Global Analysis of the Membrane Subproteome of Pseudomonas aeruginosa Using Liquid Chromatography-Tandem Mass Spectrometry Josip Blonder,† Michael B. Goshe,‡ Wenzhong Xiao,§ David G. Camp II,| Mark Wingerd,| Ronald W. Davis,§ and Richard D. Smith*,| Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352 Received September 10, 2003
Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants, or cancer. Liquid chromatography coupled online with tandem mass spectrometry was used for the large-scale proteomic analysis of the P. aeruginosa membrane subproteome. Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins. The application of these approaches resulted in the identification of 786 proteins. A total of 333 proteins (42%) had a minimum of one transmembrane domain (ranging from 1 to14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32). Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of P. aeruginosa and for prokaryotes in general. Keywords: proteome • membrane proteins • low abundance • LC-MS/MS • affinity labeling
Introduction Pseudomonas aeruginosa is a gram-negative bacterium of increasing importance because it is one of the top three opportunistic pathogens in humans causing premature death in patients with cystic fibrosis, HIV infection, organ transplants, or cancer.1 It is the most common cause of various nosocomial infections including sepsis in burned or extensively injured patients. Its intrinsic adaptation ability complicates therapeutic strategies even more, with the emerging occurrence of P. aeruginosa strains exhibiting a high level of multidrug resistance to several classes of antibiotics.2 Infections caused by this pathogen have an adverse impact on the mortality and economics of the above-mentioned diseases and clinical conditions.3-8 The complexity of pathophysiological processes involved in P. aeruginosa pathogenicity is emphasized by the diversity of the diseases associated with this organism and is * To whom correspondence should be addressed. E-mail:
[email protected]. † SAIC-Frederick Inc., Laboratory for Proteomics and Analytical Technologies, Mass Spectrometry Center, National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702. Phone: (301) 846-7211. ‡ Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Campus Box 7622, Raleigh, NC 27695. § Stanford University School of Medicine, Stanford Genome Technology Center, Stanford, CA 94305. | Environmental Molecular Sciences Laboratory, P.O. Box 999, MSIN: K898, Richland, WA, 99352.
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indicative of the multiplicity of virulence factors that it is able to produce.9-14 Consequently, there is significant interest in the proteins responsible for this virulence, particularly those of the cellular membrane.15 The phenotypic resistance of gram-negative bacteria is a direct consequence of the complex structure of their cell envelope, which acts as a barrier and prevents drug molecules from reaching their target sites or increases their active efflux, primarily mediated by various classes of integral membrane proteins.16,17 It has been indicated that antibiotic resistance is due to integral outer membrane protein channels (porins) and membrane protein complexes known as multidrug efflux pumps that transport antibiotic and biocide molecules out of the cell.18 Currently, about seven hundred genes are implicated in the infection process with a significant number of them expected to code for membrane and secretory proteins.19 Large-scale analysis of hydrophobic integral membrane proteins from complex protein mixtures is an important and challenging aspect of mass spectrometry-based membrane proteomics. Although, large-scale proteomic studies based on in-gel digestion of proteins separated using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) have recently shown significant improvements in protein coverage, very hydrophobic integral membrane proteins are generally not amenable using this approach primarily due to the issues 10.1021/pr034074w CCC: $27.50
2004 American Chemical Society
P. aeruginosa membrane subproteome by LC-MS/MS
related to the insolubility of these proteins and precipitation occurring at their isoelectric points.20,21 In-solution digestion of membrane proteins and consecutive large-scale mass spectrometric analysis of chromatographically separated peptides offer an effective alternative to gel-based approaches. Although several in-solution based approaches have been recently developed22 more generally applicable methodologies need to be explored and improved. Recently, we have reported a method to extract, enrich, and solubilize highly hydrophobic integral membrane proteins to enable large-scale proteome analysis using microcapillary liquid chromatography-tandem mass spectrometry (µLC-MS/MS)23 which can be coupled to an affinity labeling technique to effectively target this protein class.24 In this study, we have combined these two approaches for a large-scale proteomic analysis of the membrane subproteome of P. aeruginosa.
Experimental Procedures Materials. P. aeruginosa strain PAO1 was obtained from Laurence G. Rahme at Massachusetts General Hospital, Boston, MA. (+)-Biotinyl-iodoacetamidyl-3, 6-dioxaoctanediamine (iodoacetyl-PEO-biotin) and Coomassie assay reagents were obtained from Pierce (Rockford, IL). Sequencing grade-modified trypsin was from Promega (Madison, WI). Phenylmethylsulfonyl fluoride (PMSF) was from Sigma (St. Louis, MO). Acetonitrile (HPLC grade) and formic acid (ACS reagent grade) were from Aldrich (Milwaukee, WI). Water was purified using a Barnstead Nanopure Infinity water purification system (Dubuque, IA). Bacterial Growth. Overnight culture of P. aeruginosa PAO1 strain was diluted 1:100 and grown to mid-exponential phase (OD600 ) 0.7) in Laura-Bertini liquid media at 37 °C with shaking (200 rpm). To stabilize the P. aeruginosa outer membrane structure, 3 mM MgCl2 was added to the culture. Cells were harvested by centrifugation (6000 × g, 15 min), washed three times with buffer (50 mM NH4HCO3, 1 mM PMSF), and frozen immediately using liquid nitrogen. Preparation of the Unlabeled and Labeled Membrane Protein Samples. Aminco French Press was used to lyse cells resuspended in 50 mM NH4HCO3 (14 000 psi, 3×), and unbroken cells were removed by centrifugation (2 500 × g, 20 min). A crude membrane was isolated by ultracentrifugation at maximum force of 151 000 × g for 1 h (Beckman Type 70 Ti rotor). The membrane pellet was thoroughly resuspended in 50 mM NH4HCO3 and was divided in two parts for the labeled and unlabeled sample preparations. The enrichment, extraction and solubilization of the membrane proteins were performed as described elsewhere.23 Briefly, the membranes were prefractionated using Na2CO3 treatment and pelleted by ultracentrifugation. The pellet was washed twice with double distilled water and resolubilized in 50 mM NH4HCO3. The membranes were pelleted again by ultracentrifugation, the supernatant was discarded, and then the membrane proteins were extracted and solubilized using intermittent sonication and vortexing in 60% methanol/40% 50 mM NH4HCO3. The Coomassie assay was used to determine membrane protein concentration. A standard curve for BSA protein standards prepared in the organic solution was generated between 0 and 0.5 µg/µL and used to determine the membrane protein sample concentration. Tryptic digestion in the same organic-aqueous buffer was immediately performed, then quenched by freezing the sample using liquid nitrogen. Prior to µLC-MS/MS analysis the peptide sample was purified
research articles and preconcentrated using a C18 solid-phase extraction column (Alltech, Deerfield, IL) in accordance to the manufacturer’s instructions, then lyophilized and resuspended in 60% methanol/ 40% water to a final concentration of 0.8 µg/µL. For the labeled sample, the enrichment, extraction, and solubilization of the membrane proteins were conducted as described above. Solubilized membrane proteins in the organicaqueous buffer were labeled with iodoacetyl-PEO-biotin and the biotinylated peptides were affinity isolated using immobilized avidin as previously described.24 The acetyl-PEObiotin labeled peptides were lyophilized and resuspended to a final concentration of 0.4 µg/µL for µLC-MS/MS analysis. µLC-MS/MS Analysis of Unlabeled and Labeled Peptide Mixtures. The unlabeled sample was analyzed using an inhouse manufactured, automated, high-pressure LC system described elsewhere.25 Briefly, a reversed-phase capillary column was made by slurry packing 5 µm Jupiter C18 stationary phase (Phenomenex, Torrence, CA) in a 150 µm i.d. × 360 µm o.d. × 65 cm capillary (Polymicro Technologies Inc., Phoenix, AZ). The mobile phases consisted of (A) 0.05% trifluoroacetic acid (TFA) in water and (B) 0.1% TFA in 90% acetonitrile/10% water. The LC system was equilibrated at 5000 psi with 100% mobile phase (A) prior to the sample injection. Each peptide sample was thawed to ambient temperature and 10 µL were loaded on the LC column. The exponential gradient was initiated 10 min after sample loading with a column flow of approximately 1.8 µL/min. The capillary LC system was coupled to an LCQ ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) using an in-house manufactured ESI interface without sheath gas or makeup liquid. The capillary temperature was 220 °C and electrospray voltage was 2.2 kV. The precursor ions were dynamically selected based on their intensity in the MS scan and subjected to collision-induced dissociation (CID) using the normalized collision energy setting of 45%. Each sample was analyzed using the data-dependent MS/MS mode over the m/z range 400-2000 followed by narrower m/z ranges (400-700, 700-900, 900-1100, 1100-1300, 1300-1500, 15001700, and 1700-2000). The labeled samples were analyzed using the Agilent 1100 Series capillary LC system (Agilent Technologies, Inc., Palo Alto CA) coupled to an LCQ Deca XP ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) using the in-house manufactured ESI interface described above. The reversed-phase separation was performed using an in-house slurry packed capillary column with the same stationary phase and dimensions as described above. The mobile phases consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in 90% acetonitrile/10% water. The lyophilized peptide sample was solubilized in 5% B/95% A to produce 0.4 µg/µL. After injecting a sample volume of 8 µL onto the column, a gradient program held the mobile phase at 5% B for 20 min, followed by a linear gradient to 70% B over 80 min, then a linear gradient to 85% B over 45 min. Each run was followed by reequilibration of the column at 5% B for 60 min prior to the next injection. The LCQ Deca XP was operated in the data-dependent MS/MS mode as described above using the following m/z ranges: 400-2000, 400-700, 700-900, 900-1100, 1100-1300, and 1300-2000. Peptide Identification. Both labeled and unlabeled peptides were identified by searching the MS/MS spectra against the complete annotated P. aeruginosa database (available at http:// www.pseudomonas.com/current_annotation.asp) using SEQUEST26 (Thermo Finnigan, San Jose, CA). All spectra generated from the labeled sample were analyzed using a dynamic mass Journal of Proteome Research • Vol. 3, No. 3, 2004 435
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Figure 1. Membrane protein sample preparation for µLC-MS/ MS analysis. The first step involves cell disruption, high pH carbonate treatment, and membrane isolation. Once the membranes have been isolated, the sample is divided into two aliquots. For one part, the membrane proteins are solubilized using methanol extraction followed by proteolysis and µLC-MS/ MS. For the other aliquot the membrane proteins are solubilized in the same way and cysteinyl-containing proteins are labeled using iodoacetyl-PEO-biotin and then digested with trypsin. The biotinylated peptides are captured using avidin affinity chromatography, eluted, and then analyzed by µLC-MS/MS.
modification on cysteinyl residues (i.e., both modified and unmodified forms of the residues) equal to the additional mass of the acetyl-PEO-biotin label (414.194 Da). Only tryptic peptides displaying a cross-correlation score (Xcorr) of at least 2.0 and delta-correlation score (∆Cn) of at least 0.1 were accepted for protein identification. For labeled peptides, the additional constraint of the presence of at least two label-specific fragment ions in the MS/MS spectrum27 (manually confirmed) was required for positive identification. Hydropathicity Calculations and Transmembrane Mapping. All identified proteins of P. aeruginosa strain PAO1 were analyzed using the ProtParam program (available at http:// www.expasy.ch/sprot/sprot-top.html) in order to calculate the grand average of hydropathicity (GRAVY) for each protein and selected peptides.28 The PSORT algorithm (available at http:// psort.nibb.ac.jp/form.html) was used to map the putative transmembrane domains (TMDs) of the identified proteins.29
Results and Discussion Membrane Protein Identification. The membrane subproteome of P. aeruginosa was probed using two techniques we developed specifically for MS identification of highly hydrophobic integral membrane proteins23,24 and is outlined in Figure 1. Both techniques were used concomitantly in the current investigation in order to achieve a more comprehensive analysis of membrane proteins than could be obtained by using only one approach. The first step involved cell disruption and membrane isolation. At this point, the sample was divided and one aliquot was prepared using the nonlabeling method and the other aliquot was prepared using the affinity labeling 436
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technique. The labeling strategy permits a reduction in sample complexity by enriching for only cysteinyl modified peptides and thus, increases the coverage of lower abundant membrane proteins. The number of membrane proteins identified using our approach is dependent on several factors. Enrichment of membrane proteins from the global cell lysate is critical and has been discussed in detail elsewhere.23,24 Once the membrane proteins are extracted and solubilized, a tryptic digestion is performed in the same buffer. Although, trypsin activity is attenuated in the 60% methanol-aqueous buffer (determined by N-benzoyl-L-arginine ethyl ester hydrolysis) the overall protein digestion efficiency is unaltered (as determined by SDSPAGE).30 The use of a one-dimensional chromatographic separation (reversed-phase liquid chromatography) to separate peptides of a complex mixture may limit the number of peptides identified by tandem mass spectrometry performed with a conventional ion trap. To alleviate this limitation and increase peptide coverage, MS/MS spectra were acquired using narrow m/z ranges to selectively target peptide ions for CID that were not captured using typical m/z precursor scanning ranges.31 Undoubtedly, incorporating a multidimensional chromatographic approach to separate the peptides prior to reversed-phase LC-MS/MS will further increase the number of identified proteins. A summary of the overall results using our approach on P. aeruginosa is presented in Table 1. When analyzed by SEQUEST all the collected MS/MS spectra of both experiments produced 62 511 dta files, yielding a total of 9951 fully tryptic peptides of which 2727 peptides were unique. From these data, a total of 786 protein identifications were observed: 623 proteins from the unlabeled sample and 163 proteins from the labeled sample (a comprehensive list of all identified proteins from both samples is available as Supporting Information). As illustrated in the Venn diagram in Figure 2, the set of 786 identified proteins contains 544 identified from the unlabeled sample and 84 from labeled sample, corresponding to 707 unique proteins. Using results presented in a previous report describing a global analysis of yeast proteins using multidimensional chromatography and database searching of MS/MS spectra using SEQUEST,32 the percentage of false positives associated with our study was determined to be no greater than 10%. This is based on (1) the similar size of the P. aeruginosa and the S. cerevisiae genomes (5770 and 6183 open reading frames, respectively) used in the database searching, and (2) the error associated with identifying peptides using our SEQUEST cutoff criteria. To increase the confidence of positive identification of cysteinyl-labeled peptides to greater that 90%, we added the additional requirement of the presence of at least two labelspecific ions in the MS/MS spectrum.27 When examining all identified proteins, a significant number of identifications of hydrophobic membrane proteins, especially those containing multiple TMDs, were observed. A total of 195 (25%) were classified as hydrophobic based on positive GRAVY values (ranging from 0.01 to 1.31) and 333 (42%) had at least one mapped TMD (ranging from 1 to 14). The PSORT algorithm analysis of the entire P. aeruginosa genome revealed that 1933 proteins contain at least one mapped TMD, representing 34% of all open reading frames. On the basis of these numbers, the overall enrichment of proteins containing at least one TMD relative to all those identified was 42% and 44% for the labeled and unlabeled sample, respectively. However, this percentage of enrichment using our approach may actually be
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P. aeruginosa membrane subproteome by LC-MS/MS Table 1. Proteins Identified from the Enriched Membrane Sample of P. aeruginosa Strain PAO1 identifications from unlabeled membrane samplea
identifications from labeled membrane sampleb
total
2087 623 161 0.01-1.31 274 1-14
640 163 34 0.01-1.02 59 1-12
2727 786 195 0.01-1.31 333 1-14
no. of detected peptides no. of identified proteins no. of hydrophobic proteins identifiedc GRAVY range of hydrophobic proteins no. of proteins with one or more mapped TMDsd range of mapped TMDs in identified proteins
a SEQUEST search parameters were set for the trypsin enzyme rule allowing two miscleavages. Only fully tryptic peptides producing SEQUEST results of Xcorr > 2 and ∆Cn of >0.1 were considered for conclusive protein identifications. b SEQUEST search parameters were set using a dynamic mass modification for acetyl PEO-biotin labeled cysteinyl residues. Only fully tryptic peptides producing SEQUEST results of Xcorr > 2 and ∆Cn of >0.1 were considered for identification. MS/MS spectra meeting these criteria and displaying at least two-label specific product ions (performed manually) were used for conclusive protein identification. c GRAVY value is calculated using ProtParam algorithm. Proteins and peptides showing positive values were classified as hydrophobic d Number of mapped transmembrane domains calculated by the PSORT algorithm.
producing an Xcorr of 3.9 and a ∆Cn score of 0.656. As shown in Figure 3, the peptide is completely embedded within the second mapped TMD. The detection of this peptide is possible due to disruption of phospholipid bilayer and solubilization of the entire membrane-spanning domain permitting enzymatic cleavage.
Figure 2. A Venn diagram illustrating the relationship between the total numbers of unique proteins identified for P. aeruginosa strain PAO1 from the unlabeled and labeled membrane protein samples.
higher since not all proteins containing a mapped TMD in the genome are necessarily expressed under the given experimental conditions. These levels of enrichment are similar to those we obtained with Deinococcus radiodurans as previously reported.23,24 Further analysis of the data show that 79 proteins were identified using both the labeled and unlabeled approach (Figure 2). This represents 48% of all labeled proteins identified and clearly indicates the efficiency of the labeling method to target membrane proteins. The labeling technique can be used with a variety of stable isotope and/or biotinylation reagents, including ICAT and ICAT-like reagents enabling quantitative profiling of very hydrophobic membrane proteins as well.32-34 Using other stable isotope labeling techniques, like 14N/15N metabolic labeling, will enable quantitation to be performed on both nonlabeled and labeled peptides.35,36 Thus, stable isotope labeling techniques coupled with our method outlined in Figure 1 can be a viable approach for comparative membrane proteomics of pathogenic bacteria.34,37 The performance of the applied techniques to identify membrane proteins is exemplified in Figure 3, which shows the MS/MS spectra of two (out of four) peptides identifying succinate dehydrogenase, sdhC (PA1581). This protein has not been previously identified in P. aeruginosa, but its homologue has been experimentally verified in Escherichia coli exhibiting a 72% sequence similarity.38 It is a very hydrophobic protein (calculated GRAVY of 0.82) containing 158 residues with three TMDs. PA1581 participates in the aerobic electron-transport pathway, which generates energy via oxidative phosphorylation and is the sole membrane-bound enzyme of the tricarboxylic acid cycle.39 The MS/MS spectrum in Figure 3A was generated from the CID of the [M+2H]2+ ion of the highly hydrophobic, fully tryptic peptide LVIWGLLSALLYHLVAGVR present in the unlabeled sample. The peptide has a GRAVY value of 1.70, and 22 out of 36 predicted y and b ions (61% coverage) were identified
The MS/MS spectrum in Figure 3B is the hydrophobic, membrane spanning peptide AC*LTSPLAK ([M+2H]2+ ion, Xcorr 2.8, ∆Cn 0.5, Gravy 0.257) from the labeled aliquot. This peptide spans the same TMD as the unlabeled peptide in Figure 3A, contains a cysteinyl-tagged residue, sequenced from the doubly charged parent ion, and displays 13 out of 16 predicted (80% coverage) y and b fragment ions. In addition, all labelspecific fragment ions, due to the partial CID of the acetylPEO biotin label, are observed and confirm affinity labeling. Importantly, all four subunits of succinate dehydrogenase sdhA (PA1583), sdhB (PA1584), sdhC (PA1581), and sdhD (PA1582) predicted by the genome sequence were confidently identified in the unlabeled sample out of which sdhC and sdhD are very hydrophobic. Subunits sdhA, sdhB, and sdhC were also identified in the labeled aliquot. Together these results reflect the efficiency of the techniques used in extracting, solubilizing and labeling, highly hydrophobic transmembrane peptides which lead to unambiguous identification of highly hydrophobic integral membrane proteins from complex mixtures using conventional one-dimensional µLC-MS/MS analysis. In an effort to better understand the scope of coverage of the membrane subproteome of P. aeruginosa using our approach (Figure 1), each identified protein was examined according to its classification, location, and role in antibiotic resistance and protection. As discussed below, many of these proteins have not been experimentally verified, but their ability to be probed using our membrane subproteomic approach holds promise for future comparative studies. Functional Classification of Identified Proteins. The 707 unique proteins identified from the nonlabeled and labeled samples were classified according to their function and are listed in Table 2.19 The highest percentage of identifications (35.5%) belongs to the category of hypothetical and conserved hypothetical proteins with 251 identifications, which is expected since 50% of the open reading frames of P. aeruginosa PAO1 have not been characterized. Out of the 251 hypothetical proteins identified, 97 (39%) have at least one mapped TMD (ranging from 1 to 14) and 47 (19%) are recognized as hydrophobic based on their positive GRAVY values (ranging from 0.1 to 1.2). The hypothetical membrane proteins identified in this study may be further investigated using biological techniques such as mutational analysis in an attempt to Journal of Proteome Research • Vol. 3, No. 3, 2004 437
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Figure 3. Mass spectrometric identification of succinate dehydrogenase (C subunit), the sdhC gene product of P. aeruginosa. (A) The MS/MS spectrum of the doubly charged peptide LVIWGLLSALLYHLVAGVR that produced an Xcorr of 3.9, a ∆Cn of 0.656, and displayed 22/36 ions (identified y and b ions/theoretical). This peptide is completely embedded within the second mapped TMD and exhibits a high positive GRAVY value of 1.684. (B) The MS/MS spectrum of the doubly charged cysteinyl-labeled peptide AC*LTSPLAK which produced an Xcorr of 2.81, a ∆Cn of 0.473, and displayed 13/16 ions. This peptide is hydrophobic as well (positive GRAVY value of 0.747) and partially covers the same TMD as the peptide in (A). In addition, all the label-specific product ions of the affinity label are observed in the MS/MS spectrum indicating labeling of the cysteinyl residue. This protein was also identified with two other peptides underlined in the protein sequence (MS/MS spectra not shown). This very hydrophobic integral membrane protein possessing a positive GRAVY value of 0.82 has three TMDs (bold italics). Table 2. All Unique Proteins Identified from the Enriched Membrane Sample of P. aeruginosa Strain PAO1 Sorted According to Their Functional Classes protein function
no.
percentage
hypothetical and conserved hypothetical transport of small molecules energy metabolism translation, post-translational modification, degradation putative enzymes transcriptional regulators cell wall/LPS/capsule two-component regulatory systems amino acid biosynthesis and metabolism adaptation, protection biosynthesis of cofactors, prosthetic groups and carriers motility and attachment chemotaxis carbon compound catabolism protein secretion/export apparatus cell division fatty acid and phospholipid metabolism DNA replication, recombination, modification and repair central intermediary metabolism secreted factors (toxins, enzymes, alginate) antibiotic resistance and susceptibility chaperones & heat shock proteins nucleotide biosynthesis and metabolism related to phage, transposon, or plasmid transcription, RNA processing and degradation total
251 94 52 42
35.5% 14.0% 7.3% 5.9%
41 23 22 20 17 16 16
5.7% 3.2% 3.1% 2.8% 2.4% 2.3% 2.3%
15 13 13 12 11 10 8
2.1% 1.9% 1.9% 1.6% 1.5% 1.4% 1.1%
7 5 5 5 4 3 2 707
0.9% 0.7% 0.7% 0.7% 0.5% 0.4% 0.2% 100%
characterize significant novel membrane proteins and discover potential pharmacological targets. The second largest class of identified proteins is a group of small molecule transporters (14%), which corresponds to 92 438
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identifications and represents remarkable enrichment of this protein class which comprises 4.5% of the entire complement of predicted open reading frames of the P. aeruginosa genome.19 Of these 92 identified proteins (which represents 36% of all predicted transporters in the sequenced genome database), 17 are annotated as outer membrane proteins and have been previously identified in P. aeruginosa or other bacteria using nonmass spectrometric-based studies.19 The remaining 75 proteins are annotated as integral inner (cytoplasmic) membrane proteins involved in the transport of small molecules. The most numerous transporter families identified are (1) the ABC transporters with 17 distinct identifications, (2) the MFS transporters, and (3) the transporters involved in iron metabolism. The third largest class belongs to those proteins involved in energy metabolism with 53 identifications, of which 44 proteins were previously identified and 9 are identified for the first time. In fact, of the 707 proteins identified in this study, 265 of them were previously identified using nonmass spectrometric-based experimental methods,19 indicating that 442 (63%) were identified (perhaps for the first time) using an MS-based approach. Integral Cytoplasmic Membrane Proteins. Despite the abundance and biological significance of membrane proteins, the proteome-wide analysis of highly hydrophobic, integral cytoplasmic membrane proteins has remained a challenge for MS-based investigations,22 particularly for 2-D PAGE based approaches.21 Regardless, whether they are single-pass or multiple-pass, the extraction and solubilization of transmembrane spanning proteins can only be achieved by disruption of the phospholipid bilayer. Besides, it is equally critical to keep the extracted proteins solubilized throughout the digestion in order to prevent their aggregation and consequent loss. On the basis of the results listed in Table 3, our technique accomplishes both goals.
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Table 3. Selected Subset of Identified Integral Inner (cytoplasmic) Membrane Proteins Identified from the Enriched Membrane Sample of P. aeruginosa Strain PAO1 PA no.
protein description & peptide sequence
classa
GRAVYb
PA0341
prolipoprotein diacylglyceryl transferase, lgt K.SFFQLMDFIAPLVPIGLGAGR.I sodium/proline symporter, putP R.VQTEHNGNALTLPDYFTNR.F probable cytochrome oxidase subunit (cbb3-type) R.AVNEDGTLTYSFVEALVASHPGFIVR.M probable cytochrome oxidase subunit R.AVNEDGTLTYSFVEALAAGHPGFIVR.M probable transporter (membrane subunit)** R.NPYPLVLTC*LR.E NADH dehydrogenase I chain H, nuoH R.VGPFGAFQLGADMVK.M heat shock protein, htpX K.MPEVGIFPAYEANAFATGWNKNDALVAVSQGLLER.F probable chemotaxis transducer R.SNLLPLEQAASLGGTAAQALGK.G probable amino acid permease K.LPTFDASQFPEVQQK.L homoserine dehydrogenase, hom** K.VGIC*GLGTVGGGTFNVLER.N probable MFS transporter R.IPFLLSAALVVVGLYVR.L apolipoprotein N-acyltransferase, lnt R.GLIAFFDLPMSDFAR.G secretion protein, secY R.QNEGTILSLFNMFSGGALER.M probable cytochrome b K.QIISLATIQSPLGNQFR.I probable permease of ABC transporter R.STLFLGHALLGR.R probable two-component sensor** R.YQC*LGLGSAPR.L probable permease of ABC transporter R.HLILPAIVLGTIPLAVIAR.M probable permease of ABC transporter R.FLLGTDELGRDLLSR.L conductance mechanosensitive channel, mscL K.REEAVAPSEPPVPSAEETLLTEIRDLLK.A ribose-phosphate pyrophosphokinase, prs** K.SVLDELVVTNTIPLSAAAQAC*GR.I chemotaxis protein, motA R.FTQTYYLEVLGMLYEILNK.S glucans biosynthesis protein, mdoH K.AAVDPLLNALAC*AMGTAR.H two-component sensor, envZ** R.VC*LPLGLLLPR.G ATP-binding/permease fusion ABC transporter** R.GEIFGFLGSNGC*GK.S proton-glutamate symporter, gltP K.AWWIANVLQPAGDIFIR.L
2
0.526 1.038 0.749 -0.932 0.563 0.442 0.588 0.365 1.024 0.445 0.867 0.640 0.417 0.066 0.061 0.227 0.748 -0.713 0.135 0.774 0.809 2.018 0.364 0.780 0.576 -0.015 0.562 0.171 0.762 0.817 0.195 -0.55 0.656 1.842 0.739 0.000 0.693 -0.475 0.097 0.639 0.449 0.263 0.019 0.922 -0.085 1.600 0.101 0.193 0.851 0.641
PA0783 PA1554 PA1557 PA2042 PA2643 PA2830 PA2867 PA3641 PA3736 PA3749 PA3984 PA4243 PA4430 PA4455 PA4494 PA4503 PA4504 PA4614 PA4670 PA4954 PA5077 PA5199 PA5231 PA5479
2 3 3 3 2 2 3 3 1 3 2 2 3 3 3 3 3 2 2 2 2 2 3 2
TMDsc
nd
4
4
11
2
9
3
9
3
9
2
8
2
2
2
2
3
8
5
1
2
10
2
6
2
10
2
9
5
4
2
4
2
5
5
7
3
2
3
1
3
4
2
6
3
2
2
6
4
10
2
chge
Xcorrf
2
2.6
2
3.3
3
3.9
2
4.4
2
2.3
2
4.5
3
3.9
2
4.4
2
3.6
2
3.5
2
2.9
2
3.5
2
4.3
2
4.7
2
2.9
2
3.0
2
4.6
2
3.1
3
4.9
3
3.7
2
3.6
2
4.1
2
2.3
2
2.3
2
3.7
a
Class assigned according to the pseudoCAP database. 1-Function experimentally demonstrated in P. aeruginosa. 2-Function of highly similar gene experimentally demonstrated in another organism. 3-Function proposed based on presence of conserved amino acid motif, structural feature or limited sequence similarity to an experimentally studied gene. 4-Homologues of previously reported genes of unknown function, or no similarity to any previously reported sequences. b GRAVY value is calculated using ProtParam algorithm. Proteins and peptides showing positive values were classified as hydrophobic. c Number of mapped transmembrane domains (TMDs) predicted by the PSORT algorithm. d Number of peptides detected/identified protein. e Charge state of the precursor ion selected for CID. f Cross-correlation score (Xcorr) of the peptide is based on the match of the obtained MS/MS spectra to the theoretical ion distribution for the corresponding peptide contained in the database. * Labeled cysteinyl residue. ** Proteins identified from the labeled sample only.
The majority of integral cytoplasmic membrane proteins listed in Table 3 was identified by hydrophobic peptides mapped by the PSORT algorithm. These transmembrane spanning peptides typically possess high GRAVY values, indicating their remarkably high hydrophobicity. The corresponding SEQUEST results and the multiple number of peptides identified for each protein indicate unambiguous identification of these hydrophobic integral membrane proteins. The evidence presented in Figure 3 and in the supporting tables indicates that the methodology used in this study represents a simple and effective technique for qualitative analysis of this protein class, and once coupled with one of the quantitative techniques
involving stable isotope labeling,37 it will permit a more comprehensive proteomic analysis of membrane protein abundance. Transferases and Cytochromes. Prolipoprotein diacylglyceryl transferase (PA0341) is a hydrophobic integral membrane protein with four mapped TMDs. This protein, not previously identified in P. aeruginosa, was characterized by identifying four peptides and is listed in Table 3. The peptide SFFQLMDFIAPLVPIGLGAGR is very hydrophobic and completely covers the second mapped TMD. PA0341 is an essential factor for the growth, division, and viability of bacterial cells at nonpermissive temperatures. Homologues were identified in Escherichia coli, Journal of Proteome Research • Vol. 3, No. 3, 2004 439
research articles Salmonella typhymurium, and Haemophilus influenzae.40 PA0341 is encoded by the lgt gene and is involved in fatty acid and phospholipid metabolism, translation, and post-translational modifications.41 As a cytoplasmic membrane enzyme, it is a part of the sequential catalysis that involves prolipoprotein signal peptidase and the lnt gene which encodes for apolipoprotein N-acyl transferase (PA3984)40 which is also listed in Table 3. PA3984 has six mapped TMDs and is involved in the formation of lipid-modified proteins. The protein modification is achieved by transferring the diacylglycerol group from phosphatidyl-glycerol to the sulfhydryl group of a cysteinyl residue present at the C-terminal portion of the signal sequence of the protein.40 MdoH and mdoG genes are necessary for the synthesis of membrane-derived oligosaccharides that occupy the periplasmic space of gram-negative bacteria and for glucosyl transferase activity. MdoH codes for an integral membrane protein expressing 76% homology with the mdoH gene of E. coli whose protein spans the cytoplasmic membrane and is required for the expression of disease symptoms and development of hypersensitive reaction on nonhost plants.42 The mdoH protein of P. aeruginosa (PA5077) was detected by three peptides from both the unlabeled and labeled samples, where the labeled peptide is listed in Table 3. Three proteins (PA1554, PA1557, and PA4430) from the cytochrome super family were also identified by hydrophobic peptides (Table 3). These proteins were not previously identified in P. aeruginosa or other prokaryotes according to the current P. aeruginosa Community Annotation Project Database. In addition 14 other proteins from this super family (presented in the Supporting Information) were identified. Additional proteins, not previously identified in P. aeruginosa until applying our MS-based approach, include NADH dehydrogenase I chain H (PA2643) and heat shock protein htpX (PA2830) whose homologues were characterized previously in E. coli exhibiting 84% and 63% sequence similarity, respectively.19,43,44 Proteins Involved in Chemotaxis and Osmostasis. P. aeruginosa is able to swim using a single polar flagellum and exhibits chemotaxis toward various chemical stimuli including L-amino acids.45 PA2867 is annotated as a probable chemotaxis transducer (Table 3) and was identified by three peptides. This protein belongs to the group of methyl-accepting chemotaxis proteins, which are the best-characterized signal transducers of the chemosensory apparatus. However, the physiology of these mechanisms is still poorly understood at the molecular level. It has been found, using mutant phenotypic complementation experiments, that the presence of this protein is necessary for chemotaxis toward glycine, L-serine, L-threonine, and L-valine.45 The chemotaxis protein motA (PA4954) is essential for motor rotation and the topology of its homologue (65% sequence similarity) was experimentally determined in E. coli. This protein has four mapped TMDs, each of them approximately 20 residues in length46 and was identified by two peptides (Table 3). The conductance mechanosensitive channel (mscL) (PA4614) belongs to the family of proteins that are able to transduce changes in membrane tension due to an ion flux across the membrane and achieve optimal osmostasis. PA4614 has 97% similarity to the mscL gene product of Pseudomonas fluorescens and is considered a low abundance protein of significant importance in the adaptation and protection of bacteria against adverse conditions resulting in either sudden desiccation or other osmotic insult.47 This 137-residue long integral membrane 440
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protein contains two mapped TMDs and was confidently identified by three peptides exhibiting high Xcorrs (Table 3). Transporters. A probable amino acid permease (PA3641) and a probable MFS transporter (PA3749), not previous identified in P. aeruginosa, are presumed to be involved in the transport of small molecules. PA3749 permits the utilization of citrate as a sole carbon source by facilitating the uptake of citrate across the membrane with concomitant proton export. The function of these two proteins was annotated based on homology analysis.48,49 Both proteins are very hydrophobic and contain multiple TMDs. PA3749 was identified by one of its transmembrane spanning peptides (Table 3). Gram-negative bacteria must transport proteins to and from different cell wall compartments including the inner membrane, the outer membrane, and the periplasmic space. Several proteins catalyze these processes and the secretion protein secY (PA4243) identified in this study is an important factor in the translocation cascade, based on its 79% homology to secY gene product of E. coli.50 The presence of this protein was not previously verified in P. aeruginosa. The ABC (ATP-binding cassette) transporters are an essential part of the active transport system of the bacterial cell and are also present in archaea and eukaryota.51 They are multicomponent systems consisting of two integral membrane proteins (permeases) each having a variable number of transmembrane segments: two peripheral membrane proteins that bind and hydrolyze ATP, and a periplasmic substrate-binding protein. Overexpression of certain ABC transporters is a frequent cause of resistance to antibiotics and biocides, and thus, there is significant interest in their identification.52 In our study, 21 proteins from this class have been identified with three of them listed in Table 3 (PA4455, PA4503, and PA4504). As observed with many of the proteins described thus far, all three proteins were detected by hydrophobic peptides. Symporters. Integral membrane proteins that regulate the influx of a wide variety of molecules with the concomitant uptake of ions (symporters) belong in a number of distinct families. In P. aeruginosa the proton-glutamate symporter (PA5479) was unambiguously identified by two peptides (Table 3). This protein has an 87% sequence similarity with the experimentally identified E. coli proton-glutamate transporter and has 10 predicted TMDs.53 The sodium/proline symporter (putP) (PA0783) listed in Table 3 was previously characterized in E. coli and P. fluorescens with 86% and 91% homology, respectively, and causes proline accumulation which is coupled with the Na+ electrochemical gradient across the cytoplasmic membrane.54 PA0783 is composed of 70% nonpolar amino acids and contains 11 putative TMDs. This very hydrophobic protein was identified by two peptides with the one listed in Table 3 displaying a remarkably high Xcorr of 3.3 for the [M+2H] 2+ precursor ion. Integral Outer Membrane Proteins. The integral outer membrane proteins of gram-negative bacteria that are embedded in the phospholipid bilayer are major regulators of the integrity and selective permeability of the outer membrane. They contribute approximately 50% of the mass of the outer membrane, mediating the flux of small molecules across the outer membrane,16 and function as a permeability barrier against various hydrophilic antibiotic molecules as well. Porins are trimeric channel complexes that facilitate transport of hydrophilic molecules through the membrane. The expression and size of these channels are often influenced by external stimuli.55 Because these membrane proteins are not very
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Table 4. Selected Subset of Identified Integral Outer Membrane Proteins Identified from the Enriched Membrane Sample of P. aeruginosa Strain PAO1 PA no.
protein description and peptide sequence
classa
GRAVYb
nc
PA0291
anaerobically induced outer membrane porin, oprE R.LLPVTFEGGQVTSTDLKDFTLVAGQLEHSKGR.N outer membrane protein, oprM precursor R.SLRDQALEQYLATEQAQR.S organic solvent tolerance protein, ostA precursor R.TESGSAIFHYQPEVDPGK.V R.C*EPSSNAWTLK.G basic peptide and imipenem outer membrane porin, oprD R.YDLNLASYGVPGLTFMVR.Y outer membrane protein, oprL precursor R.YLVLQGVSPAQLELVSYGK.E probable outer membrane protein R.LSITGHTDSVGSDAYNQK.L conserved hypothetical protein K.TPIGSIAGAGVGGVAGSAVGGGK.G probable outer membrane lipoprotein R.ALLAVDIDKVRLEGHTDNYGDEGYNQK.L outer membrane protein, oprF precursor K.VKENSYADIKNLADFMK.Q K.C*PDTPANVTVDANGC*PAVAEVVR.V outer membrane lipoprotein, oprI precursor K.FSALALAAVLATGC*SSHSK.E probable outer membrane protein R.AQAVADVLADLGVDPAR.M general secretion pathway protein D, xcpQ K.TNFANTGLSIGTLLQSLESNK.A probable peptidyl-prolyl cis-trans isomerase K.QQLTDEELTEAFAFLQK.R probable outer membrane protein R.YNFGFVDPYWTVDGVSLGYNAFYR.K R.TVLFWDVGSTFDTDC*PTK.T probable outer membrane protein R.NIQQLAEFLQQNPER.Q Fe(III) dicitrate transport protein, fecA R.SQALDGEYTLEEALAALLVGSGLEAR.A Fe(III)-pyochelin receptor precursor, fptA R.GSNGLLHGTGNPAATVNLVR.K outer membrane lipoprotein, omlA R.VSLFFNDSDQLAGLNGDFMPGVSRDEAILGK.E type 4 fimbrial biogenesis protein, pilO R.IVTLHDFEIKPVAPGSTSK.L
1
-0.436 -0.159 -0.215 -1.803 -0.599 -0.856 -0.691 -0.466 -0.383 -0.432 0.453 -0.070 -0.706 0.108 0.757 -0.315 -0.874 -0.443 -0.618 0.200 -0.541 0.974 -0.391 0.512 -0.123 -0.114 -0.348 -0.541 -0.368 -0.096 -0.033 -0.574 -1.160 -0.489 0.219 -0.554 -0.010 -0.517 0.000 -0.004 0.063
5
PA0427 PA0595 PA0958 PA0973 PA1041 PA1053 PA1119 PA1777 PA2853 PA2900 PA3105 PA3262 PA3648 PA3692 PA3901 PA4221 PA4765 PA5042
1 2 1 1 3 4 3 1 1 3 1 3 3 3 2 1 1 1
chgd
Xcorre
3
4.0
2
6.4
2 2
4.2 2.4
2
3.3
2
4.8
2
4.9
2
6.0
3
4.2
2 2
5.6 4.3
2
3.7
2
4.7
2
4.9
2
4.9
2 2
4.4 3.9
2
4.6
2
3.9
2
4.3
3
4.5
2
4.6
8 9 3 10 5 5 3 12 8 4 4 5 7 7 6 2 5 4
a
Class assigned according to the pseudoCAP database. 1-Function experimentally demonstrated in P. aeruginosa. 2-Function of highly similar gene experimentally demonstrated in another organism. 3-Function proposed based on presence of conserved amino acid motif, structural feature or limited sequence similarity to an experimentally studied gene. 4-Homologues of previously reported genes of unknown function, or no similarity to any previously reported sequences. b GRAVY value is calculated using ProtParam algorithm. Proteins and peptides showing positive values were classified as hydrophobic c Number of peptides detected/identified protein. d Charge state of the precursor ion selected for CID. e Cross-correlation score (Xcorr) of the peptide is based on the match of the obtained MS/MS spectra to the theoretical ion distribution for the corresponding peptide contained in the database. * Labeled cysteinyl residue.
hydrophobic, they are readily identified in 2-D PAGE based studies20,56 and were also identified using our approach. Table 4 contains a subset of the identified integral outer membrane proteins from both the labeled and unlabeled samples. Each identified protein was characterized by multiple peptides, producing remarkably high Xcorr values, indicating unambiguous SEQUEST based protein identification. We have confidently identified 7 (53%) (oprC, oprD, oprE, oprF, oprI, oprL, and oprM) out of all the predicted porins in the annotated genome of P. aeruginosa representing the highest number of identified porins from P. aeruginosa obtained from a single proteomic study. OprI and oprF were identified from both samples while oprC was identified only from the labeled sample. A total of 9 proteins (labeled as class 2, 3, and 4 in Table 4) were not previously experimentally verified in P. aeruginosa.19 For example, Fe (III) dicitrate transport protein (fecA, PA3901) is an outer membrane receptor involved in iron metabolism and its homologue (exhibiting 75% sequence similarity) was formerly characterized in E. coli.57 These examples of unequivocal identification of integral outer mem-
brane proteins demonstrate the ability and applicability of the reported technique for qualitative and potential quantitative analyses of less hydrophobic integral membrane proteins with similar or better efficiencies than 2-D gel-based proteomic studies. Antibiotic Resistance, Adaptation, and Protection Proteins. Since the most important targets of antibacterial agents are located within the cytoplasmic membrane or cytoplasm, the outer membrane presents a natural barrier to antibiotics and biocides preventing their access to target sites.58 Porins or channel-type proteins are important factors of antibiotic resistance of P. aeruginosa because they prevent the passage of hydrophilic antibiotics via a size exclusion mechanism, whereas the dense lipopolysaccharride bilayer of the outer membrane slows down the penetration of hydrophobic macromolecules inside the cell.16,59 However, the lower permeability of the outer membrane alone cannot completely protect the microbe since the periplasmic concentration of certain antibiotics quickly reaches 50% of their extracellular concentration. Therefore, additional cellular mechanisms are necessary to Journal of Proteome Research • Vol. 3, No. 3, 2004 441
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Table 5. Partial List of Identified Antibiotic Resistance Proteins and Their Corresponding Peptides from P. aeruginosa Strain PAO1 protein description and peptide sequences
PA0425
PA0426
PA0427
PA1032
RND multidrug efflux membrane fusion protein, mexA R.VIGDKWLVTEGLNAGDKIITEGLQFVQPGVEVK.T K.AGQQLYQIDPATYEADYQSAQANLASTQEQAQR.Y R.IAEVRPQVNGIILK.R R.AVFPNPNNELLPGMFVHAQLQEGVK.Q RND multidrug efflux transporter, mexB K.GVGDFQVFGSQYSMR.I R.YNGVPAMEILGEPAPGLSSGDAMAAVEEIVK.Q R.QTIANLEPFMPQGMK.V R.LQTAEQFENILLK.V major intrinsic multiple antibiotic resistance efflux outer membrane protein precursor, oprM R.SLRDQALEQYLATEQAQR.S R.RPDILEAEHQLMAANASIGAAR.A K.AIQTAFQEVADGLAAR.G R.QLSGLFDAGSGSWLFQPSINLPIFTAGSLR.A probable penicillin amidase R.LIVDFGQSEPMIGVNSSGQSGNPASPHYADGIDAWLK.G R.SIQQEADKTLDGFFDLSR.A R.HGPLLNSALGER.K K.SLMANDTHLPLSMPSVWNYVQIR.S
chga
Xcorrb
∆Cnc
ionsd
3 3 2 3
5.2 2.8 2.7 2.6
0.587 0.359 0.312 0.409
42/128 28/128 14/26 27/96
2 2 2 2
3.0 2.8 2.7 2.6
0.596 0.513 0.454 0.299
13/28 23/60 14/28 14/24
2 3 2 2
6.4 6.2 4.2 4.0
0.632 0.693 0.623 0.657
24/34 24/42 23/30 19/58
3 2 2 2
4.5 4.2 2.4 2.0
0.513 0.529 0.400 0.179
40/144 21/34 14/22 13/44
GRAVYe
TMDf
-0.229 0.148 -1.036 0.471 -0.084 0.274 -0.213 0.252 -0.367 -0.123 -0.215
10
-1.083 -0.145 0.294 0.370 -0.410 -0.230 -0.700 -0.392 -0.013
0
0
0
a Charge state of the precursor ion selected for CID. b Cross-correlation score (Xcorr) of the peptide is based on the match of the obtained MS/MS spectra to the theoretical ion distribution for the corresponding peptide contained in the database. c A calculated “difference” between the top two Xcorr values for the given peptide. d The total number of identified b and y ions/theoretical. e GRAVY value is calculated using ProtParam algorithm. Proteins and peptides showing positive values were classified as hydrophobic. f Number of mapped transmembrane domains (TMDs) predicted by the PSORT algorithm.
Table 6. Partial List of Identified Proteins from both Unlabeled and Labeled Samples Belonging to the Adaptation and Protection Functional Class PA no.
protein description and peptide sequences
classa
GRAVYb
PA0595
organic solvent tolerance protein, ostA precursor R.ISDPYYFQDLDTDLGVGSTTYVNQR.G R.C*EPSSNAWTLK.G heat shock protein, htpX K.MPEVGIFPAYEANAFATGWNKNDALVAVSQGLLER.F R.TRHEQWLLQTVEELSR.E bacterioferritin, bfrB R.EAIVHC*EQVHDYVSR.D K.ILGNELIAINQYFLHSR.M bacterioferritin, bfrA K.GIALC*EQHKDFVSR.D K.AQLADTEEDHAYWLEQQLGLIAR.M conserved hypothetical protein K.IFDSNPAVGFVALGEK.Y K.YATALDAGQVLAPAK.D osmotically inducible lipoprotein. osmE R.LMKPGSC*NSYILNK.D R.LMKPGSC*NSYILNKDGQQQPFYVSFDGSGK.V
2
-0.599 -0.628 -0.691 0.417 0.066 -1.075 -0.520 -0.373 0.312 -0.504 -0.264 -0.526 0.792 0.519 0.393 -0.489 -0.229 -0.683
PA2830 PA3531 PA4235 PA4606 PA4876
2 2 1 4 2
TMDc
chgd
Xcorre
2 2
4.6 2.3
3 2
3.9 2.9
2 2
3.0 2.8
2 2
3.3 2.1
2 2
2.3 2.3
2 3
2.8 2.2
0 2 0 0 14 0
a Class: according to pseudoCAP database. 1-Function experimentally demonstrated in P. aeruginosa. 2-Function of highly similar gene experimentally demonstrated in another organism. 3-Function proposed based on presence of conserved amino acid motif, structural feature or limited sequence similarity to an experimentally studied gene. 4-Homologues of previously reported genes of unknown function, or no similarity to any previously reported sequences. b GRAVY value is calculated using ProtParam algorithm. Proteins and peptides showing positive values were classified as hydrophobic. c TMD Number of mapped transmembrane domains by PSORT algorithm. d Charge state of the precursor ion selected for CID. e Cross-correlation score (Xcorr) of the peptide is based on the match of the obtained MS/MS spectra to the theoretical ion distribution for the corresponding peptide contained in the database. * Labeled cysteine residue.
create the high level of antibiotic resistance exhibited by P. aeruginosa.59,60 Hydrolytic enzymes possessing β-lactamase activity are omnipresent within the periplasmic space, and as shown in Table 5, we confidently identified a probable penicillin amidase (PA1032) by detecting multiple peptides. Notably, this enzyme was not previously characterized in P. aeruginosa and could serve as a viable pharmacological target. Active transport of antibiotics and biocides to the outside of the cell is another known mechanism that further increases the resistance ability of gram-negative bacteria.55 Particular types of multidrug transporters are assembled as membrane complexes and may belong to a major facilitator family of transporters or to the resistance-nodulation-division (RND) 442
Journal of Proteome Research • Vol. 3, No. 3, 2004
family. When highly expressed, these drug efflux pumps produce an impressive resistance to a variety of antibiotics. For example, the RND family can excrete drug molecules directly into the extracellular space and consequently cause a decrease in the intracellular drug concentration.13 Each membrane protein complex contains at least three subunits (a transporter protein located within the membrane, an outer membrane channel, and a periplasmic linker) and characteristically displays extremely broad substrate specificity. The mexB gene encodes for a transporter protein containing 10 mapped TMDs and is a part of the mexA-mexB-oprM protein complex system.13 The mexB transporter was unambiguously identified by 4 peptides using our approach. As shown in Table 5, we
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have confidently identified the entire mexA-mexB-oprM efflux system. To our knowledge, this is the first report of an unambiguous mass spectrometric characterization of a transmembrane protein complex from a compound protein mixture using one-dimensional µLC-MS/MS. Table 6 shows a partial list of the adaptation and protection proteins identified from both the unlabeled and labeled protein preparations. The majority of the peptides listed contain cysteinyl residues and thus, their expression level under various conditions or insults can be quantified using ICAT or ICAT-like alkylating reagents. Those proteins that change in relative abundance may be potential targets for pharmacological agents. This strategy is particularly applicable to bacterioferritins bfrA (PA4235) and bfrB (PA3531) since they belong to the small molecule transporter class and are involved in iron transport, a function that is an absolute requirement for gram-negative bacterial survival.
Conclusion This work presents a global qualitative analysis of the membrane subproteome of P. aeruginosa strain PAO1 in which hydrophobic membrane proteins are confidently identified on a comprehensive, large-scale basis using µLC-MS/MS. Several biologically and medically significant classes of integral membrane proteins involving antibiotic resistance, adaptation, and susceptibility were identified in this investigation regardless of their hydrophobicity and membrane location. Many of these identifications represent the first time these proteins have been detected experimentally. Although the results reported here are subject to further confirmation, the set of proteins identified in this study suggest specific candidate proteins for various mutational analyses to target genes encoding for integral membrane proteins of potential interest. A follow-up study is currently in process to systematically screen for protein factors important in virulence, biofilm formation, and antibiotic resistance using a similar strategy applied to highly purified outer membrane samples isolated by sucrose density gradients. Importantly, the techniques presented in this work to target highly hydrophobic membrane proteins can be extended and incorporated into multidimensional chromatographic separations and quantitative profiling using various stable isotope labeling proteomic strategies.
Acknowledgment. We thank Drs. L.G. Rahme, M. Mindrinos, and M. Bains for helpful discussions. This work is funded in part by NIH P01 HG00205 to R.W. Davis. A portion of the research described in this manuscript was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the U.S. Department of Energy under Contract No. DEAC06-76RLO 1830. Supporting Information Available: Supporting Table 1: Comprehensive list of all identified proteins of P. aeruginosa strain PAO1 from the unlabeled membrane protein sample. Supporting Table 2: Comprehensive list of all identified proteins of P. aeruginosa strain PAO1 from the labeled membrane protein sample. This material is available free of charge via the Internet at http://pubs.acs.org.
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