SDS polyacrylamide gel electrophoresis in the identification of Streptomyces coelicolor cytoplasmic protein complexes

J. Biochem. Biophys. Methods 70 (2007) 565 – 572 www.elsevier.com/locate/jbbm

Sample preparation for two-dimensional blue native/SDS polyacrylamide gel electrophoresis in the identification of Streptomyces coelicolor cytoplasmic protein complexes Zhi-Jun Wang, Xin-Ping Xu, Ke-Qiang Fan, Cui-Juan Jia, Ke-Qian Yang ⁎ State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, PR China Graduate School of Chinese Academy of Sciences, PR China Received 14 October 2006; received in revised form 28 November 2006; accepted 7 January 2007

Abstract Ammonium sulfate precipitation was tested as a sample preparation step for BN-PAGE analyses of S. coelicolor cytoplasmic protein complexes. A procedure of sample preparation compatible with two-dimensional BN/SDS-PAGE was established and used to visualize protein complexes. To validate the sample preparation procedure, representative protein complexes were identified. Several previously characterized protein complexes were rediscovered and their reported oligomeric states reconfirmed. In addition, we identified new but plausible interactions that have never been reported before. Our work provides useful reference for the wide application of BN-PAGE in protein interaction study. © 2007 Elsevier B.V. All rights reserved. Keywords: BN-PAGE; Ammonium sulfate precipitation; Peptide mass fingerprint; Protein identification; Streptomyces coelicolor

1. Introduction Identification of multi-protein complexes is an important step toward understanding cellular behavior at an integrative level. Methods are needed to achieve high resolution separation and efficient identification of intact protein complexes. Recently, blue native polyacrylamide gel electrophoresis (BN-PAGE) coupled with a second dimensional SDS-PAGE and mass spectrometry has emerged as a powerful tool for this purpose. Compared to other protein complex identification methods, such as affinity purification and immunoprecipitation, the BN-PAGE method holds several advantages, the most important being it provides a global view and a direct visual comparison of relative abundance of protein complexes. Therefore, it has great potential to be applied on a proteomic scale. To establish this technology for a specific organism, sample preparation and fractionation techniques need to be developed. ⁎ Corresponding author. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, P. O. Box 2714, Beijing, 100080, PR China. Tel./fax: +86 1062652318. E-mail addresses: [email protected] (Z.-J. Wang), [email protected] (X.-P. Xu), [email protected] (K.-Q. Fan), [email protected] (C.-J. Jia), [email protected] (K.-Q. Yang). 0165-022X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2007.01.001

BN-PAGE is a charge shift method first developed by Schägger et al. [1]. It differs from other native gel electrophoresis mainly because the electrophoretic mobility of a protein is determined by the negative charge of the bound Coomassie blue dye, while separation is still achieved by the molecular sieve effect provided by the polyacrylamide gradient of descending pore size. BN-PAGE, when coupled with a second dimensional SDS-PAGE and mass spectrometry, was used successfully to characterize subunit composition of protein complexes [2–6] and proved very effective to separate purified or fractionated protein samples. For instances, it had been used to study the green algae Chlamydomonas reinhardtii thylakoid membrane proteins [7], to purify chloroplast FoF1-ATP synthase [8] and to investigate E. coli protein-translocation complex SecYEG [9]. Recently, it was applied at a proteomic scale, in the analysis of mammalian whole cell lysates [10] and E. coli cell envelope protein complexes [11] and in the survey of protein complexes in Methanothermobacter thermautotrophicus [12]. Streptomyces coelicolor is a high G + C Gram-positive bacterium known for its complex life cycle and ability to produce secondary metabolites. When analyzing protein samples of S. coelicolor using BN/SDS-PAGE, we found samples of whole cell lysates showed severe smear on second dimensional SDS-PAGE even after fractionation into cytoplasmic and membrane portions. To

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overcome this problem, and also to detect low abundance proteins, we tested ammonium sulfate precipitation as a sample treatment step. A sample preparation procedure compatible with BN-PAGE was developed. Using this procedure, cytoplasmic protein complexes of S. coelicolor were consistently separated and visualized. As a show of principal, representative protein complexes were identified and their observed oligomeric states reported in this paper. 2. Materials and methods 2.1. Strain and growth conditions S. coelicolor A3(2) spores were collected, pregerminated as described by Kieser et al. [13]. To prepare cells for proteomic analyses, pregerminated spores were inoculated into 1 liter MM supplemented with 0.2% casamino acids (SMM) [14], in a 5 l baffled flask and cultivated at 250 rpm and 30 °C for 24 h. Mycelia were harvested by 8000 g centrifugation for 10 min.

for 30 min. To the resulting supernatant, DNaseI and MgCl2 were added to reach a final concentration of 25 μg/ml and 10 mM, respectively, and the supernatant was kept at 4 °C for 1 h to digest DNAwhich may interfere with the performance of BN-PAGE. The DNaseI digested supernatant was then subjected to ultracentrifugation at 200,000 g for 1 h. The resulting sediment (cell membrane) was collected for other use and the new supernatant was collected and subjected to further ammonium sulfate precipitation. Protein sediments precipitated by ammonium sulfate were resolubilized in 10 ml sample buffer containing 50 mM Bis–Tris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl) propane1,3-diol) pH 7.0, 250 mM n-aminocaproic acid and 0.5 mM PMSF. The samples were then placed in Millipore Amicon® Ultra-15 column and centrifuged 45 min at 5000 g and 4 °C. The flow-through was discarded, and 10 ml of cold sample buffer was added to the column and spun again. This wash step was repeated for four times. After the final spin, the protein samples were collected. To these protein samples, glycerol was added to 25% and protein concentrations were adjusted to 10 mg/ml. The samples were stored at − 70 °C until use.

2.2. Preparation and fractionation of water soluble cytoplasmic proteins

2.3. Two dimensional BN/SDS PAGE

All operations were conducted at 4 °C. About 10 g cell pellet was washed twice with 50 ml cell lysis buffer containing 50 mM Tris pH7.5, 100 mM NaCl, 50 mM NaF, 0.1 mM NaVO3, and resuspended in the same buffer provided with 1 mM PMSF (phenylmethylsulfonyl fluoride), 2.5 μg/ml leupeptin, 2 μg/ml aprotinin and 1 μM pepstatin A. Resuspended cells were disrupted by passing through French Press (Thermal Electronics) three times at 15000 psi. Cell debris was removed by centrifugation at 10000 g

BN-PAGE and SDS PAGE were performed using a DYY-23A apparatus (product of Beijing WoDeLife Sciences Instrument Company). In the first dimensional BN-PAGE, a stacking gel of 4% and a separating gradient of 5–16% or 10–20% were used. Anode and cathode electrophoresis buffers were the same as described by Schägger et al. [1]. Gel buffer was modified to 250 mM n-aminocaproic acid, 50 mM Bis–Tris, pH 7.0. 100 μg cytoplasmic proteins were loaded to each lane. Electrophoresis

Table 1 Protein spots of S. coelicolor cytoplasmic protein complexes identified by peptide mass finger printing Protein spots

Annodated functions

Accession numbers

A1 A2 A3 B1 B4 B5 B2 B3 C1 C2 D1 D2 E1 E2 F1 F2 G1 G3 G2 G4 H1 H2

Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase NiSOD NiSOD NiSOD Succinyl-CoA synthetase β chain Phosphoglycerate mutase FeSOD II FeSOD II Ketol-acid reductoisomerase 1 Ketol-acid reductoisomerase 2 Phosphoglycerate kinase Unkown Putative thiosulfate sulfurtransferase Peptidyl-prolyl cis–trans isomerase Putative phenylalanyl-tRNA synthetase β chain Putative phenylalanyl-tRNA synthetase α chain Unknown Putative bacterioferritin Putative 2-oxoglutarate dehydrogenase Putative 2-oxoglutarate dehydrogenase

SCO1947 SCO1947 SCO1947 SCO5254 SCO5254 SCO5254 SCO4808 SCO4209 SCO0999 SCO0999 SCO5514 SCO7154 SCO1946 SCO2634 SCO4164 SCO7510 SCO1594 SCO1595 SCO2524 SCO2113 SCO5281 SCO5281

Predicted MW (Da)

pI

36246

5.24

14694 a 14694 41577 28262

7.79 7.79 4.76 6.08

23585 36231 36325 41740 23971 31752 17878 89930 40694 68660 19208 139121 139121

5.27 4.93 4.93 4.87 4.82 4.74 5.53 5.00 4.79 5.17 4.75 5.91 5.91

Mowse scores

Sequence coverage

53 186 114 45 82 56 61 216 110 104 188 185 106 160 190 80 263 137 194 101 492 442

32% 50% 42% 57% 65% 52% 15% 50% 46% 46% 39% 35% 30% 53% 51% 46% 40% 36% 42% 38% 44% 42%

Observed complex sizes (kDa)

Reported complex sizes (kDa)

110

84

70 [19,20]

90 90 75

120 [23]

b60 b60 b60350 350 350 N1500

a Predicted MWs were calculated based on the full length of annodated monomeric proteins. In case of H1 and H2, which spot represents the intact monomer is unknown, so both spots were assigned the same predicted MW.

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was conducted at 100 V for 2 h, and then adjusted to 200 V to run for another 10 h. Ferritin, catalase and BSA from Amersham Biosciences (Sweden), were used as markers to indicate the sizes of 880, 440, 250, 132 and 66 kDa. For the second dimensional SDS-PAGE, strips of the first dimensional BN-PAGE were cut and soaked in 2% (w/v) SDS, 250 mM Tris–HCl, pH 6.8 for 20 min or boiled in the same buffer for 4 min. SDS-PAGEs were performed using a 4% stacking and a 12% separating gel according to standard protocols. Gels were fixed in 50% (v/v) methanol and 12% (v/v) acetic acid for 1 hour and then stained with 0.25% (w/v) Coomassie Blue R250 in10% (v/v) acetic acid and 45% (v/v) methanol or silver stained [15]. A series of proteins (Tiangen Company, China) with the sizes of 94, 66, 45, 35, 24, 20 and 14 kDa were used as markers.

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SDS-PAGE (Fig. 1A). As shown in Fig. 1, the resolution of total lysate samples on the second dimensional SDS gel was poor, making protein spot picking very difficult. We suspected that the problem was due to cell membrane fragments presented within total lysate which precipitated during electrophoresis on

2.4. Trypsin treatment and sample preparation for mass spectrometry These procedures were performed by GeneCore BioTechnologies Co., Ltd. (Shanghai, China). Briefly, protein spots excised from SDS gel were rinsed in dd H2O. Gel slices were destained with 50% acetonitrile and 25 mM NH4HCO3 until they became transparent. Destained gel splices were soaked in 100% acetonitrile, placed in a clean container then dried in vaccum. In gel trypsin digest was performed in 25 mM NH4HCO3, pH 8.0 at 37 °C overnight. For mass spectrometry, 0.75 μl of trypsin digested sample was mixed with an equal volume of freshly prepared 10 mg/ ml α-cyano-4-hydroxycinnamic acid and placed on sample plate. 2.5. MALDI-TOF Mass spectrometry and MASCOT search parameters MALDI-TOF measurements were performed by GeneCore BioTechnologies Co., Ltd. on an Applied Biosystems Voyager DE Pro6192 mass spectrometer (ABI, USA), set to reflectron mode. Mass spectra were collected as the sum of 150 measurements. Monoisotopic peaks were manually selected, excluding background peaks. Resulting peptide masses were used to perform MASCOT search (Peptide Finger Printing: http://www. matrixscience.com) against S. coelicolor proteome. Searches were set for a mass accuracy of 1 ppm, one missed cleavage of trypsin in matching peptides, oxidation of methionine and carbamidomethylation of cysteine. A Mowse score of 45 was adopted as the minimum for protein identification. Although most of the identifications obtained score higher than 45, the cutoff point was set to this score to include the identifications of small proteins, such as NiSOD (Table 1). Protein annotations followed those of S. coelicolor proteome and putative complex organization referred to literatures. 3. Results 3.1. Sample pre-treatments and separation effects on two dimensional BN/SDS-PAGE Total protein lysate of S. coelicolor and samples prepared after further treatments were analyzed by two dimensional BN/

Fig. 1. Effects of sample treatments on the resolution of S. coelicolor proteins by two dimensional BN/SDS PAGE. Total proteins (A), cytoplasmic proteins (B) and ammonium sulfate precipitated cytoplasmic proteins (C) were separated by a first dimensional 6%–15% gradient BN-PAGE and a second dimensional 12% SDS-PAGE. Polyacrylamide gels were silver stained.

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BN-PAGE. Total protein lysate was thus subjected to ultracentrifugation as described in material and method to obtain cell membrane fraction (collected and stored for other usage) and cytoplasmic fraction, and the latter was subjected to BN/SDS-PAGE (Fig. 1B). As shown in Fig. 1B, this operation greatly reduced background caused by membrane fragments. Protein spots on SDS-PAGE became clearer (compare the right half of Fig. 1A with that of Fig. 1B). However, high background still existed around the left half of the gel. It is likely that ultracentrifuation at 100000 g for 2 h was not enough to sediment all membrane fragments. To remove residual membrane fragments and to further fractionate and concentrate cytoplamic proteins, we treated cytoplasmic sample by ammonium sulfate precipitation. As shown in Fig. 1C, ammonium sulfate treatment further reduces the background (compare Fig. 1B with Fig. 1C). 3.2. Treatments of BN-PAGE gel strips for SDS-PAGE Before running the second dimensional SDS-PAGE, ammonium sulfate precipitated protein complexes were run on BNPAGE of varying gradients. The gradient of 10–20% for the first dimensional BN-PAGE was found to separate protein complexes between 40 and 150 kDa with high resolution, the size distribution of protein complexes fractionated within 60– 90% ammonium sulfate saturation fell within this range. As a result, a combination of first dimensional BN-PAGE gradient of 10–20% and second dimensional SDS-PAGE of 12% was adopted. For protein complexes of larger sizes, a BNPAGE separating gradient of 5–16% was used. Interestingly, when gel strips from BN-PAGE were soaked in 2% (w/v) SDS, 250 mM Tris–HCl, pH 6.8 for 20 min and then run on SDSPAGE, protein spots of different sizes distributed along the diagonal line (Fig. 2A). For example, protein spots A1, B1 and C1 were located along the diagonal line (Fig. 2A). We suspected that although some of these protein spots observed along the

diagonal line were indeed monomeric proteins, some may represent partially denatured protein complexes. In line with our reasoning, protein spots A1, A2 and A3 were later shown to be the same protein, glyceraldehydes-3-phosphate dehydrogenase (G3P, SCO1947) (Fig. 2A, Table 1). And if the same gel strip was boiled for 4 min in 2% SDS buffer, spots A1 and A2 disappeared while A3 remained, which indicates that A3 is the monomer while A1 and A2 represented partially denatured complex (compare Fig. 2B and A). Similar observations were made with NiSOD complex (SCO5254, protein spots B1, B4, B5) and FeSOD II complex (SCO0999, protein spots C1 and C2), protein spots B1 and C1 disappeared when gel strip was boiled before loading on SDS-PAGE (compare Fig. 2A and B). 3.3. Cytoplasmic protein complexes of S. coelicolor identified by MALDI-TOF After procedures to prepare protein samples and conditions to perform 2D electrophoresis were established, samples were resolved on BN/SDS-PAGE, selective protein spots were cut from SDS PAGE and subjected to peptide mass finger printing analyses. The identified proteins were listed in Table 1. Protein spot A3 is the monomeric form of glyceraldehydes-3-phosphate dehydrogenase (G3P). Complex of A3 migrated at an estimated size of 110 kDa on BN-PAGE, smaller than the calculated size of homotetrmer (144 kDa). G3Ps from many prokaryotes and eukaryotes have been crystallized, and they all form homotetramers [16,17]. Both protein spots B4 and B5 represent the same Ni superoxide dismutase ( NiSOD, SCO5254) whose three dimensional structure was solved recently [18,19] and shown to be a homohexamer. The estimated size of 84 kDa of B4 and B5 complex on BN-PAGE agrees with its reported homohexameric size (78 kDa) [18]. It is interesting to note that both B4 and B5 (Fig. 2A and B) are monomeric form of NiSOD. B5 is most likely processed from B4 by a proteolytic mechanim, as

Fig. 2. Comparison of SDS-PAGE patterns of same gel strips after different treatments: (A) soaked in 2% SDS buffer for 20 minutes; (B) boiled in the same buffer for 4 minutes. Proteins precipitated between 60–90% saturation ammonium sulfate were separated by 10–20% gradient BN-PAGE in duplicate lanes, two gel strips were cut from BN-PAGE and treated under A or B conditions and resolved on 12% SDS-PAGE. Protein spots selected for peptide mass finger printing were indicated with an arrow and labeled with a numbered alphabet. Same alphabet indicates proteins dissociated from the ‘same’ spot observed on BN-PAGE, while the spot numbering increases when protein sizes decrease. Sizes estimated for the first dimensional gel were labeled on the top of the gels, size markers of the second dimensional gel were labeled beside the gels.

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Table 2 Comparison of peptides of D1 and/or D2 identified by peptide mass finger printing Common peptides for D1 and D2

Peptides unique to D1

Peptide unique to D2

AELFYDADADLSIIQGR AKAEEQGLR AEEQGLR GPGHLVR RQYEEGR DGDALFFGHGLNIR LIVDLMYEGGLEK QYEEGR KVLAEIQDGTFAK VLAEIQDGTFAK NWMDEYHGGLK NWMDEYHGGLKK QDSEHLLETTGK WSISETAEWGDYVTGPR

DNLKDGDALFFGHGLNIR YGFIKPPAGVDVCMVAPK TTFTEETETDLFGEQAVLCGGTAALVK

AEMRK

was observed before [20]. Although previous report [20] suggested that functional NiSOD of S. coelicolor Müller is a homohexamer of the processed B5, our results suggest that the native complex of NiSOD observed here actually contained both B4 and B5. Protein spot B2 is succinyl-CoA synthetase β chain (SCO4808). Succinyl-CoA synthetase of E. coli was shown to be a complex of α2β2 tetramer [21]. On S. coelicolor genome, the genes encoding succinyl-CoA synthetase α and β chain are adjacent (SCO4808 and SCO4809). Kim et al. [22] identified the α chain on S. coelicolor membrane, probably the membrane location of the α chain prevented isolation of the intact complex in cytoplasmic fraction. Protein spot B3 is phosphoglycerate mutase (SCO4209), with a monomeric MW of 28.3 kDa. This enzyme has been purified from S. coelicolor and estimated to be a homotetramer, with a MW of 120 kDa [23]. On BN-PAGE, the B3 complex migrated at an estimated size of 90 kDa, smaller than the calculated size of 113 kDa. B2 and B3 migrated at different sizes under altered BN-PAGE gradient (data not shown), ruling out the possibility that they form a complex. Protein spot C2 represents Fe superoxide dismutase II (FeSOD II, SCO0999) [24]. Its homologous protein in S. coelicolor Müller was shown to be homotetramer [25]. Its estimated complex size of 75 kDa on BN-PAGE is slightly smaller than the calculated tetrameric size of 88 kDa. Protein spots D1 (SCO5514) and D2 (SCO7154) are both ketol-acid reductoisomerases. These proteins belong to a short form (Class I) ketol-acid reductoisomerase family [26] and are highly homologous to each other (only 9 amino acid residues differ between the two proteins along 332 residues). Due to their high similarity, MASCOT searches always assign the two spots to the same protein. Close examinations provided clues that they may represent two different proteins: first, they migrated as two distinctive protein spots on SDS gel; second, mass spectrometry identified peptides unique to D1 and D2 with high abundance (Table 2). The data as presented therefore strongly indicate that D1 and D2 are different proteins. A class I ketol-acid reductoisomerase from Pseudomonas aeruginosa was crystallized as a homododecamer (12 mer) [27] in contrast to the class II ketolacid reductoisomerase from E. coli and spinach [26], which forms homotetramer and homodimers, respectively. The identification of D1 and D2 as distinctive proteins indicates the ketol-acid reductoisomerase of S. coelicolor is heteromeric. But the estimated complex size is smaller than 66 kDa, suggesting a heteromer of just D1 and D2 (calculated to be 72 kDa). E1 is phosphoglycerate kinase (SCO1946). In bacteria, phosphoglycerate kinase exists as a monomer or it forms a fusion protein with triosephosphate isomerase and the fusion

proteins exist as homotetramers [28]. The estimated size of E1 on BN-PAGE is smaller than 66 kDa, which suggests that E1 exists as a monomer (calculated size 42 kDa). E2 is a small protein (SCO2634) of unknown function. F1 is a putative thiosulfate sulfurtransferase (SCO4164). It co-migrated with F2 on BN-PAGE, a putative peptidyl-prolyl cis–trans isomerase (PPIase, SCO7510, Fig. 1A and B). PPIases assist protein folding by catalyzing the cis–trans isomerization of proline imidic peptide bonds in oligopeptides [29]. It is interesting to note the broad band shapes of F1 and F2 on SDSPAGE, which may indicate the progressive dissociation of F1/F2 complex during the first dimensional BN-PAGE. We also identified two protein complexes (G and H) with high molecular weights. Protein spots G1, G2, G3 and G4 co-migrated at 350 kDa on BN-PAGE (Fig. 3A). G1 is the putative phenylalanyl-tRNA synthetase β chain (SCO1594) while G3 is the putative phenylalanyl-tRNA synthetase α chain (SCO1595). On S. coelicolor genome, the genes encoding phenylalanyl-tRNA synthetase α and β chains are adjacent (SCO1594 and SCO1595). Phenylalanyl-tRNA synthetase of Thermus thermophilus was

Fig. 3. Protein spots from selected large protein complexes (G and H) visualized on SDS-PAGE. (A) protein spots from G complex; (B) protein spots from H complex. Proteins precipitated with ammonium sulfate and separated by 5–16% gradient BN-PAGE were cut and ran on second dimensional 12% SDS-PAGE. Protein complex sizes estimated from the first dimensional BN-PAGE was labeled on the top. Protein size markers for the second dimensional SDS-PAGE were labeled on the left. Selected protein spots were labeled similarly as described in Fig. 2.

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shown to be an α2β2 tetramer [30]. The observed size of 350 kDa of the putative G1/G3 complex is much larger than the calculated size of an α2β2 tetramer (260 kDa). G2 represents a protein of unknown function (SCO2524). While G4 is a putative bacterioferritin (SCO2113). Bacterioferritin was reported to exist as a homooligomer of 24 identical monomers [31]. A 24 mer complex of G4 is expected to show a size larger than 480 kDa. H1 and H2 (Fig. 3B) represent the same proteins: 2-oxoglutarate dehydrogenase E1o and E2o fusion protein (SCO5281). The H complex migrated on BN-PAGE with a size larger than 1500 kDa. The E. coli 2-oxoglutarate dehydrogenase [32,33] exemplifies super large complexes: the so-called multienzyme “machine”. It consists of 3 component enzymes: E1o (2-oxoglutarate dehydrogenase/decarboxylase), E2o (lipoate succinyltransferase) and E3o (lipoamide dehydrogenase). The E2o component forms the 24 mer core of the whole complex. In many other prokaryotes, including Gram-positive bacteria, E1o and E2o are fused into a single large protein, the oligomeric state of which is unknown. Our results suggest that the S. coelicolor 2-oxoglutarate dehydrogenase also forms a very large complex. Interestingly, the E3o component was not observed in our experiment. H1 and H2 appeared as two distinct spots on SDS-PAGE that maybe the result of post-translational modification, which is common in S. coelicolor [34–36]. 4. Discussion Previously, proteomic analyses of S. coelicolor were conducted using standard two-dimensional electrophoresis and protein mass fingerprinting [34–39]. We recently attempted a preliminary inner membrane proteome analysis using a gel independent method [40]. Protein complexes in S. coelicolor have not been studied on a global scale. Sub-fractionation of total cell lysates into membrane and water soluble proteins is commonly used in proteome analyses: it reduces sample complexity, improves sample separation on gel electrophoresis and also serves a functional grouping purpose. For example, sub-fractionation of total protein into membrane and cytoplasmic fractions was applied in the analysis of M. thermautotrophicus protein complexes [12]. However, membrane components may be not the only source of interference on BN-PAGE, small molecules of unknown identities were often part of the problem. Previously, ultra-filtration had been used to eliminate suspected interfering small molecules from human total cell lysates for BN/SDS-PAGE [10]. We attempted similar ultra-filtration treatment for total protein lysates of S. coelicolor, but encountered protein precipitation problem (data not shown). We therefore tested ammonium sulphate precipitation as a sample treatment and fractionation method. A procedure to prepare cytoplasmic protein samples for BNPAGE was thus developed in this work, which allows clear and reproducible visualization of protein complexes on BN-PAGE. Treatment of water soluble proteins by ammonium sulfate precipitation serves multiple purposes: it is expected to remove interfering small molecules therefore improve sample separation on downstream electrophoresis, it helps to generate con-

centrated samples and thus to detect low abundance proteins, and it could also be used as a crude fractionation method to reduce sample complexity and facilitate protein spot identification on SDS-PAGE. Although additional sample handling steps will always increase the risk of complex dissociation, but due to the mild and antichaotropic [41] nature of ammonium sulfate precipitation, this risk could be reduced to minimum. In fact, at high ammonium sulfate concentration, proteins tend to coagulate [41], thus stabilizing protein complexes. Several complexes identified from BN-PAGE showed sizes that agree with those reported (i.e. size of the NiSOD complex agrees well with its reported homohexameric size of 78 kDa) indicating these complexes remained intact in our sample preparation process. Most complexes were observed at sizes larger than their corresponding monomeric sizes, again indicating these complexes remained intact in the sample preparation process. The complex sizes and components identified from one experiment could always be verified by additional experiments with more stringent conditions. Evidence exists that our results present an integrative view of protein function. For example, based on previous reports, phenylalanyl-tRNA synthetase of S. coelicolor is expected to be a complex consisting of α and β chain. The α chain was identified by Novotna et al. [36], and the β chain identified by Hesketh et al. [35]. For the first time, we identified both chains together with an apparent size of 350 kDa, representing a plausible synthetase complex. Similar observations include the association of ketolacid reductoisomerases SCO5514 and SCO7154, the association of the putative thiosulfate sulfurtransferase (SCO4164) with the peptidyl-prolyl cis-trans isomerase (PPIase SCO7510). They may all represent true functional relationships not identified before. Consistent with previous reports [34–36], glyceraldehyde-3phosphate dehydrogenase, succinyl-CoA synthetase β chain, phosphoglycerate mutase, ketol-acid reductoisomerase, phosphoglycerate kinase, the putative thiosulfate sulfurtransferase, NiSOD and 2-oxoglutarate dehydrogenase were present in cytoplasmic protein fractions. One technical challenge with BN-PAGE is the accurate estimation of protein complex size. Unlike traditional native PAGE gel, the apparent sizes of the protein complexes on BN-PAGE are greatly influenced by the bound Coomassie brilliant blue (CBB) molecules, which bind nonspecifically to the hydrophobic surface of proteins. CBB has a molecular weight of 854.04 Da, a mass should not be ignored especially when we calculate sizes of proteins between 50 kDa and 150 kDa. The differential dye binding (due to differences in exposed hydrophobic surfaces between commercial marker proteins and those of sample proteins) will cause false estimation of molecular weight. In fact, CBB binding was reported to cause a factor of 1.8 fold difference in molecular weight estimation [42]. Thus, care must be taken when one try to calibrate the molecular weight of the identified protein complex. In general, native gel electrophoresis is not considered an accurate measure for molecular weight. In our analyses, the predicated homotetrameric G3P should have a molecular weight of about 144 kDa (monomeric subunit is 36.2 kDa), but the observed molecular weight on BN-PAGE was about 110 kDa. The calculated size of

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phosphoglycerate mutase homotetramer is 113 kDa, but its estimated size on BN-PAGE was only 90 kDa. These inconsistencies between the molecular weights observed on BNPAGE and those calculated can not be explained simply by posttranslational modification. The most likely reason is differential CBB binding. Partial complex dissociation should also be carefully considered. In conclusion, our protein interaction data observed on BN/SDS-PAGE are solid qualitatively, particularly after considering the fact that the protein complexes survived relatively rigorous sample handling. However, further inference of complex size and organization is risky. Like other gel based methods, protein identification is limited by the detection limit of staining [35]. But even with the low sensitivity Coomassie Blue R250 staining method (detection limit about 40 ng), we identified proteins that have not been seen before. These include proteins SCO2524 and SCO2634 of unknown function, the putative bacterioferritin (SCO2113) and the ketol-acid reductoisomerase (SCO7154). By further improving detection limit, for example, using the highly sensitive stain Lightning Fast [43], and improving sample preparation techniques, it is possible to visualize more protein complexes on BN-PAGE, including those low abundance protein complexes. 4.1. Simplified description of the method

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13] [14]

Blue native polyacrylamide gel electrophoresis (BN-PAGE) coupled with a second dimensional SDS-PAGE and mass spectrometry has emerged as a powerful tool for protein complex analysis. For optimized use of this method in S. coelicolor, total cell lysates should first be divided into membrane and cytoplasmic fractions by ultracentrifugation. The cytoplamic fraction can be further treated by ammonium sulfate precipitation and ultrafiltration. With these treatments, BN/SDS PAGE could be used effectively to identify cytoplasmic protein complexes of S. coelicolor.

[15]

[16]

[17]

[18]

Acknowledgments This work was supported by the Knowledge Innovation Fund of Chinese Academy of Sciences. The authors wish to acknowledge the kind assistance of Associate Professor Xiu-Fen Kou, Hui Han and Jian-Ting Zheng during the course of this study. References [1] Schagger H, von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 1991;199:223–31. [2] Eubel H, Braun HP, Millar AH. Blue-Native PAGE in plants: a tool in analysis of protein–protein interactions. Plant Methods 2005;1:11. [3] Schagger H. Quantification of oxidative phosphorylation enzymes after blue native electrophoresis and two-dimensional resolution: normal complex I protein amounts in Parkinson's disease conflict with reduced catalytic activities. Electrophoresis 1995;16:763–70. [4] Schagger H. Electrophoretic techniques for isolation and quantification of oxidative phosphorylation complexes from human tissues. Methods Enzymol 1996;264:555–66. [5] Schagger H, Ohm TG. Human diseases with defects in oxidative phosphorylation. 2. F1F0 ATP-synthase defects in Alzheimer disease revealed by

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