obscure membrane proteins in the SDS-PAGE. Our prior aim was to separate and identify integral membrane proteins, and therefore, it was necessary 1050
Journal of Proteome Research • Vol. 6, No. 3, 2007
to further reduce the complexity of the membrane fraction by removing cytosolic and membrane-associated proteins. To obtain a fraction enriched in membrane integral proteins, two washing conditions were tested. The membranes were washed with 5 mM EDTA in 20 mM Tricine-Tris, pH 7.5, or with 2 M NaBr in 20 mM Tricine-Tris, pH 7.5. Absorption spectroscopy was used to characterize the obtained soluble protein fraction. Figure 1A (spectrum I) shows the absorption spectrum of the washed proteins with EDTA, while Figure 1B with NaBr (spectrum I). The maximum of 420 nm indicates the presence of heme-containing protein, whereas in the other case, the maxima of 420 and 750 nm indicate the presence of BChlc-containing proteins. In addition, the spectra of the NaBrwashed membranes showed and additional peak at 810 nm indicating the detachment of chlorosome from the cytoplasmic membrane (Figure 1A,B, spectra II). The purity of the isolated membrane, as well as of the washed proteins, was investigated, by separating the protein samples from the four fractions using 1-D Tricine-SDS-PAGE (Figure 1C). The protein pattern of the membrane fractions differs only in the low molecular weight area (Figure 1C, lanes 1 and 2). In contrast, the differences in the soluble fractions extended to the whole molecular weight range (Figure 1C, lanes 3 and 4). To estimate the nature of soluble and integral membrane proteins in the membrane fractions, as well as in the wash fraction, proteins were separated using 1-D TricineSDS-PAGE (Figure 1C, lanes 1 and 2). and the bands were analyzed with MALDI-TOF-peptide mass fingerprinting (PMF). Soluble and membrane-associated proteins remain in the membrane fraction, as for example, the ribosomal proteins L5, S1, S3, L10, and others. In the EDTA-washed membranes, about 145 proteins were identified, where in the case of NaBr, 106
Membrane Proteome Analysis of C. tepidum
Figure 2. Effect of different detergents on the solubilization of C. tepidum membrane proteins. A defined amount of protein was solubilized at 4 °C for 1 h. Solubilized and nonsolubilized proteins were separated by ultracentrifugation at 100 000g. (A) TricineSDS-PAGE of membrane proteins solubilized with various detergents and various detergents/protein rations (lane 1, DDM/ protein 1; lane 2, DDM/protein 5; lane 3, Triton X-100/protein 1; lane 4, Triton X-100/protein 3; lane 5, Triton X-100/protein 5; (B) BN-PAGE of solubilized proteins as in panel A.
proteins were identified from 49 and 34 protein bands, respectively. Furthermore, different ratios of inner membrane and outer membrane, as well as unknown proteins, were obtained in the membrane fractions. Additionally, chlorosomal proteins were identified only in the EDTA-washed membrane. After the wash, which was followed by separation of the membrane fraction and Tricine-SDS-PAGE (Figure 1C, lanes 3 and 4), we could detect 45 bands in the supernatant of EDTA and 23 bands in the NaBr supernatant. We have identified 106 proteins in the EDTA samples and 60 proteins in the NaBr samples, respectively. While the inner and outer membrane proteins represent only 8% of the EDTA supernatant, an increase to 21% is observed in the case of NaBr samples. In addition, the soluble proteins (periplasmic and cytoplasmic) represent about 72% of the EDTA supernatant (Figure 5A), while only 62% in NaBr (Figure 5B). 3.2. Protein Solubilization. The washed membrane fraction has to be solubilized by a detergent which is compatible with BN-PAGE conditions. Two nonionic detergents were tested, and their solubilization efficiency was analyzed using Tricine-SDSPAGE (Figure 2A). The best result with respect to the amount
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Figure 3. Separation protein complexes and supercomplexes of C. tepidum by 1-D BN-PAGE. Proteins were solubilized by DDM in a ratio of DDM/protein of 1. (A) Solubilized EDTA-washed membranes; (B) solubilized NaBr-washed membranes. Numbers indicate the proteins bands which were analyzed by MALDI-TOF MS.
of solubilized proteins and solubilization selectivity was obtained with the detergent DDM in a ratio of 1:1 to the protein (Figure 2A, lane 1) and Triton X-100 in a ratio 5:1 to the protein (Figure 2A, lane 5). Further characterization of the solubilized membrane protein complexes was achieved by BN-PAGE (Figure 2B). It is clear that only in the case of the membrane solubilization with DDM in a 1:1 ratio it was possible to obtain distinct complexes (Figure 2B, lane 1). In contrast, with higher concentration of DDM as with Triton X-100 it was not possible to obtain distinct bands in the BN-PAGE separation. Therefore, for the solubilization of the washed membranes, DDM was used in a DDM/protein ratio of 1. In the NaBr-washed membranes, more protein complexes were observed than in the EDTAwashed membranes (Figure 3, lanes 1 and 2). MALDI-TOF analysis of about 30 obtained protein bands of the NaBrwashed membranes allowed the identification of only 33 proteins (Table 1). While about 37% of them represent soluble proteins (periplasmic, cytoplasmic), the rest correspond to inner membrane, outer membrane, and unknown proteins which contain at least one transmembrane helix (TMH) (Figure 5E). 3.3. Characterization of Membrane Complexes by 2-D BN/ Tricine-SDS-PAGE. 1-D BN-PAGE was combined with TricineJournal of Proteome Research • Vol. 6, No. 3, 2007 1051
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Table 1. List of the Identified Proteins Separated in 1-D ΒΝ-PAGE, 1-D Tricine-SDS-PAGE, and 2-D BN/Tricine-SDS-PAGEa
no.
protein definition
pept. accession predicted no. of cov. no./gene MW/pI peptides % TM GRAVY
1 magnesium-chelatase, subunit I Q93SW1/ 42799/8.2 bchI 2 magnesium-chelatase 67 kDa Q93SW0/ 67014/5.1 subunit bchD 3 magnesium-chelatase, subunit Q8KFA7 72104/7.7 D/I family 4 magnesium-chelatase, subunit Q8KBZ1 55909/7.3 D/I family 5 magnesium-transporting Q8KEQ5/ 97910/5.7 ATPase, E1-E2 family mgt 6 iron(III) ABC transporter, Q8KBM6/ 28634/8.5 ATP-binding protein fecE 7 iron compound ABC Q8KDV1 46795/5.9 transporter, ATP-binding protein 8 Ferric siderophore receptor, Q8KB41 93821/5.9 putative, TonB receptor family 9 phosphate ABC transporter, Q8KDZ9 28378/9.5 periplasmic phosphate-binding protein, putative 10 ABC transporter, ATP-binding Q8KFE9 70359/8.6 protein 11 ABC-type export system, Q8KEY0 26710/5.5 ATP-binding subunit 12 ABC-type export system, Q8KEX9 34024/9.9 membrane fusion protein 13 ABC-type export system, outer Q8KEX8 53358/8.7 membrane channel protein 14 glutathione-regulated Q8KEH3 66658/6.8 potassium-efflux system protein KefC, putative 15 ArsA ATPase family protein Q8KDR5 44541/5.0 16 ArsA ATPase family protein Q8KB52 42959/6.0 17 ArsA ATPase family protein Q8KFH8 49523/6.2 18 proton transporting ATPase, Q8KBU9 96714/5.8 E1-E2 family 19 outer membrane efflux protein, Q8KED1 52717/8.9 putative 20 AcrB/AcrD/AcrF family protein Q8KAV4 118868/8.7 21 Na+/H+ antiporter, putative Q8KDB0 87355/9.1 22 sodium:solute symporter family Q8KBI1 63830/8.8 protein 23 multidrug resistance protein, Q8KCX1 51903/9.3 FusA/NodT family 24 membrane fusion efflux protein, Q8KCR5 46300/9.1 putative 25 photosystem P840 reaction center protein PscD 26 photosystem P840 reaction center cytochrome c-551 27 photosystem P840 reaction center iron-sulfur protein 28 photosystem P840 reaction center, large subunit 29 cytochrome b6-f complex, iron-sulfur subunit 30 pyruvate flavodoxin/ferrodoxin oxidoreductase 31 ATP synthase F1, alpha subunit 32 ATP synthase F1, beta subunit 33 ATP synthase F0, B subunit 34 ATP synthase F1, gamma subunit 35 ATP synthase F1, epsilon subunit 36 bacteriochlorophyl A protein 37 NADH dehydrogenase I, 49 kDa subunit 38 NADH dehydrogenase I, 30 kDa subunit 39 NADH dehydrogenase I, 23 kDa subunit 40 NADH dehydrogenase I, 20 kDa subunit 41 NADH dehydrogenase I, subunit 3 42 cytochrome b-c complex, cytochrome b subunit 43 chlorosome envelope protein A 44 chlorosome envelope protein B 45 chlorosome envelope protein E
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Q8KEP5/ 16607/9.6 pscD O07091/ 22715/9.5 pscC Q8KAY1/ 23506/9.4 pscB Q8KAY0/ 81713/8.7 PscA Q9F722/ 18863/9.1 PetC Q8KC02/ 130174/6.4 hifJ Q8KAW8/ 56847/6.3 atpA Q8KAC9/ 50124/4.9 atpD Q8KGE9/ 19437/7.7 atpF Q8KAW9/ 32052/9.5 atpG Q8KAC8/ 9341/5.7 atpC Q46393/ 40383/7.1 fmoA Q8KEC0/ 41764/5.5 ndhK Q8KEC1/ 19882/5.1 ndhJ Q8KEB8/ 23569/7.4 ndhI Q8KEC2/ 20925/8.9 ndhH Q8KEC3/ 16001/9.1 ndhC Q9F721/ 47495/9.5 petB Q46467/ 8289/6.5 csmA Q46383/ 8903/8.1 csmB Q46386/ 8954/9.5 csmE
Journal of Proteome Research • Vol. 6, No. 3, 2007
9 12
PL
Transport and Binding Proteins 42 No -0.197 OM 37
4
-0.204 OM
Washed Washed wash 2-D BN 1DBN membranes membranes NaBr EDTA NaBr NaBr NaBr EDTA -
18
-
-
-
-
-
-
+
-
-
12
13
23
1
-0.579 Unkn
-
-
-
12
-
-
13
36
5
0.014 Unkn
-
-
+
-
-
-
12
19
9
0.197 CM
-
-
-
21
5
-
8
37
1
0.076 CM (peripheral)
-
-
+
-
-
-
14
42
No
-0.121 CM
-
-
+
-
-
-
5, 9, 10
5, 6, 8, 9 32
16
22
5
-0.378 OM
-
-
-
15
59
1
0.020 CM
6
30
-
-
10
28
5
0.145 CM
-
-
+
-
-
-
15
49
1
-0.205 CM
-
-
+
-
25
-
23, 24
25
10
43
2
-0.076 Unkn
-
-
+
-
21
27
12
38
4
-0.113 OM
-
-
-
-
13
-
8
34
14
0.692 CM
-
-
+
-
-
-
11 14 7 19
43 39 29 25
1 No No 10
-0.117 -0.043 -0.283 0.196
-
-
+ + + -
-
-
23 5
-0.163 OM
7
-
-
-
-
-
0.235 CM 0.351 CM 0.578 CM
-
-
-
9, 11 16 -
3 17
3 21
CM Unkn CM CM
8
16
3
20 14 8
28 14 18
12 13 13
10
28
4
-0.168 OM lipoprotein
-
-
-
-
6
-
10
31
1
-0.368 CM
-
-
-
-
-
13
5
Energy Metabolism 33 No -0.558 CM
9, 10
-
+
-
-
41, 42
5
34
9
-
+
-
30
40
-
29
3
0.301 CM -0.202 CM
-
-
-
-
0.329 CM
-
-
+
2-9
7
29
1
14
20
14
12
57
3
-
-
+
-
15
16
5
-0.187 Unknown
-
3, 4
-
-
2
2
12
25
2
-0.051 CM
22
14
+
-
11
15
16
42
1
-0.130 CM
2
-
+
29
14
19
8
42
1
-0.225 CM
12
-
+
-
-
-
16
57
No
-0.177 CM
-
-
+
-
4
47
No
0.253 CM
-
-
-
22
56
No
-0.288 CM
3
22
+
0.060 CM
2-4, 6-19 21,22
11, 12, 13, 14 30
21, 22
40
28
-
47
18
24
9
21
No
-0.054 Unkn
21
-
+
17
22
5
35
No
-0.380 Unkn
-
-
-
-
-
39
8
34
1
-0.388 Unkn
-
-
+
-
-
-
8
24
1
-0.052 Unkn
8
-
+
-
-
-
4
74
3
0.601 CM
-
38
-
-
-
-
7
13
8
7
63
No
6
61
1
4
43
No
4
-
+
-
-
-
19, 24
-
+
-
-
49
0.272 Chl
18
-
+
-
-
-
-0.563 Chl
-
-
-
-
-
48
0.406 CM -0.047 Chl
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Membrane Proteome Analysis of C. tepidum Table 1 (Continued)
no.
protein definition
46
chlorosome envelope protein I
47
chlorosome envelope protein J
48
Light-independent protochlorophyllide reductase subunit N (LI-POR subunit N) (DPOR subunit N) magnesium-protoporphyrin methyltransferase
49
50 51 52 53 54
preprotein translocase SecA subunit signal peptidase I LipD protein, putative type II secretion system protein type II secretion system protein CAAX prenyl protease 1, putative aminopeptidase
accession predicted no. of no./gene MW/pI peptides
pept. cov. % TM GRAVY Energy Metabolism 18 2 0.082
PL
wash 2-D BN NaBr EDTA NaBr
1DBN NaBr
Washed Washed membranes membranes NaBr EDTA
Chl
6
-
-
-
-
0.131
Chl
7
-
-
-
-
-
4
0.063
Unkn
-
-
-
-
-
21
5
-0.246
CM
-
-
-
5, 12
-
1
31
Protein Fate 34 1 -0.553
CM
-
2, 3, 4
-
-
2
2
31802/8.8
14
50
1
-0.196
CM
-
-
-
-
23
30
50088/8.8 52852/6.0
30 13
43 33
1 No
-0.355 -0.196
OM Unkn
--
-
+ +
4 -
15 -
20, 21 -
Q8KF97
46867/7.7
19
59
4
O68988/ csmI O68983/ csmJ Q9F716/ bchN
25929/7.6
5
23895/8.7
5
35
3
46643/5.3
9
36
Q8KB39/ bchH-1
142815/5.5
12
12
Q8KD18/ secA Q8KCH1/ lepB Q8KAV2 Q8KF93
118003/5.7
32, 34
0.056
OM
-
-
-
-
12
16, 17
Q8KCB5
47156/9.0
11
33
7
0.382
CM
-
-
+
-
-
--
Q8KD74/ pepA
52994/5.9
11
31
2
-0.073
OM
-
-
-
-
-
17
acetolactate synthase, small subunit acetolactate synthase, large subunit glutamate synthase, large subunit
Q8KER6/ ilvN Q8KER5/ ilvB Q8KFC6/ gltB
17862/9.0
20
11
-
-
-
-
41, 42
Q8KE24
43789/8.8
Central Intermediary Metabolism 11 28 6 0.434 CM
Q8KE25
82601/5.7
14
27
3
-0.265
Q8KDM1
45492/7.1
11
28
1
-0.137
64
heterodisulfide reductase, putative heterodisulfide reductase, subunit A/hydrogenase, delta subunit, putative sulfide dehydrogenase, flavoprotein subunit, putative Sulfide-quinone reductase, putative DsrM protein
65 66
cell division protein, putative cell division protein FtsH
67
cell division protein FtsZ
68
55 56
57 58 59
60 61 62
Amino Acid Biosynthesis 80 No -0.065 Unkn
62874/5.8
12
29
5
-0.081
Unkn
-
-
-
25
-
-
169774/6.7
16
12
7
-0.304
Unkn
-
1
-
-
-
-
-
-
+
-
-
-
Unkn
-
-
-
-
-
7, 15
Unkn
-
-
+
-
16
21
Q8KG51
53974/6.7
15
33
5
-0.215
Unkn
-
-
-
-
12
17
Q8KAB9/ dsrM
37893/9.5
8
28
6
0.431
CM
-
-
-
-
23
30
31466/9.8 78396/5.7
12 14
Cellular Processes 38 4 0.427 24 3 -0.288
CM CM
-
-
+ -
-
25 6
7
Smc family protein
Q8KFL1 Q8KG41/ ftsH-1 Q8KGD8/ FTSZ Q8KBS6
69 70
OmpA family protein outer surface protein, putative
Q8KCR0 Q8KAL2
71
63
72 73 74 75 76
46081/5.9
11
32
-0.220
CM
-
-
-
-
-
20
134314/5.9
11
CM
-
-
-
-
1
-
24334/9.4 20056/5.7
5 8
10 1 -0.560 Cell Envelope 23 3 -0.196 33 2 0.013
OM Unkn
8 8
34 -
+ +
1-3, 6 -
29 33, 34
Outer membrane protein OmpH Q8KFR8/ ompH rod shape-determining protein Q8KEY5/ MreB mreB-1 basic membrane protein A Q8KDL1/ bmpA band 7 family protein Q8KE93
20430/9.5
7
24
1
36894/7.8
11
28
37943/5.7
11
33
27873/6.0
9
Bacterial surface antigen family protein penicillin-binding protein 1
Q8KFQ6
93746/7.3
Q8KFZ4/ PONA Q8KEP8
83628/9.7
1
-0.448
OM
10
-
+
-
-
39 43, 44, 45 -
1
0.067
OM
23
-
+
-
-
-
2
-0.184
OM
-
-
+
-
-
-
47
1
0.161
OM
-
-
+
-
-
13
17
6
-0.398
OM
-
-
-
5
33, 34, 35 5
21
34
3
-0.220
CM
-
-
-
-
-
6
17850/4.8
8
59
2
-0.334
OM
-
-
-
-
31
-
7
19, 20
77
peptidoglycan-associated lipoprotein
78
sensor histidine kinase/ response regulator
Q8KF01
79025/5.8
21
Regulatory Functions 35 1 -0.332
CM
-
-
-
-
5, 6
79
Hemaglutinin-related protein TonB-dependent receptor-related protein pentapeptide repeat family protein Oxa1/60 kDa IMP family protein
Q8KGA0
22146/4.8
16
Uknown Function 56 No -0.208
OM
-
-
+
-
-
-
Q8KDV9
73908/5.5
10
16
3
-0.383
OM
-
-
+
-
9
11
Q8KDX6
40536/9.5
12
29
1
-0.436
OM
-
-
+
-
-
-
Q8KGG2/ oxaA
65489/9.1
7
11
7
-0.051
CM
-
-
-
-
11
15
conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein
Q8KG37
33752/5.9
-
-
+
-
-
-
Q8KF98
29034/10.0
9
54
3
0.219
OM
-
-
-
-
-
Q8KC70
95830/8.9
11
16
4
-0.237
OM
-
-
-
-
4
80 81 82
83 84 85
Conserved Hypothetical Proteins 16 48 1 0.043 Unkn
29, 31 -
Journal of Proteome Research • Vol. 6, No. 3, 2007 1053
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Aivaliotis et al.
Table 1 (Continued) accession no./gene
no.
protein definition
86
hypothetical protein
Q8KBI3
87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104
hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein hypothetical protein
Q8KEF0 Q8KDW9 Q8KB82 Q8KBI6 Q8KGF1 Q8KCV1 Q8KAE7 Q8KFQ0 Q8KA97 Q8KAR5 Q8KD76 Q8KD97 Q8KE01 Q8KBW3 Q8KD45 Q8KF09 Q8KAY5 Q8KCV5
a
predicted MW/pI
no. of peptides
pept. cov. %
42811/5.6
19
60
10152/9.9 13476/5.8 12045/5.7 32338/6.3 44022/9.8 19251/11.2 20975/9.7 13389/7.9 21451/8.8 9040/10.7 31828/9.4 25394/7.7 42410/8.6 83641/9.6 14207/9.7 107606/7.0 63227/6.8 54883/9.3
7 4 6 10 20 15 14 4 7 8 7 9 7 12 5 13 8 11
46 95 53 44 49 66 58 30 48 66 24 37 24 25 36 16 19 19
TM
GRAVY
PL
Hypothetical Proteins 2 -0.248 OM 1 2 1 1 2 1 3 1 1 1 1 2 3 2 1 5 6 1
0.437 0.002 -0.638 -0.178 -0.575 -0.709 0.177 -0.243 0.115 0.201 -0.322 -0.231 -0.409 -0.334 -0.260 -0.382 -0.251 -1.288
Unkn Unkn Unkn OM Unkn Unkn CM Unkn OM Unkn Unkn Unkn OM OM Unkn OM OM Unkn
NaBr
wash EDTA
2-D BN NaBr
4
24
+
16 -
37 38 -
+ + + + + + + + + + -
1DBN NaBr 14, 16-19, 22 9 -
Washed membranes NaBr
Washed membranes EDTA
19, 20
25, 26
24 21 22 19 4 10 17
31 27 37 25 6 46 -
Numbers indicate the band in the corresponding gel. Plus (+), identified; minus (-), not identified; TM, transmembrane helices; PL, predicted localization.
SDS-PAGE to elucidate the identities of the resolved protein complexes on the basis of subunit composition (Figure 4). The best separation occurred on a 20 cm gel (12% T and 1% C); gel with 15 cm separating length. For protein identification, gel bands were excised, and most of the separated proteins were identified by MALDI-TOF-MS. From 100 excised bands, we identified 120 different proteins with the combination of MALDI-TOF-MS (see Table 1). We further categorized the proteins according to their predicted subcellular location: 26%
cytosolic, 4% periplasmic, 25% integral membrane, 2% chlorosomal, and 39% unknown proteins (Figure 5 D). Our analysis was not limited to integral membrane proteins with only one or two TMHs, but we identified membrane proteins with up to 14 TMHs, such as glutathione-regulated potassium-efflux system protein KefC and photosystem P840 reaction center PscA subunit. Additionally, components of the respiratory chain were identified: the subunits NdhH, I, and K of NADH-dehydroge-
Figure 4. 2-D gel of membrane proteins from C. tepidum. Since protein complexes migrate in native form in the first dimension, their protein subunits occur on vertical lines in the 2-D gel. Information on the identified proteins are summarized in Table 1; numbers on the top indicate the number of the protein complex in the BN, and the number in the side are the apparent mass (kDa) of the molecular mass standards in the second dimension. 1054
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research articles
Figure 5. Predicted localization of the identified proteins from 1-D Tricine-SDS-PAGE of the EDTA soluble fraction (A), of the NaBr soluble fraction (B), of the EDTA membrane fraction (C), of the NaBr membrane fraction (D), of 1-D BN of the NaBr membrane fraction (E), of 2-D gel of membrane proteins of the NaBr membrane fraction (F).
nase I; subunits AtpA, B, and G of the ATP synthase F1 and AtpF of the ATP synthase F0; subunits PscA, C, D, and FmoA of the photosynthetic reaction center, subunits PetB and C of the cytochrome bc complex; and the CsmA and CsmB chlorosomal proteins. Surprisingly, two chlorosomal proteins (CsmA, B) were found in the upper region of the BN gel, where all the other subunits of the reaction center were also identified. Moreover, although the FmoA subunit has been found to be in association with the reaction center complex,22,23 the 2-D electrophoresis indicated the existence of the dissociated to the reaction center form of the FmoA. Several ABC-transport systems were identified: the ATP binding protein of the iron(III) (FecE) and iron compound transporter, the periplasmatic peptide-binding protein of peptide transporters, the ATP binding and periplasmatic substrate binding protein of a ABC transporters without a known substrate, the ATP binding, and the membrane fusion protein of an ABC-type export system. The subunit BchD and I of the
magnesium chelatase, the glutathione-regulated potassium efflux protein, along with three ArsA ATPase family proteins, were also identified. In addition, several integral membrane proteins of the cell envelope, as well as of the outer membrane, were found, such as OmpA, H, pentapeptide repeat family protein, Ton-Bdependent receptor related protein, hematoglutinin related protein, basic membrane protein, band 7 family protein, outer surface protein, and rod-shape-determining protein. Regarding the proteins involved in sulfur metabolism, sulfide reductase, two sulfide dehydrogenase flavoprotein subunits, and a putative heterosulfide were identified. Out of the 120 identified proteins, 25% are membrane proteins and 39% are proteins without known localization. The TMH contents of these proteins vary from 1 to 6. Three of these protein are clearly overrepresented: a putative outer membrane protein (Q8KAL2), a hemaglutinin related protein (Q8KGA0), and a hypothetical protein (Q8KBI3). All of them contain at their carboxyl terminal Journal of Proteome Research • Vol. 6, No. 3, 2007 1055
research articles a phenylalanine which is essential for their correct assembly to the outer membrane.24
4. Discussion It has been proposed that the ancestral photoautotroph was a green-sulfur bacterium.25 C. tepidum is a Gram-negative green-sulfur bacterium, which has unique mechanisms of photosynthesis in comparison to other phototrophs. Because of these unique features, studies on the Chlorobia are important for understanding the evolution and the mechanisms of photosynthesis and energy metabolism. Attempts to display the membrane proteome of this organism using 2-D PAGE after fractionation and solubilization in different detergents were of limited success.4,12 It is generally agreed that integral membrane proteins are highly underrepresented in 2-DE gels.2 There are three main reasons for this fact: (i) they are often low abundant, (ii) most of them have an alkalic pI which hampers separation, and (iii) most importantly, they are poorly soluble in the buffer used for IEF or become insoluble during the focusing process. Our results show that separation through ΒΝ-PAGE, followed by Tricine-SDS-PAGE, yields much better results with 23 out of 120 identified proteins being clearly integral membrane proteins. BN-PAGE is an analytical technique that can easily be applied to a wide variety of tissues, cells, and organelles to separate proteins, multisubunit membrane proteins, and membrane protein super-complexes, as well as transiently interacting proteins in their native state.4,26 In view of the relevance of membrane proteins in transport as energy transduction of C. tepidum, and the poor performance of the 2-DE technique for membrane proteins, we decided to use an alternative approach for the analysis of the membrane proteome. To enrich membrane proteins prior to BN-PAGE and thereby increase the probability of their detection, we applied a prefractionation protocol to separate the bacterial membrane from cytosolic proteins.13 Moreover, various washing buffers had to be tested regarding their ability to remove soluble proteins which are associated with the membrane. Sodium carbonate is a widely used washing agent for bacterial membranes,1,27 but its application on the membrane of C. tepidum showed partial extraction of membrane proteins (data not show). Two other agents, NaBr and EDTA, are superior to sodium carbonate. The fact that about 30% of the identified proteins from the 1-D Tricine-SDS PAGE in the membrane fraction are cytosolic and periplamatic indicates that the NaBrand EDTA-wash does not completely dissociate soluble subunits of membrane protein complexes as well as membraneassociated proteins from the membrane. Also, proteins of the protein synthesis machinery may have co-fractionated because they were still attached to nascent membrane or secreted proteins. Another reason for co-fractionation with the membrane may be the high abundance of some proteins. In contrast to the other anoxygenic photosynthetic bacteria, Chlorobia possess unique light-harvesting antenna known as chlorosomes.28 Αn average C. tepidum cell contains about 200250 chlorosomes, which contain about 200 000 BChl c and about 2000 BChl a molecules.29 The presence of these molecules with the surrounded chlorosome protein and galactolipid monolayer influences the solubilization of the membranes as the performance of 2-DE systems. A crucial first step toward the analysis of the membrane proteome of C. tepidum, is the separation of the chlorosomes from the membrane. 1056
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The nature of the identified proteins from the 1-D TricineSDS-PAGE of the supernatants from both agents shows differences. While the cytoplasmatic and periplasmatic proteins represent, in the case of the EDTA supernatant, more than 70% of the 106 identified proteins (Figure 5A), in the case of NaBr, a decrease to 55% of the 60 identified proteins (Figure 5B) is observed. In contrast, NaBr is more effective in the separation of subcellular organelles such as the chlorosome (7%), while in the case of EDTA samples, no chlorosomal proteins were detected. The absence of chlorosome proteins in the EDTA supernatant is in correlation with the absorption spectrum, where the absorption peak of BChlc at 750 nm is absent. BN-PAGE is tolerant to a large number of nonionic and zwitterionic detergents. Efficient but mild solubilization of the membrane proteins is the main prerequisite for BN-PAGE, and good starting conditions are often described in established protocols for the isolation of native membrane proteins. Nonionic detergents as DDM and Triton X-100 have been reported to be efficient in the solubilization as well as in the isolation of active membrane protein complexes from C. tepidum.13,30,31 For this reason, we focus our attempts on these detergents. According to the 1-D Tricine-SDS-PAGE analysis, both detergents in different ratios to protein can solubilize the membrane proteins, but DDM in a ratio of 1:1 to protein proved to be the best choice. It is known that long alkyl chains are the mildest detergents and preserve the membrane protein in a “native” state.32 However, the outcome of such a screening is, in our estimation, difficult to predict and will have to be optimized in each individual case. Using milder solubilization conditions should allow us to separate not only single membrane proteins, but also membrane protein complexes using BN-PAGE. Protein bands in ΒΝ-PAGE gels have to be wellseparated to be applicable for protein digestion with trypsin and subsequent identification by MALDI-TOF-MS and database search. These bands represent protein mixtures resulting in complex peptide mixtures after proteolytic cleavage. While identification from binary mixtures is sometimes successful, as shown for PscA, PscC, and FmoA subunits from the reaction center, proteins from more complicated mixtures are seldom identified unambiguously. In contrast to IEF, the efficiency of BN-PAGE is not affected by the hydropathicity of the individual proteins, since proteins migrate as multiprotein complexes in the BN-PAGE. While approaches focusing on IEF in the first dimension usually fail to observe a significant number of membrane proteins, in our study, 63 out of 120 identified proteins contain at least one membrane spanning segment. The other proteins represent hydrophilic, but still, membrane-associated subunits of the membrane protein complexes. The membrane proteins that were identified range from the small rather hydrophilic CsmB subunit with one transmembrane helix, to the hydrophobic PscA subunit of the reaction center and the putative glutathione-regulated potassium-efflux protein with 14 transmembrane helices. The GRAVY value distribution of the identified proteins in 1-D BN-PAGE and in 2-DE show enormous differences (Figure 6). In the case of the 1-D BN-PAGE, only 18% (6 proteins) of the identified proteins have positive GRAVY values, while after 2-DE, that percentage increases to 35% (42 proteins). In addition, the pI range in the case of the 2-DE shifted to a higher value. The most hydrophobic protein, which was identified in 2-DE, is a glutathione-regulated potassium-efflux system pro-
research articles
Membrane Proteome Analysis of C. tepidum
and FmoA subunits were identified in the 2-DE. In contrast, the identification of the soluble PscB was not possible. Taking in account the estimated molecular weight of the reaction center with one FmoA trimer of about 440 kDa, the present results suggest the presence of at least two FmoA trimers per reaction center.22,23 Furthermore, the presence of the chlorosomal proteins CsmA and CsmB supports the suggestion of the close association between these proteins and the FmoA trimer and correlates to the proposed model.4,38 In contrast to other bacteria, the cytochrome complex from C. tepidum contains only the cytochrome b and the Rieske subunit.11 In the 2-DE gels, both subunits were identified (PetB and PetC) in the area of the front of the BN, indicating the instability of the complex. Furthermore, two ABC transporters and the glutamine-depended proton efflux protein which belongs to high molecular weight complexes were identified in the 2-DE, but the resolution of the BN must be improved for the exact subunit identification. In addition, the presence of outer membrane proteins in the upper region of the BN allows a speculation of the supermolecular structures of these proteins. In contrast, although gel-free systems allow the identification of hydrophobic proteins, there is not access to information about protein interactions.39,40
5. Conclusions Figure 6. GRAVY value of the identified proteins by 1-D BN-PAGE (A) and 2-D BN/Tricine-SDS-PAGE (B).
tein KefC, with a GRAVY value of 0.75. The very hydrophobic integral membrane proteins usually have GRAVY values over 0.250.33 BN-PAGE is known to be tolerant to both basic and hydrophobic proteins, since the anionic Coomassie dye binds to the surface of hydrophilic and hydrophobic proteins. As a result of the excess of negative charges, even basic proteins migrate to the anode.34 This is nicely shown in the case of the hypothetical protein Q8KAR5. The protein has a theoretical pI of 10.7, one TMH, and GRAVY of 0.201, and it is both hydrophobic and basic. Moreover, the detection of a high number of basic proteins, as well as proteins with more than one TMH, underlines the separating properties of BN-PAGE for protein complexes, which are both basic and hydrophobic. High-abundance proteins often cause horizontal streaking in 2-D gels containing samples separated by BN-PAGE in the first dimension.35 Regardless of the protein identification method, streaking has to be avoided, since contaminations from high-abundance proteins prevent the detection of less abundant proteins. Although the membranes were extensively washed, the Q8KBI3, Q8KGA0, Q8KAL2, and FmoA showed streaking in the 2-D gels. FmoA is the connecting protein between the chlorosome and the reaction center complex.36 Structural analysis showed the association of two FmoA trimers per reaction center.22,23,37 The obtained streaking indicates the different aggregation states of the FmoA. The other three proteins are hypothetical proteins present in high abundance in the membrane fraction of C. tepidum and were also obtained in earlier studies.4,12 Prediction using the PRED-TMBB indicates the presence of β-barrel structure.18 This correlates with the C-terminal aromatic phenylalanine and allows the localization of these proteins in the outer membrane.24 In response to the functional state of the membrane protein complexes, the photosynthetic reaction center complex was observed in the region of 850 kDa where the PscA, PscC, PscD,
Although BN-PAGE is not capable of solving all problems linked to the investigation of membrane proteins, it allows efficient and reproducible separation of membrane proteins. The ability to display complex rearrangements in biological membranes in a single gel makes it a potent and unique tool for the investigation of the membrane-located subproteome. Furthermore, we were able to identify large, extremely hydrophobic proteins by in-gel digestion and MALDI-MS. Mapping of the membrane proteome of C. tepidum is important to elucidate the regulation of transport processes, to monitor the energy metabolism, and to understand the evolution and mechanisms of photosynthesis. Abbreviations: MALDI-TOF; matrix-assisted laser desorption ionization-time of flight; 2-DE, two-dimensional gel electrophoresis; PMF, peptide mass fingerprinting; FMO, FennaMatthews-Olson; BChl, bacteriochlorophyll; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BN, blue native.
Acknowledgment. The authors thank K. Kouyianou for the critical reading of the manuscript. The University of Crete, the Ministry of Education of Greece, and the Centre of Membrane Proteomics (CMP) in Frankfurt supported the project. M.A. thanks Alexander von Humboldt Foundation for the financial support. References (1) Molloy, M. P.; Herbert, B. R.; Slade, M. B.; Rabilloud, T.; Nouwens, A. S.; Williams, K. L.; Gooley, A. A. Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 2000, 267 (10), 2871-2881. (2) Santoni, V.; Molloy, M.; Rabilloud, T. Membrane proteins and proteomics: Un amour impossible? Electrophoresis 2000, 21, 1054-1070. (3) Luche, S.; Santoni, V.; Rabilloud, T. Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in twodimensional electrophoresis. Proteomics 2003, 3, 249-253. (4) Aivaliotis, M.; Haase, W.; Karas, M.; Tsiotis, G. Proteomic analysis of chlorosome-depleted membranes of the green sulfur bacterium Chlorobium tepidum. Proteomics 2006, 6, 217-232.
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research articles (5) Wallin, E.; von-Heijne, G. Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci. 1998, 7, 1029-1038. (6) Hippler, M.; Klein, J.; Fink, A.; Allinger, T.; Hoerth, P. Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinhardtii. Plant J. 2001, 28, 595-606. (7) Henningsen, R.; Gale, B. L.; Straub, K. M.; DeNagel, D. C. Application of zwitterionic detergents to the solubilization of integral membrane proteins for two-dimensional gel electrophoresis and mass spectrometry. Proteomics 2002, 2, 1479-1488. (8) Wilkins, M. R.; Gasteiger, E.; Sanchez, J.-C.; Bairoch, A.; Hochstrasser, D. F. Two-dimensional gel electrophoresis for proteome projects: The effects of protein hydrophobicity and copy number. Electrophoresis 1998, 19, 1501-1505. (9) Nijtmans, L. G. J.; Henderson, N. S.; Holt, I. J. Blue native electrophoresis to study mitochondrial and other protein complexes. Methods 2002, 26, 327-334. (10) Schaegger, H.; von Jagow, G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 1991, 199 (2), 223-231. (11) Eisen, J. A.; Nelson, K. E.; Paulsen, I. T.; Heidelberg, J. F.; Wu, M.; Dodson, R. J.; Deboy, R.; Gwinn, M. L.; Nelson, W. C.; Haft, D. H.; Hickey, E. K.; Peterson, J. D.; Durkin, A. S.; Kolonay, J. L.; Yang, F.; Holt, I.; Umayam, L. A.; Mason, T.; Brenner, M.; Shea, T. P.; Parksey, D.; Nierman, W. C.; Feldblyum, T. V.; Hansen, C. L.; Craven, M. B.; Radune, D.; Vamathevan, J.; Khouri, H.; White, O.; Gruber, T. M.; Ketchum, K. A.; Venter, J. C.; Tettelin, H.; Bryant, D. A.; Fraser, C. M. The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, greensulfur bacterium. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 95099514. (12) Aivaliotis, M.; Corvey, C.; Tsirogianni, I.; Karas, M.; Tsiotis, G. Membrane proteome analysis of the green-sulfur bacterium Chlorobium tepidum. Electrophoresis 2004, 25 (20), 3468-74. (13) Aivaliotis, M.; Neofotistou, E.; Remigy, H.-W.; Tsimpinos, G.; Lustig, A.; Lottspeich, F.; Tsiotis, G. Isolation and characterization of an outer membrane protein of Chlorobium tepidum. Photosynth. Res. 2004, 79 (2), 161-166. (14) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72 (1-2), 248-254. (15) Schaegger, H.; von Jagow, G. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 1987, 166 (2), 368-379. (16) Lauber, W. M.; Carroll, J. A.; Dufield, D. R.; Kiesel, J. R.; Radabaugh, M. R.; Malone, J. P. Mass spectrometry compatibility of two-dimensional gel protein stains. Electrophoresis 2001, 22 (5), 906-918. (17) Hofmann, K.; Stoffel, W. TMbasesA database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler 1993, 374, 166. (18) Bagos, P. G.; Liakopoulos, T. D.; Spyropoulos, I. C.; Hamodrakas, S. J. PRED-TMBB: a web server for predicting the topology of beta-barrel outer membrane proteins. Nucleic Acids Res. 2004, 32, W400-404. (19) Bendtsen, J. D.; Nielsen, H.; von Heijne, G.; Brunak, S. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 2004, 340 (4), 783-795. (20) Gardy, J. L.; Laird, M. R.; Chen, F.; Rey, S.; Walsh, C. J.; Ester, M.; Brinkman, F. S. PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 2005, 21 (5), 617-623. (21) Gill, S. C.; von Hippel, P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 1989, 182 (2), 319-26. (22) Remigy, H.; Fotiadis, D.; Wolpensinger, B.; Mueller, S. A.; Engel, A.; Tsiotis, G. Evidence for the association of three FMO subunits per reaction center of Chlorobium tepidum by scanning transmission electron microscopy. In Photosynthesis: Mechanisms and Effects; Garab, G., Ed.; Kluwer Academic Publishers: Dordrecht, Boston, 1998; pp 531-534.
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