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Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-Based Fractionation and LC-MS/MS Protein Identification Roslyn N. Brown, Margaret F. Romine, Athena A. Schepmoes, Richard D. Smith, and Mary S. Lipton* Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington Received March 9, 2010

A simple and effective subcellular proteomic method for fractionation based on osmotic lysis, differential centrifugation, and Sarkosyl solubilization was applied to the Gram-negative bacterium Shewanella oneidensis to gain insight into its subcellular architecture. Global differences in bacterial cytoplasm, inner membrane, periplasm, and outer membrane protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution liquid chromatography-tandem mass spectrometry. Proteins predicted to be localized to each subcellular fraction were enriched ∼2fold (on average) in each fraction compared to crude cell lysates. In addition, the Sarkosyl solubilization method facilitated separation of the inner and outer membranes, making the procedure amenable for effective probing of the subcellular proteome of Gram-negative bacteria via liquid chromatographytandem mass spectrometry. With 40% of the observable proteome represented, this study provides extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other Gram-negative bacteria. Keywords: subcellular • proteomics • Sarkosyl • fractionation • Shewanella

Introduction Combining subcellular fractionation with proteomics, dubbed “subcellular proteomics”,1 is a powerful tool for deducing physiologically relevant protein localization within cells.1,2 While crude cell homogenates have been the foundation of proteomics, fractionating these homogenates into subcellular components allows the elucidation of cellular organization and reduces sample complexity, which increases the probability of observing low abundance protein species. Moreover, the use of subcellular fractionation enables the discovery of proteins that are associated with subcellular structures only under certain physiological conditions.3 As such, subcellular proteomics has become increasingly popular for probing the cell biology of Gram-negative bacteria. While many subcellular proteomic studies have relied on 2D gels for protein separation,4–9 this approach is limited in dynamic range, and membrane proteins are grossly underrepresented.1,10,11 Some research groups have employed gel-free mass spectrometry (MS)-based methods to analyze one or more subcellular fractions of Gram-negative bacteria;3,12–17 however, few have surveyed all possible subcellular compartments at once.3,16 This tendency can be attributed to focused studies (e.g., one cellular compartment), as well as to the technical difficulties associated with achieving efficient subcellular fractionation while maintaining MS-compatibility and achieving * To whom correspondence should be addressed. Tel: 1 509 371 6589. Fax: 1 509 371 6564. E-mail: [email protected].

4454 Journal of Proteome Research 2010, 9, 4454–4463 Published on Web 07/19/2010

good proteome coverage. Toward this end, we sought to identify a simple and effective subcellular fractionation method that provided large numbers of highly confident peptide identifications for a comprehensive view of subcellular protein organization and could readily be scaled for use in highthroughput applications. In this study, we implemented an MS-amenable subcellular fractionation method based on differential centrifugation, Sarkosyl solubilization, and osmotic lysis to gain better insight into the subcellular architecture of the Gram-negative, dissimilatory metal-reducing bacterium Shewanella oneidensis. Because this organism localizes a portion of its respiratory apparatus to the outer membrane (OM) and cell surface to utilize insoluble substrates for terminal electron transfer, there is considerable interest in targeted proteomic analysis of subcellular constituents to more fully characterize these unusual respiratory systems.18–21 We also have extensive experience with this bacterium.14,22–29 After separating S. oneidensis cells into inner membrane (IM), OM, periplasmic (PERI), and cytoplasmic (CYT) fractions, we utilized SDS PAGE and liquid chromatography-tandem MS (LC-MS/MS) to assess global differences in protein content for each fraction, as well as for whole cell lysates (WCL). The results from this study represent the most extensive experimental analysis of subcellular localization in any Gram-negative bacterium to date and provide a foundation for future work on subcellular architecture and protein-membrane interactions. 10.1021/pr100215h

 2010 American Chemical Society

Subcellular Proteome of Shewanella oneidensis

Materials and Methods Bacterial Strain, Media, and Chemicals. S. oneidensis MR-1 (ATCC 700550) was maintained in Luria-Bertani (LB) broth or on LB plates containing 1.5% (wt/vol) agar. Unless noted otherwise, growth media components were obtained from Difco (Franklin Lakes, NJ) and chemicals, from Sigma (St. Louis, MO). Cell Fractionation. Five-mL overnight starter cultures were diluted 1:100 into 80 mL fresh LB broth and shaken overnight at 30 °C until cells were in the early stationary growth phase (OD600 1.0-1.2). The membrane fractionation procedure was adapted from Chart30 and Leisman et al.31 Unless otherwise noted, all centrifugations were performed at 4 °C. Fifty mL of cells were harvested by centrifugation at 10 000× g for 10 min. The supernatant was removed and the cell pellet suspended in 20 mL of 20 mM ice-cold sodium phosphate (pH 7.5) and passed four times through a prechilled French Press (8000 lb/ in2). The cell lysate was centrifuged at 5000× g for 30 min to remove unbroken cells, and an aliquot of the supernatant was saved as the WCL sample. The remaining supernatant was centrifuged at 45 000× g for 1 h. The soluble fraction (CYT) was centrifuged again (45 000× g for 1 h) to remove residual membrane components. The tube containing the crude membrane pellet was inverted to drain, after which the crude membranes were suspended in 20 mL 0.5% Sarkosyl in 20 mM sodium phosphate and shaken horizontally at 200 rpm for 30 min at room temperature. The crude membrane sample was centrifuged at 45 000× g for 1 h to sediment the OM. The supernatant containing the IM was removed and the sediment containing the OM was washed in ice-cold sodium phosphate and recentrifuged. The PERI fraction was prepared according to the method of Ross, et al.,32 starting with 30 mL of the same cells used for the membrane preparation. Centrifugation steps were performed at 10 000× g. A microscope slide was prepared to observe spheroplasts for characteristic round morphology. Removal of Sarkosyl, Concentration of Dilute Proteins, and SDS-PAGE. The very dilute PERI fraction and the dilute IM fraction that contained 0.5% Sarkosyl were cleaned and concentrated using Ultrafree 0.5 Centrifugal Filter Units with 5 kDa molecular weight cutoff (Millipore Corp., Billerica, MA). Protein concentrations were determined using a bicinchoninic acid (BCA) assay (Pierce Protein Research Products, Rockford, IL) with bovine serum albumin as the standard. Protein samples were suspended in NuPAGE LDS sample buffer, heated to 70 °C for 10 min, and resolved on NuPAGE Novex 4-12% gradient gels (Invitrogen Corp., Carlsbad, CA) at 200 constant volts for 35 min. Gels were either stained with GelCode Blue (Pierce) to observe total proteins or treated with tetramethylbenzidine-peroxide33 to observe heme-containing proteins. Gels were run at least twice, with similar patterns observed. Preparation of Tryptic Peptides from Soluble Protein Fractions. The CYT and PERI tryptic peptides were prepared as follows. To 100 µg of each protein sample, urea (7 M final) and DTT (5 mM final) were added, followed by incubation at 60 °C for 30 min. The samples were diluted 10-fold with 100 mM NH4HCO3 and CaCl2 was added (1 mM final). The samples were then digested with trypsin (1:50 trypsin:protein ratio) for 3 h at 37 °C and cleaned using 1 mL, 50 mg Discovery DSC-18 solid phase extraction (SPE) columns (Supelco, St. Louis, MO). Each SPE column was conditioned using methanol and then

research articles rinsed with 0.1% TFA in water. Samples were introduced to the columns and washed with 95:5 H2O/ACN containing 0.1% TFA. Excess liquid was removed from the columns under vacuum, and the samples were eluted using 80:20 ACN:H2O that contained 0.1% TFA and concentrated in a SpeedVac (Thermo-Savant) to a final volume of 50-100 µL. Final peptide concentrations were determined using a BCA protein assay. Preparation of Tryptic Peptides from Insoluble Protein Fractions. Tryptic peptides from the IM, OM, and WCL samples were prepared as follows. To 100 µg of each sample, urea (7 M final), CHAPS (1% final), and DTT (10 mM final) were added and incubated at 60 °C for 30 min. The samples were then diluted 10-fold with 50 mM NH4HCO3, and CaCl2 was added (1 mM final). Digestion was performed as described for the soluble samples. Samples were cleaned using 1 mL 50 mg Discovery DSC-SCX strong cation-exchange SPE columns (Supelco). Each column was conditioned using methanol and rinsed in varying sequences and amounts with 10 mM ammonium formate in 25% ACN (pH 3.0), 500 mM ammonium formate in 25% ACN, and with Nanopure water. Samples were acidified to a pH e 4.0 by adding 20% formic acid, and centrifuged at 10 000× g for 5 min. Samples were injected into the columns and washed with 10 mM ammonium formate in 25% ACN (pH 3.0). After removing excess liquid from the columns under vacuum, the samples were eluted using 80:15:5 MeOH/H2O/NH4OH, and then concentrated to 50-100 µL using a SpeedVac. In-Gel Digests. Gel bands of interest were excised from the GelCode Blue-stained gel and cut into 1-1.5 mm pieces. Destaining solution (400 µL of 40% Acetonitrile, 200 mM NH4HCO3) was added to the gel pieces, which were then incubated for 30 min at 37 °C. The solution was removed and the destaining step was repeated, after which the gel pieces were dried for 15-20 min in a SpeedVac (Thermo Savant). Trypsin (1.6 µg in 80 µL of 0.1 mM CaCl2 with 8.1% acetonitrile and 36 mM NH4HCO3) was added to the samples along with an additional 200 µL of 9% acetonitrile and 40 mM NH4HCO3. The samples were incubated for 4 h at 37 °C. Following incubation, 200 µL of peptide extraction solution (0.1% TFA in 50% acetonitrile) was added to the samples, which were then incubated for 30 min at 37 °C and concentrated to ∼25 µL prior to analysis. Capillary Liquid Chromatography-Mass Spectrometry Analysis. The HPLC system and method used for capillary LC have been described in detail elsewhere.34 MS/MS analysis was performed using an LTQ orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) with electrospray ionization. The HPLC column was coupled to the mass spectrometer by using an inhouse manufactured interface. The heated capillary temperature and spray voltage were 200 °C and 2.2 kV, respectively. Data acquisition began 20 min after sample injection and continued for 100 min over a mass (m/z) range of 400-2000. For each cycle, the six most abundant ions from MS were selected for MS/MS using a collision energy setting of 45%. A dynamic exclusion time of 60 s discriminated against previously analyzed ions. Data Analysis. Peptides were identified by using SEQUEST to search the mass spectra against in silico tryptic sequences derived from an in-house S. oneidensis MR-1 protein FASTA protein sequence file previously described.26 A standard parameter file with no modifications to amino acid residues was employed. The searches allowed for all possible peptide termini, that is, not limited by tryptic terminus state. The large Journal of Proteome Research • Vol. 9, No. 9, 2010 4455

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Figure 1. Fractionation scheme. A fractionation scheme based on differential centrifugation and Sarkosyl solubilization of membranes was combined with spheroplasting to obtain OM, IM, CYT, and PERI samples from S. oneidensis strain MR-1. Subcellular fractions were further processed prior to highresolution LC-MS/MS analysis.

sets of tentative peptide identifications were subsequently combined and binned by subcellular fraction. These results were filtered,35 and a discriminant approach36,37 with a score of at least 0.9 was used to increase confidence in identified peptides. An estimate of the false positive rate was obtained by searching against a reversed FASTA database, as described elsewhere.38 A calculated false discovery rate of 1 mg of protein for each subcellular fraction with the exception of the PERI fraction (30 mL starting volume), which yielded ∼0.5 mg, which indicates that this procedure can be further scaled down if desired. To observe the protein profiles and to obtain a rough sense of fraction separation, proteins from each subcellular fraction were resolved by either SDS PAGE, and the gels stained with 4456

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Figure 2. Protein profiles of subcellular fractions. Proteins from each subcellular fraction (10 µg) were (A) resolved on NuPAGE Novex 4-12% Bis-Tris gels and stained with GelCode Blue or (B) treated with tetramethylbenzidine-peroxide to observe hemecontaining proteins. Invitrogen SeeBlue Plus2 prestained std was used as an indicator of protein molecular weight, and protein contents of indicated regions (indicated by asterisks to the right of each) were confirmed by in-gel digests followed by LC-MS/ MS analysis.

GelCode Blue to detect proteins (Figure 2A) or tetramethylbenzidine-peroxide (TMBZ) to detect c-type cytochromes (Figure 2B). Each subcellular fraction displayed, in general, a unique total protein profile (Figure 2A), indicating that our procedure separated proteins into distinct fractions. The CYT fraction was almost identical to the WCL, which is not surprising as most abundant proteins are typically found in the cytoplasm. It should be noted that TMBZ staining indicates heme peroxidase activity and is not a quantitative measure of heme-containing proteins. The identity of proteins stained by TMBZ in Figure 2B was assessed by analysis of in-gel digests of the corresponding bands from the GelCode Blue-stained gel (mass spectrometry compatible) for the presence of known c-type cytochromes. Eleven -c-type cytochromes were identified in the excised gel bands (Supplemental Table 1, Supporting Information). Outer membrane decaheme cytochromes OmcA and MtrC were observed in the OM fraction, and to a lesser extent in the IM fraction, while the periplasmic tetraheme fumarate reductase, FccA, was found mainly in the PERI fraction, although some (∼20-fold less) was detected in the OM and IM fractions following MS/MS analysis of in-gel digests (Supplemental Table 1, Supporting Information). FccA appeared as a faint band in the WCL and was almost imperceptible in the CYT fraction (Figure 2B). The PERI fraction also showed high concentration of a 36 kDa heme protein (Figure 2B). In-gel digest revealed this band to contain the decaheme cytochrome MtrA, the diheme cytochrome c5 peroxidase CcpA, and a SoxA-like diheme cytochrome c. A third, low molecular weight hemecontaining protein band was observed exclusively in the PERI fraction (Figure 2B). This band contained the monoheme cytochrome c-prime (16 kDa) and, to a lesser extent, the PERI tetraheme cytochrome c, CctA (12 kDa). In most cases, the proteins observed in the heme-stained gel were among the top 20 most abundant proteins detected in the corresponding subcellular fractions by LC-MS/MS. Assessment of Subcellular Fractionation by LC-MS/MS. A total of six technical replicates from two sets of cell fractions that were collected from independent experiments yielded

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Figure 3. Distribution of proteins observed in subcellular fractions via LC-MS/MS. (A) Protein composition of each subcellular fraction, based on number of proteins observed in each fraction sorted according to annotated/predicted subcellular location (based on curated database). Parentheses indicate the total number of proteins observed in each fraction. (B) Combined absolute abundance of proteins observed in subcellular fractions. Parentheses indicate the combined spectral counts of all proteins observed in each fraction. Pie charts categorize proteins by annotated/predicted subcellular location based on curated database.

14 311 unique peptides that represented 1698 proteins (40% of the 4198 proteins in the S. oneidensis FASTA). After filtering to remove those proteins not observed in two of three technical replicates in either biological replicate, 1248 proteins remained. For statistical analyses, proteins were considered present in a fraction only when represented by g2 peptides in that fraction (n ) 1203). When these final filtering criteria were applied, the CYT fraction yielded 777 proteins; the IM fraction, 580; OM, 248; PERI, 424; and the WCL, 704. It was noteworthy that the CYT fraction yielded more proteins than the WCL. This could result from an improvement in dynamic range or could be due to the criterion that proteins be identified by at least two peptides in a fraction to be considered “present” for statistical analysis. Over 80% of the CYT fraction was composed of predicted/ known CYT proteins (n ) 632) (Figure 3A). Proteins predicted/ known to be in other locations were present at very low levels (IM, 5%; PERI, 10%; OM/surface/extracellular, 4%). Despite the fact that the CYT fraction was actually a “soluble” fraction that could potentially capture both CYT and PERI proteins, the predominance of CYT proteins (almost 10-fold greater than the number of PERI proteins) indicates that the CYT fraction truly represents the cytoplasmic proteome. In addition, the total abundance levels (summed spectral counts) of all PERI proteins in the CYT fraction were generally low compared to the levels observed in the PERI fraction (Figure 3B). For the two most highly observed known/predicted PERI proteins (fumarate reductase, SO_0970 and ABC tungstate transporter, SO_4719), the ratio in the CYT to PERI fractions was 2-fold higher peptide coverage compared to the WCL, and for 458 proteins, percent coverage increased by >4-fold. Conversely, peptide coverage increased by >2-fold for only 18 proteins in the WCL compared to the combined fractions. A significant increase in coverage via fractionation (mean 15%) was observed for the subset of proteins with >2 transmembrane domains (IM proteins) compared to the WCL (mean 4%; P < 0.001), with ∼70% of the 4458

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proteins having peptides detected exclusively in the IM. These findings substantiate the use of subcellular fractionation to improve protein coverage (in terms of the number of proteins and the number of unique peptides per protein) in MS-based proteomics. Agreement of Observed Protein Localization with Predicted Localization. Observed localization of proteins in the four subcellular fractions was compared to various subcellular localization prediction models, that is, PSORTb v.2.0,39 Cello v.2.5,40 and a comprehensive, curated subcellular localization database specific to Shewanella spp. (Romine M. et al., in preparation). Note that of the 1203 proteins being analyzed, 500 (42%) had no PSORT prediction associated with them (unknown or unknown due to multiple possible localizations). Agreement with predicted localization was assessed separately for proteins observed in a single fraction and those observed in multiple fractions. Of the proteins observed exclusively in a single fraction (n ) 617, excluding the 32 observed exclusively in the WCL), 79% agreed with the manually compiled localizations, 67% agreed with Cello, and 83% agreed with PSORTb (Table 2 and Supplemental Table 2, Supporting Information). For those proteins observed in multiple fractions (n ) 554), a hierarchical clustering approach was used to determine the most likely subcellular location based on relative protein abundances. Z-scores were calculated for the relative abundances (spectral counts) of the 554 proteins. These scores were clustered using OmniViz v.6.0 and represented as a heat map (Figure 4 and Supplemental Table 3, Supporting Information). On the basis of this approach, proteins were assigned to the CYT (n ) 240), PERI (n ) 55), IM (n ) 140), and OM (n ) 54). Some proteins that exhibited similar abundances in multiple fractions were not resolved into a distinct location (Figure 4, inset). This may reflect the reality that some proteins are present in more than one subcellular location, including those large proteins that span multiple subcellular fractions. Of the proteins that could be resolved into a single primary localization by hierarchical clustering, there was 76% agreement between observed and predicted localization using the manually compiled prediction database, 65% agreement with Cello, and 77% agreement with PSORTb (based on 298 proteins with available PSORTb predictions). Predicted CYT Proteins Observed in the IM Fraction. A total of 274 proteins with predicted CYT localization were observed in the IM fraction (Supplemental Table 4, Supporting Information). Of these, 140 were observed in the IM fraction at levels that were equal to or greater than those observed in the CYT fraction. Sixty-nine proteins with predicted CYT localization were observed exclusively in the IM fraction. Observed proteins were searched against the Uniprot (www. uniprot.org) and the Bacterial Protein Interaction (www. bacteriome.org) databases to further investigate localization.

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Twenty-one of the proteins were assigned a subcellular location of “inner membrane”, or “peripheral membrane” by Uniprot (Supplemental Table 4, Supporting Information). The Bacterial Protein Interaction Database, based on the model Gramnegative bacterium Escherichia coli, showed potential interacting partners for 13 of the IM-localized, predicted CYT proteins. In many cases, some of the interacting partners were predicted and observed in membrane fractions, which suggests that protein-protein interactions may have caused colocalization of CYT proteins and their interacting membrane proteins. In total, 57 of the predicted CYT proteins were justifiable in their IM localization because of potential IM associations (Supplemental Table 4). Notable examples were the alternative RNA polymerase sigma factor, σE (SO_1342) and the signal recognition particle (SO_1356). σE (rpoE gene product) is a cytoplasmic protein that controls expression of an extensive regulon of genes involved in responding to external stress.44,45 In unstressed cells, σE is bound by the IM protein RseA, which, in turn, is bound by periplasmic RseB. We observed peptides from RseA, σE, and RseB mainly in the IM fraction, which suggests that in the absence of envelope stress, the CYT sigma factor was peripherally associated with the IM via RseA. The second example of note was the signal recognition particle, SRP, composed of the protein Ffh and 4.5S RNA.46–48 This CYT protein is involved in cotranslational protein translocation into and through membranes and associates with the membranebinding receptor FtsY.49,50 The SRP has been shown or predicted to associate with ribosomal proteins RplW, RplS, RplU, RpmA, and RpsP51,52 (and http://www.compsysbio.org/ bacteriome/graphapplet.php?data set)functional&layer)1& view)profile&genestr)ffh). FtsY and all of the Ffh-associated ribosomal proteins were observed in the IM fraction at levels equal to or greater than CYT levels, which suggests IMassociation via protein-protein interaction. Other observed multiprotein complexes known to assemble or interact at the IM included four components of the NADH dehydrogenase complex involved in energy metabolism (NuoI, NuoB, NuoC, NuoF, NuoG), as well as PspA and PspB of the phage shock protein (PSP) response system. In addition, all five subunits of ATP synthase F1 (AtpA, AtpC, AtpD, AtpG, and AtpH) were enriched in the IM fraction (Supplemental Table 4, Supporting Information). Other predicted CYT proteins observed primarily in the IM fraction may represent potential novel protein-protein or protein-membrane interactions. Identification of Envelope Proteins. Because of its ability to reduce metals via OM cytochromes,53 S. oneidensis envelope proteins have been the focus of active research.9,19–21,54–56 In this study, 475 proteins whose predicted and/or annotated localization was IM, OM, PERI, surface, or extracellular were observed in the envelope fractions (IM, PERI, OM). The observed localization of the envelope proteins was in good agreement with the manually curated predictions, Cello, and PSORTb (not shown). Transmembrane R-helices, a feature of integral membrane proteins, were inferred57 from primary protein structures. Of the proteins whose primary observed localization was the IM, 99 (29%) had >2 transmembrane helices. Conversely, only 24 (4%) CYT, 3 (2%) PERI, and 5 (6%) OM-localized proteins had two or more transmembrane Rhelices (Figure 5A). The IM fraction was also significantly enriched for integral membrane proteins compared to the WCL, further supporting the notion that global preparations tend to undersample hydrophobic proteins. While 18% of all proteins observed in the IM fraction had g2 transmembrane domains,

Figure 5. Distribution of proteins with membrane-associated characteristics. (A) Proteins with multiple transmembrane R-helices. For the proteins whose primary observed localization (see text) is indicated in the figure key, the number of proteins with TM helices (according to ref 57) is shown. (B) Grand average of hydropathy of proteins. GRAVY scores of proteins were calculated according to ref 58. Only proteins whose primary observed localization was the IM and OM had GRAVY scores above 0.5 (very hydrophobic). (C) Predicted OM β-Barrel proteins. The presence of OM β-barrel motifs was predicted using BOMP (http://services.cbu.uib.no/tools/bomp). The number of proteins in each BOMP category is shown. BOMP categories 3, 4, and 5 (confident β-barrel predictions) were predominated by proteins whose primary observed localization was the OM.

only 8% of the WCL contained multiple transmembrane domains, and of these, the abundances in the WCL were usually much lower than in the IM (data not shown). As an additional indicator of membrane proteins, the hydropathy characteristics of the observed proteins were examined. Average hydropathy (GRAVY) scores were calculated according to the method of Kyte and Doolittle58 (Figure 5B). Of the Journal of Proteome Research • Vol. 9, No. 9, 2010 4459

research articles proteins whose primary observed localization was the IM, 42 (12%) had GRAVY scores >0.2. In contrast, 28 (5%) CYT-, 0 (0%) PERI-, and 3 (4%) OM-localized proteins had GRAVY scores >0.2. Using a GRAVY cutoff of 0.5,58 only proteins whose primary localization was the IM or OM were very hydrophobic (Figure 5B), and the WCL fraction was a poor source of hydrophobic proteins. No proteins with GRAVY scores >0.2 were observed exclusively in the WCL, and of the 53 proteins with GRAVY scores >0.2 that were detected in the IM fraction, only 21 were also present in the WCL, but with an average relative abundance one-third of those observed in the IM (data not shown). To further characterize the observed OM proteins, we employed the β-barrel OM protein predictor, BOMP.59 The distribution of the 53 potential OM β-barrel proteins (grouped by primary observed location) is depicted in Figure 5C. The BOMP program classifies proteins into five categories (1-5) in order of increasing reliability of the prediction. Proteins in categories 1 and 2 were observed across the four subcellular locations, while proteins in categories 3, 4, and 5 almost uniformly had the OM as their primary observed location (Figure 5C). Similar to the other two indicators of membrane proteins (transmembrane domain and hydropathy), predicted OM β-barrel proteins were under represented in the WCL. Only one predicted OM β-barrel protein SO_0130 (BOMP category 3) was exclusive to the WCL (Figure 5C). Of the proteins highly predicted to form β-barrels (BOMP categories 4 and 5), only five were observed in the WCL compared to 25 observed in the OM (data not shown). Additionally, these five proteins were 8- to 34-fold higher in abundance in the OM than in the WCL. These results suggest that information may be lost by using global protein preparations in studies of biological processes involving integral membrane proteins. Results confirmed the localization of several envelope proteins of potential importance for energy production and bioremediation. These proteins included 24 ABC transporters; 4 OM porins; transporters of ferrous iron, mercury, biopolymers, magnesium/cobalt, and two acraflavine/multidrug transporters; 26 Cytochromes, cytochrome-associated, or hemeassociated proteins; 16 methyl-accepting chemotaxis proteins; 11 flagellum-associated proteins, and 7 members of the type II secretion pathway required for correct localization of OM proteins involved in iron and DMSO reduction. Figure 6A summarizes the observed subcellular distribution and relative abundance of a subset of cytochromes and cytochromeassociated proteins involved in electron transport. Of 68 observed proteins functionally categorized as transport and binding proteins, 23 involved in cation transport were graphically represented, including multiple siderophores, TonBdependent receptors, iron transporters, heavy metal efflux pumps, divalent cation transporters, and a mercury transporter (Figure 6B). These proteins are likely to be key players in the energy production and bioremediation capabilities of this organism. In addition to the many expected proteins dedicated to energy metabolism, 10 proteins with JCVI subrole “Pathogenesis” (http://cmr.jcvi.org) were observed, primarily in the envelope fractions. In most cases, these proteins were missed or were present in much lower abundances in the WCL. Among the detected proteins were seven mannose-sensitive hemeagglutinin (MSHA) biogenesis proteins (MshA, MshB, MshE, MshI, MshJ, MshL, and MshQ); an agglutination protein, AggA; a virulence regulator, BipA, and an expressed lipoprotein, SO_0135. 4460

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Brown et al. In addition, several proteins belonging to the JCVI subgroup “Toxin production and resistance” could have roles in pathogenesis, including a periplasmic β-lactamase, AmpC, two acraflavine/multidrug efflux proteins, SO_3484 and SO_4014 observed in the IM, and RtxB, a member of a putative RTX toxin operon predicted in the genome sequence60 and observed exclusively in the IM. Although there have been no reports of S. oneidensis having pathogenic roles, closely related Shewanella species S. putrefaciens and S. alga are opportunistic human pathogens.61,62 The fact that S. oneidensis has been shown to express many putative virulence factors, including attachment and colonization factors and drug resistance determinants, is noteworthy for remediation strategies that plan to use this bacterium. Subcellular fractionation is a useful technique for investigating the roles of such proteins on a global scale, as they are underrepresented and often absent in global sample preparations.

Discussion Assessment of Membrane Separation Using Sarkosyl Solubilization and Differential Centrifugation. Detergents such as SDS, NP-40, Triton X-100, and CHAPS have been widely used for solubilizing membrane components for proteomic analysis via 2D electrophoresis.6 However, their use in LC-MS/MSbased proteomics can be problematic due to constraints of the reversed phase chromatographic method of peptide separation10 and the tendency of ionic detergents to interfere with electrospray ionization. In addition, the amount of detergent must be permissive to in-solution enzymatic protein digestion, although advances are currently being made toward the efficient removal of detergents for enzymatic digests and downstream processing.63 While alternatives to detergents, such as organic solvents, organic acids, and high pH have been used to study total membrane proteins,12,15,35,64 we employed the detergent Sarkosyl because of its ability to preferentially solubilize the IM,65 thus facilitating not only enrichment of hydrophobic proteins, but also allowing for efficient separation of the IM and OM by centrifugation.66–68 Similar to the “total membrane” methods mentioned above, the surfactants Triton X-100 and SDS lack the specificity of selecting for the IM65 and thus require tedious sucrose density gradients or rely on differential centrifugation alone to separate the two membrane compartments. Some research groups have used alkaline solutions such as sodium carbonate (pH 11) to remove loosely associated proteins from cell membranes.6 While this approach is useful to those who are interested solely in integral membrane proteins, the complement of peripheral membrane proteins is not captured in its physiological context. As such, carbonate extraction can be incorporated into the methods used here, but information on potential protein-protein and protein-lipid interactions at the membrane interface would be lost. Caveat of Sarkosyl-Soluble OM Proteins. While there were relatively few predicted/known IM proteins observed in the OM fraction (n ) 36), the IM fraction contained large number of OM proteins (n ) 106). Some of these proteins were likely to be transiently localized to the IM en route to the OM. Using a hierarchical clustering approach based on relative protein abundances, many such proteins were designated a primary localization of OM (Figure 4). Still, 13 predicted OM proteins could not be confidently resolved between the IM and OM based on relative abundance, including the flagellar basal body protein FliF (SO_3228), the decaheme cytochrome lipoprotein

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Figure 6. Subcellular distribution and relative abundance of a subset of proteins involved in transport. (A) Subset (18) of 38 observed proteins with JCVI role “Energy metabolism: electron transport”. (B) Twenty-three observed proteins in cation transport role category. Predicted localization is indicated above each graph, and observed localization is indicated on the z axis.

OmcA (SO_1779), seven uncharacterized lipoproteins (SO_4080, SO_1173, SO_1060, SO_1061, SO_0923, SO_0135, and SO_3376), three lipoproteins with predicted peptidase activity (SO_0429, SO_1137, and SO_2753), and an uncharacterized OM protein (SO_4473) (Figure 4, as well as data not shown). In addition, 24 proteins annotated as OM lipoproteins were observed primarily in the IM. OM lipoproteins may have increased solubility in Sarkosyl compared to other OM proteins, which could lead to increased fractionation into the IM. Because lipoproteins are not integral membrane proteins, but tethered to the membrane by lipolyl modifications, it is not surprising that they could easily be separated

from the OM during fractionation. Another consideration is that wall-bound OM proteins may have a close association (via proximity) with the IM and may have preferentially fractionated to the IM. The use of lysozyme (0.64 mg/mL final) to release peptidoglycan-bound proteins and thus potentially decrease the number of OM proteins observed in the IM did not significantly decrease OM proteins in the IM fraction tested (12 vs 17%), although the number of IM proteins in the IM fraction decreased by 53%, arguing against the use of lysozyme in this method (data not shown). In light of the fact that the majority of OM proteins appearing in the IM fraction were OM lipoproteins, care should be taken Journal of Proteome Research • Vol. 9, No. 9, 2010 4461

research articles to consider functional annotation and lipoprotein sorting signals when ascribing a likely localization to proteins predicted to be OM-localized that fractionate with the Sarkosyl-soluble IM. Utility of Subcellular Localization Data for Shewanella and Other Bacteria. Knowledge of subcellular localization and interaction are important for structural, biochemical, and biotechnological applications, such as cloning, expression, and purification of proteins where special targeting sequences may be required to achieve proper expression in the correct location. While subcellular localization prediction tools, genomic annotation, and homology of proteins with known localization have great utility in assigning putative subcellular locations to proteins, experimental confirmation is necessary for validation. Global analyses of subcellular localization via proteomics can be used to confirm or refine the predicted localization of proteins, especially those proteins that are confidently identified in a single subcellular location (Supplemental Table 2, Supporting Information) or predominantly in one fraction (Figure 4). Corroborating the results by alternate means further increases confidence in assignments. The addition of methods to distinguish surface-exposed OM proteins, such as surface shaving, proteinase-K treatment, and the use of chemical probes,56,69–71 is also useful for fine-tuning observed protein localization. The use of amino acid sequence alone to predict location suffers from neglecting protein-protein or protein-membrane interactions that may change the subcellular location of a protein. A notable example in this study was MtrA that is predicted, annotated, and reported72 to be periplasmic, but was observed to a large extent in the OM fraction (Figure 6A). This observation agrees well with the recent discovery that MtrA interacts with OM proteins MtrB and MtrC and thus fractionates with the OM, as indicated by Western blot and heme staining of cell fractions.32 Other examples were the many predicted CYT proteins that were concentrated in the IM fraction, likely due to interactions with IM proteins or lipids, or in some cases, incorrect predictions (Supplemental Table 4, Supporting Information). Taken together, these observations support the notion that some of the proteins we detected in unexpected fractions were not due to contamination, but rather due to yet undiscovered interactions. Thus, the results reported here may be used to infer potential protein-protein and protein-membrane interactions. Investigation of these potential interactions by secondary methods will be required for validating these inferences. Global confirmation of subcellular localization and further confirmatory experiments provide data that can be used to train subcellular localization prediction tools for more accurate and useful predictions. The methods presented herein represent a promising platform for probing global protein localization under different conditions (mutant vs WT, different growth conditions, etc.) because a large proportion of the proteome was represented (Supplmental Tables 2 and 3, Supporting Information). Additionally, semiquantitative analyses were made possible by counting the number of high-resolution LC-MS/MS spectra observed for each protein (Figure 6). The ability to infer not only subcellular localization, but also relative expression levels of proteins could be particularly informative when choosing expression or purification technologies for proteins of interest. To this end, we conducted our study under standard laboratory growth conditions wherein the majority of cellular proteins are expressed. The methods and results of this study should be of 4462

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Brown et al. great utility to those interested in Shewanella biology and to the scientific community, in general.

Conclusion We have presented a global view of subcellular protein localization in S. oneidensis MR-1 that, to the best of our knowledge, is the most extensive experimental analysis of subcellular localization of proteins for any Gram-negative bacterium. High resolution and good depth of coverage were achieved by coupling subcellular fractionation via osmotic shock, Sarkosyl solubilization, and differential centrifugation with high-performance LC-MS/MS analysis. The methods described are scalable, feasible for high-throughput analyses, and easily extendable to other Gram-negative organisms. Abbreviations: IM, inner membrane; OM, outer membrane; CYT, cytoplasmic; PERI, periplasmic; WCL, whole cell lysate; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Acknowledgment. This research was supported by the U.S. Department of Energy Office of Biological and Environmental Research (DOE/BER) GtL:Genomes to Life program at the Pacific Northwest National Laboratory (PNNL). We gratefully acknowledge the contributions of Samantha Bree Reed, Catherine Reardon, Therese Clauss, Anuj Shah, and Penny Colton to this publication. Proteomic analyses were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility on the PNNL campus in Richland, Washington. PNNL is a multiprogram national laboratory operated by Battelle for the DOE under Contract DE-AC05-76RL01830. Supporting Information Available: Supplemental Tables 1-4 containing results of the in-gel digest and lists of proteins observed in subcellular fractions are available in Excel file format. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Dreger, M. Mass Spectrom.Rev. 2003, 22, 27. (2) Huber, L. A.; Pfaller, K.; Vietor, I. Circ. Res. 2003, 92, 962. (3) Callister, S. J.; Dominguez, M. A.; Nicora, C. D.; Zeng, X.; Tavano, C. L.; Kaplan, S.; Donohue, T. J.; Smith, R. D.; Lipton, M. S. J. Proteome Res. 2006, 5, 1940. (4) Baik, S. C.; Kim, K. M.; Song, S. M.; Kim, D. S.; Jun, J. S.; Lee, S. G.; Song, J. Y.; Park, J. U.; Kang, H. L.; Lee, W. K.; Cho, M. J.; Youn, H. S.; Ko, G. H.; Rhee, K. H. J. Bacteriol. 2004, 186, 949. (5) Hu, W. S.; Lin, Y. H.; Shih, C. C. Biochem. Biophys. Res. Commun. 2007, 361, 694. (6) Molloy, M. P.; Herbert, B. R.; Slade, M. B.; Rabilloud, T.; Nouwens, A. S.; Williams, K. L.; Gooley, A. A. Eur. J. Biochem. 2000, 267, 2871. (7) Pieper, R.; Huang, S. T.; Robinson, J. M.; Clark, D. J.; Alami, H.; Parmar, P. P.; Perry, R. D.; Fleischmann, R. D.; Peterson, S. N. Microbiology 2009, 155, 498. (8) Qi, S. Y.; Moir, A.; O’Connor, C. D. J. Bacteriol. 1996, 178, 5032. (9) Ruebush, S. S.; Brantley, S. L.; Tien, M. Appl. Environ. Microbiol. 2006, 72, 2925. (10) Rabilloud, T. Subcell. Biochem. 2007, 43, 3. (11) Santoni, V.; Molloy, M.; Rabilloud, T. Electrophoresis 2000, 21, 1054. (12) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. J. Proteome Res. 2002, 1, 351. (13) Coldham, N. G.; Woodward, M. J. J. Proteome Res. 2004, 3, 595. (14) Elias, D. A.; Monroe, M. E.; Marshall, M. J.; Romine, M. F.; Belieav, A. S.; Fredrickson, J. K.; Anderson, G. A.; Smith, R. D.; Lipton, M. S. Proteomics 2005, 5, 3120. (15) Wu, C. C.; Yates, J. R., III Nat. Biotechnol. 2003, 21, 262. (16) Zeng, X.; Roh, J. H.; Callister, S. J.; Tavano, C. L.; Donohue, T. J.; Lipton, M. S.; Kaplan, S. J. Bacteriol. 2007, 189, 7464. (17) Zhang, N.; Chen, R.; Young, N.; Wishart, D.; Winter, P.; Weiner, J. H.; Li, L. Proteomics 2007, 7, 484.

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