Characterization of the Outer Membrane Protein Profile from Disease-Related Helicobacter pylori Isolates by Subcellular Fractionation and Nano-LC FT-ICR MS Analysis Elisabet Carlsohn,†,* Johanna Nystro1 m,† Hasse Karlsson,† Ann-Mari Svennerholm,† and Carol L. Nilsson‡ Institute of Biomedicine, Sahlgrenska Academy, Go¨teborg University, Sweden, and National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida Received April 20, 2006
Abstract: Because of the important role of membrane proteins in adhesion, invasion, and intracellular survival of pathogens in the host, membrane proteins are of potential interest in the search for drug targets or biomarkers. We have established a mass spectrometry-based method that allows characterization of the outer membrane protein (OMP) profile of clinical isolates from of the human gastric pathogen Helicobacter pylori. Subcellular fractionation and one-dimensional gel electrophoresis (1D-GE) analysis was combined with nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) and tandem mass spectrometry (MS/MS) analysis of fifteen H. pylori strains associated either with duodenal ulcers, gastric cancer, or isolated from asymptomatic H. pylori infected carriers. Over 60 unique membrane or membrane-associated proteins, including 30 of the 33 theoretically predicted OMPs, were identified from the strains. Several membrane proteins, including Omp11 and BabA, were found to be expressed by all strains. In the search for clinical markers we found that Omp26 was expressed by all disease-related strains but was only present in one out of five strains from asymptomatic carriers, which makes Omp26 a potential target for further investigation in the search for proteins unique to disease-related H. pylori strains. In addition, presence of Omp30 and absence of Omp6 seemed to be associated with H. pylori strains causing duodenal ulcer. Keywords: OMP • H. pylori • nano-LC • FT-ICR MS • biomarkers • vaccine antigen • outer membrane
Introduction Proteomics, the study of the proteome, is the scientific discipline that is used to bridge the gap between the understanding of the genome sequence of an organism and cellular processes. Traditionally, proteomics is performed by protein * Corresponding author:
[email protected], Phone +46-31-7733482, Fax +46 31 41 61 08. † Institute of Biomedicine. ‡ National High Magnetic Field Laboratory. 10.1021/pr060181p CCC: $33.50
2006 American Chemical Society
separation with 2-D gel electrophoresis (2D-GE) and protein identification by mass spectrometry (MS). The 2D-GE approach, however, suffers from several drawbacks, such as discrimination against membrane proteins and limited detection of low-abundance proteins, and it is therefore a high priority in proteome research to overcome these restrictions by developing alternative separation methods. The coupling of nano-LC to mass spectrometers equipped with an electrospray ionization (ESI)1 ion source has dramatically improved the sensitivity, the number of detected peptides,2 the reproducibility, and the dynamic range for analysis of peptides from complex biological samples.3 Mass spectrometric analysis of heterogeneous peptide mixtures requires high resolving power mass accuracy, sensitivity, and mass range. The FT-ICR4 mass spectrometer fulfills all of these criteria5-7 and is an excellent tool for analysis of complex samples from biological sources, especially when it is combined with LC coupled to an ESI source. The human gastric pathogen Helicobacter pylori can cause acute chronic gastritis, duodenal ulcers (DU) or gastric cancer in a subpopulation of infected individuals.8-11 To establish colonization and maintain infection in the human gastric mucosa, the bacterium expresses a variety of different types of virulence factors, which include a number of outer membrane proteins and adhesins. The best characterized adhesins are the blood group antigen-binding adhesin (BabA) and the sialic acid-binding adhesin (SabA), which bind to Lewisb and sialyl-Lewisx receptors, respectively.12,13 Three additional proteins have been proposed to be involved in adherence of H. pylori to gastric epithelial cells. These are the closely related adherence-associated proteins AlpA and AlpB and the outer membrane protein HopZ.14-16 The H. pylori adhesin A (HpaA) was first identified as a sialic acid-binding adhesin, but was subsequently identified as a lipoprotein. A recent study did however show that HpaA is an important colonization factor essential for H. pylori colonization in mice.17 This finding has made HpaA an interesting candidate in the search for potential vaccine antigens that can be used to prevent H. pylori infection. The genome sequence of the H. pylori strains 26695 and J99 contain one major outer membrane family of thirty-three (thirty-two in J99) genes, which encode for H. pylori outer membrane proteins,18,19 Figure 1. This family is divided into two subfamilies; the H. pylori outer membrane protein (Hop) and the Hop-related Hor families.20 The five adhesins menJournal of Proteome Research 2006, 5, 3197-3204
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Outer Membrane Protein Profile of Helicobacter pylori Isolates
Figure 1. Phylogenetic tree of the two major outer membrane protein families, Hop and Hor. The Hop family consists of 21 members including all identified adherence-related proteins (BabA, SabA, AlpA, AlpB, and HopZ). The Hor family consists of 12 proteins with unknown function. The HopJ/HopK and HopM/ HopN are proteins with identical amino acid sequence. The phylogenic tree is modified from ref 6.
tioned above are all Hop proteins and three of these are also located within the same gene cluster, see Figure 1. Several additional virulence factors have been associated with the development of H. pylori related disease, such as the cytotoxinassociated gene A (CagA) and a vacuolating toxin (VacA).21,22 However, despite the accumulating knowledge about the mechanisms involved in the pathogenesis, it is still not completely understood why some H. pylori infected individuals remain asymptomatic, while others develop diseases. Most likely it depends on a combination of several factors including microbial, host-genetic, and environmental. Because of the clinical importance of many surface proteins, numerous methodological approaches have been implicated in the identification of bacterial outer membrane proteins (OMPs) of H. pylori.23-27 2D-GE analysis of bacterial OMPs has previously been proved to be impractical. However, in a recent work Baik and co-workers identified 16 OMPs from the H. pylori strain 26695 by combining subcellular fractionation with sodium lauryl sarcosine (SLS) and 2D-GE analysis.28 The aim of this study was to establish a method that enables characterization of outer membrane protein profiles of clinical isolates of H. pylori in the search for new protein markers for disease-related H. pylori strains and identification of potential vaccine antigens. This was achieved by combining subcellular fractionation of the bacterial outer membrane (OM) with highsensitivity nano-LC FT-ICR MS analysis.
Experimental Procedures Bacterial Isolation and Growth. H. pylori strains isolated from asymptomatic carriers (AS), DU, and gastric adenocarcinoma patients (Can) in Go¨teborg, Sweden, were used in the study. The AS group was composed of one woman and four men (mean age, 49 years), the DU group contained five men (mean age, 39 years), and the cancer group contained one 3198
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technical notes woman and four men (mean age, 77 years). The AS subjects were recruited from blood donors who had been screened for H. pylori infection by serology as previously described.29 The DU patients had chronic relapsing DU disease, as confirmed by endoscopy, but were, at the time of the investigation, in clinical remission and had not been taking antisecretory medication for at least 5 days before the study. The cancer patients were recruited from patients undergoing gastrectomy as previously described.30 All subjects were H. pylori positive as determined by culture and serology.29 To prepare antigens from the strains, the bacteria were grown on Colombia-Iso agar plates to confluency for 3 days at 37 °C under microaerophilic conditions (10% CO2, 6% O2 and 84% N2). Enrichment of H. pylori OMPs. The SLS-insoluble outer membrane fraction of H. pylori was prepared as described previously28 with minor modifications. In brief, the H. pylori cells were harvested and washed in 20 mM Tris-HCl (pH 7.5) and pelleted twice by centrifugation (4000g for 5 min). Approximately 1 g (wet weight) of bacteria was suspended in 5 mL of 20 mM Tris-HCl (pH 7.5). DNAse (Benzonase, Sigma) and protease inhibitors (Complete EDTA-free, Roche Diagnostics, Mannheim, Germany) were added to the cell suspension, and the bacteria cells were broken by repeated ultrasonication (5 × 20 s). The mixture was incubated at room temperature for 30 min. Cell debris was removed by centrifugation (9000g, 20 min, 4 °C). Total membrane pellet was collected by centrifugation (50000g, 40 min, 4 °C), resuspended in 20 mM Tris-HCl (pH 7.5) containing 2.0% (wt/vol) sodium lauryl sarcosine (USB, Cleveland, OH), and incubated at room temperature for 30 min. The sarcosine-insoluble membrane pellet was collected by centrifugation (50000g, 30 min, 4 °C, washed in 20 mM Tris-HCl (pH 7.5), and dissolved in XT Sample Buffer (Bio-Rad, Hercules, CA) with 50 mM DTT. Protein Quantification and Solubilization. The protein concentration was measured by use of the A280 method (Nanodrop technologies, Wilmington, DE) using Bovine serum albumin as standard and XT Sample Buffer as blank. The proteins were subsequently solubilized by repeated vortexing and finally denaturized by boiling at 100 °C for 2-3 min. This step was repeated twice. One-Dimensional SDS-PAGE and Gel Staining. A 50 µg protein portion of the SLS-insoluble OM fraction from each of the 15 H. pylori isolates was loaded onto the gel and separated by SDS-PAGE (12% Bis-Tris gel, Novex, San Diego, CA) at 200 V for 55 min. The gel was fixed in 7% CH3COOH, 50% C2H5OH for 20 min and washed three times in ultrapure water. After staining the gel with Bio-Safe Coomassie (Bio-Rad) for 1 h and destaining with ultrapure water, the entire gel lane was excised and cut into equal-sized pieces approximately 1 × 3 mm in size. In-Gel Protein Digestion. The method for in-gel protein digestion with trypsin described by Shevchenko et al. 31 was applied with some minor modifications. Briefly, the gel pieces were destained by washing three times in 25 mM NH4HCO3 in 50% CH3CN. Gel pieces were dried in a vacuum centrifuge and incubated with digestion buffer (50 mM NH4HCO3, 10 ng/µL trypsin) at 37 °C overnight. Peptides were extracted in 50% CH3CN/2% CH3COOH, and the supernatant was evaporated to dryness in a vacuum centrifuge. Prior to MS analysis, the peptides were reconstituted in 0.2% HCOOH. On-Line Nano-LC Separation. The tryptic peptides were separated in a 17 cm × 50 µm i.d. fused silica column packed
technical notes in-house with 3 µm ReproSil-Pur C18-AQ porous (120Å) C18bonded particles (Dr. Maisch GmbH, Ammerbuch, Germany). Two-microliter sample injections were made with an HTCPAL autosampler (CTC Analytics AG, Zwingen, Switzerland) equipped with a Cheminert valve (0.25 mm bore, C2V-1006DCTC, Valco Instruments Co, Schenkon, Switzerland), connected to an Agilent 1100 binary pump (Agilent Technologies, Palo Alto, CA). The peptides were trapped on a precolumn (4.5 cm × 100 µm i.d.) packed with 3 µm C18-bonded particles (Hydrosphere (120 Å), YMC Co. Ltd., Kyoto, Japan) located between two microvolume teeconnectors (0.15 mm bore, Valco) in a valve-switching configuration. A 100 µm fused-silica capillary was connected between the injector and the first micro-volume tee where an 80 cm × 50 µm fused-silica capillary (Polymicro Technologies, Phoenix, AZ) was used as a splitter to waste via a switchable Rheodyne valve (Nanoseparations, Nieuwkoop, Netherlands) controlled by the mass spectrometry software (Xcalibur, Thermo, San Jose, USA). Between the second tee and the Rheodyne valve, another 100 µm fused-silica was connected to waste and from that tee also the analytical column was connected. During the injection, the split restrictor on the first tee was closed while the other split was open and a flow rate of 5 µL/min of 0%B went through the trap column and out to waste. After 3 min the flow rate was increased to 250 µL/min, the gradient started at 10% B, the first split was opened, the second split was closed, and the trapped peptides were eluted onto the analytical column with a gradient from 10% to 50% B during 40 min, with a flow rate of 100 nL/min through the analytical column. The nano-LC-ESI-MS interface was constructed in-house and is similar to the one described by Shen and co-workers.3 The analytical fused-silica column was sealed by PEEK tubing (400 µm i.d., Upchurch Scientific, Oak Harbor, WA) in a 1/16 in. through-bore union (Valco) against a steel screen (1 µm pores, 50 µm thickness, Valco). A tapered emitter tip made of fused-silica capillary (20 µm i.d., 150 µm o.d., Polymicro) was connected against the steel screen using 150 µm i.d. PEEK tubing (Upchurch). The voltage applied to the union was +1.4 kV, and the distance between the emitter and the heated capillary was less than 1 mm. ESI-FT-ICR MS and MS/MS Analysis. Mass analyses were performed in a hybrid linear ion trap-FT-ICR mass spectrometer equipped with a 7T ICR magnet (LTQ-FT, Thermo Electron, Bremen, Germany). The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey mass spectra (from m/z 4001600) were acquired in the FT-ICR mass spectrometer with a resolving power of 60,000 at m/z 600 and a target value of 500,000. Automatic gain control (AGC)32 was used for sending in an accurate number of ions to the ICR cell. The three most intense doubly or triply protonated ions in each FT-scan were sequentially isolated, fragmented using collision-induced fragmentation, and analyzed in the linear ion trap. Already fragmented target ions were excluded for MS/MS analysis for 12 s. Database Analysis. Proteins were identified by automated database searching (MatrixScience, London, United Kingdom) of all tandem mass spectra using the NCBI database. The search parameters were set to MS accuracy 5 ppm, MS/MS accuracy 0.5 Da, one missed cleavage by trypsin allowed, fixed propionamide modification of cysteine, and variable modification of oxidized methionine. For protein identification, the minimum criteria were one tryptic peptide matched at or above
Carlsohn et al.
Figure 2. Visualization of the protein pattern of the different subcellular fractions from H. pylori. Lys; total cell lysate, cyt; cytoplasmic fraction, OM; sarcosine-insoluble outer membrane fraction, IM; sarcosine-soluble inner membrane fraction.
the 99.9% level of confidence and one additional peptide match at the 95% level.
Results and Discussion Enrichment of H. pylori OM Fractions and Evaluation of the Nano-LC FT-ICR Approach. Outer membrane proteins of H. pylori play critical roles in the pathogen-host interactions which are essential for colonization, adherence, and virulence of the microbe. The study of outer membrane proteins during the past decade has been intense; however, the results obtained so far have been limited, mainly because of the difficulties associated with protein purification and separation. It is therefore of great importance to both refine existing methods and develop new techniques which will allow studies of membrane proteins. The detergent SLS has previously been shown to very effectively solubilize the inner membrane of Gram-negative bacteria, leaving an intact outer membrane,28,33,34 and was therefore used to isolate the outer membrane fraction of H. pylori. The quality and purity of the SLS-insoluble OM sample was determined by analyzing the protein content of the different subcellular fractions from the OM enrichment by small-size SDS-PAGE. As visualized in Figure 2, the protein pattern between the inner membrane and outer membrane fraction differ considerable. Moreover, the figure also illustrates the enrichment of a number of proteins in the OM fraction compared to the total cell extract. The major proteins in the OM fraction is the flagellar protein Flagellin A and B (Figure 2). The nano-LC setup was constructed in-house, and the usefulness of the 1D-GE nano-LC MS approach was verified by analysis of the OM fraction of one H. pylori isolate. By separating 50 µg protein of the OM fraction from strain Can13 and performing FT-ICR analysis of the fractionated tryptic peptides, we identified 37 membrane and membrane-associated proteins from this strain (Table 1). Several of the identified OMPs were proteins known to be expressed by the bacteria in low copy numbers, including the BabA and SabA adhesins and a number of previously undetected OMPs. Due to the low resolution of 1D-GE separation, the analyzed samples consisted of a complex mixture of peptides from different proteins with similar molecular weights. Figure 3 illustrates the complexity of the nano-LC chromatogram of the peptide mixture eluted from one 1D-gel band. Because the linear ion trap-FT-ICR Journal of Proteome Research • Vol. 5, No. 11, 2006 3199
technical notes
Outer Membrane Protein Profile of Helicobacter pylori Isolates Table 1. Selected Subset of Identified Proteins in the OM Fraction of 15 Clinical H. pylori Isolatesa
clinical H. pylori isolates asymptomatic carriers protein name
gene no.
MW
pI
709
adhesin-thiol peroxidase (tagD) cell division protein cytotoxin-associated protein CagA ferric uptake regulator flagellar hook FlgE flagellar hook-associated protein 1 (HAP1) flagellar hook-associated protein 2 (HAP2) flagellar hook-associated protein 3 (HAP3) flagellin A flagellin B HofB HofC HofG H. pylori adhesin A (HpaA) iron-regulated outer membrane protein membrane-associated lipoprotein lpp20 membrane-bound endonuclease neutrophil activating protein (Nap) Omp 1 (HopZ) Omp 2 Omp 3 Omp 4 Omp 5/ Omp 29 Omp 6 Omp 7 (Hop F) Omp 8 Omp 9 Omp 10 Omp 11 Omp 12/ Omp22 Omp 13 Omp 14 Omp 15 (HopE) Omp 16 Omp 17 (SabA) Omp 19 (BabB) Omp 20 (AlpA) Omp 21 (AlpB) Omp 23 Omp 25 Omp 26 Omp 27 (HopQ) Omp 28 (BabA) Omp 30 Omp 31 Omp 32 (HopW) Omp P1 peptidoglycan-associated lipoprotein prec. (PAL) protective surface antigen D15 putative outer membrane protein putative outer membrane protein putative outer membrane protein putative outer membrane protein putative outer membrane protein putative outer membrane protein putative outer membrane protein rare lipoprotein rod shape-determining protein secreted protein involved in flagellar motility urease R urease β
HP0390 HP0979 HP0547 HP1027 HP0870 HP1119 HP0752 HP0295 HP0601 HP0115 JHP0342 HP0486 JHP0850 HP0797 HP1512 HP1456 HP0323 HP0243 HP0009 HP0025 HP0078/ 0079 HP0127 HP0227/ 1342 HP0229 HP0252 HP0253 HP0317 HP0324 HP0472 HP0477/ 0923 HP0638 HP0671 HP0706 HP0722 HP0725 HP0896 HP0912 HP0913 HP1107 HP1156 HP1157 HP1177 HP1243 HP1395 HP1469 HP1501 HP0839 HP1125 HP0655 JHP1472 JHP1084 JHP0725 JHP1094 JHP1261 JHP0429 JHP0649 HP1571 HP0743 HP1462 HP0073 HP0072
18.4 41.7 131.3 18.1 76.2 68.3 74.1 92.2 53.3 53.8 53.0 59.5 58.9 28.3 97.5 19.1 20.3 16.8 76.6 78.0 63.7 32.0 76.0 53.2 52.2 47.5 81.6 28.7 21.0 41.7 34.1 30.5 30.2 70.6 69.3 77.7 56.1 57.2 25.7 76.7 132.6 70.0 80.1 26.7 28.2 43.1 63.9 20.2 102.9 30.3 133.4 57.1 52.8 75.8 41.8 73.1 35.6 37.4 20.6 26.7 61.8
5.54 5.32 8.75 8.50 5.04 5.06 5.19 5.29 6.36 6.10 9.50 9.35 9.15 7.88 9.05 9.52 9.50 5.69 8.71 9.16 5.01 9.56 8.51 8.51 9.55 9.18 8.67 7.85 9.16 8.94 9.78 9.17 8.98 9.31 9.12 8.64 9.14 9.20 9.12 9.12 8.86 9.21 8.95 9.65 9.50 9.51 9.50 5.84 9.05 8.89 8.81 8.91 9.55 6.10 8.87 8.27 9.36 5.52 6.54 8.78 5.64
+ + + + + + + + + +
703
386
707
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gastric cancer
231 524 516 230 267 333 13 14 12 51 21
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duodenal ulcer
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a Positive identification requires one peptide match at the 99.9% level of confidence (Mowse score >47) and one additional peptide match at the 95% level of confidence (Mowse score >30). The RMS error shown in the table is the mean mass error for the matched peptides. Several potential targets for vaccine discovery and potential clinical markers were identified.
instrument consists of two mass analyzers which can work in tandem, it is possible to perform high mass measurement in the ICR cell and fragmentation analysis in the linear ion trap simultaneously. By use of nano-columns, a large increase of sample concentration is achieved and this induced sample concentration can be essential for obtaining data for protein 3200
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identification. Because of the low-resolution separation from the 1D-GE, it is not likely to isolate unique proteins. Instead, each protein band will contain a mixture of proteins. We demonstrate that the combination of nano-LC and FT-ICR MS analysis makes it possible to obtain significant protein identification, even when analyzing complex peptide mixtures
technical notes
Carlsohn et al.
cancer, duodenal ulcer, and asymptomatic carriers were selected for analysis. Whereas the small size 1D-SDS-PAGE analysis only revealed minor differences in the protein patterns of the OM fractions (Figure 6), the difference in protein expression among the strains was significant. In total, 60 unique membrane or membrane-associated proteins were identified from the different strains. In addition, we identified several hypothetical proteins and a small number (less than 10%) of cytoplasmic proteins (data not shown). Moreover, of the 33 theoretical predicted OMPs in the 26695 genome (Figure 1), all OMPs were identified, except Omp18, Omp24, and the hypothetical protein HorD. The major criteria for a candidate vaccine antigen are that it is conserved, i.e., expressed by all strains, surface-localized, and immunogenic (capable of stimulating the immune system). The OMPs BabA, Omp15, and Omp11 were identified in all strains. The finding of a conserved BabA expression is in contrast to earlier studies which have demonstrated BabA expression in approximately 60% of the analyzed strains.12,35 While the biological functions of both the BabA adhesin and the Omp15 (HopE) porin have been demonstrated in several studies, the function of Omp11 is still unclear.12,36,37 The expression of the omp11 gene was however, found to be downregulated by H. pylori adhering to AGS cells,38 indicating a nonadhesive role of Omp11. The protein was recently determined to be immunogenic.28 Moreover, Omp11 is one of the 1,281 core proteins that recently was found to be conserved among diverse clinical isolates,39 indicating an essential function for Omp11. Omp11 is thus an interesting candidate in the search for novel targets for drug and vaccine discovery. However, further studies need to be performed in order to evaluate the potential of Omp11 as vaccine antigen. Figure 3. Nano-LC-FT-ICR MS analysis of a single 1D-GE band (**) from the clinical H. pylori isolate Can13. (A) The complexity of the nano-LC chromatogram is illustated. The insert shows an enlarged picture of a seleted fraction of the chromatogram. (B) The doubly protonated peptide at m/z 1131.591 [M ) 2261.167] was automatically selected for ion trap dissociation. The parent ion at m/z 1131.591 was mass measured in the ICR cell, which resulted in a mass error of less than 1 ppm. (C) CID spectrum of the ion m/z 1131.591. The assigned amino acid sequence is unique for the SabA adhesin in H. pylori. Only fragment ions corresponding to the amino acids marked in bold are shown. **The protein band is shown in Figure 4.
(Figure 4). The identification of the low-abundance protein SabA as one out of seventeen membrane or membraneassociated proteins present in this sample illustrates the usefulness of this method to analyze membrane proteins expressed in low amounts. Furthermore, the nano-LC separation, the mass measurement in the ICR cell, and the fragmentation efficiency in the ion trap are very reproducible (Figure 5), and the entire process, from protein fractionation to OMP profiling of a unique H. pylori strain, can be completed in less than 24 h. Search for Novel Markers for Clinical Outcome and Potential Target Proteins for Vaccine Development. To screen the outer membrane of clinical H. pylori isolates in search for potential vaccine antigens or proteins associated with disease, we used the proteomic approach described above. H. pylori strains isolated from three different patient groups, gastric
There are today no proteins known to be unique for diseaserelated H. pylori strains. Our study demonstrates, however, that Omp26, an outer membrane protein with unknown function, is expressed by all disease-related H. pylori strains tested, but only in one isolate from an asymptomatic carrier (Table 1). Moreover, the expression of Omp30 was specific for the duodenal ulcer isolates, and the lack of expression of Omp6 was related to H. pylori-induced duodenal ulcer. Taken together this makes Omp26, Omp6, and Omp30 potential targets for further investigation in the search for proteins unique for disease-related H. pylori strains. The main reason for using analytical 2-D GE in proteomic research is the ability to visualize and separate large numbers of proteins including isoforms into unique spots for identification of differences in protein pattern between, for example, bacterial strains. The combined 1-D gel LC-MS/MS approach does not enable analysis of different isoforms nor for any quantitative difference in protein expression between the examined strains; instead, it allows profiling of the total protein expression in the membrane, i.e., which proteins are expressed at a detectable level and which are not. To perform quantitative analysis of the OM proteins it might be possible to use the high quality data obtained from the nano-LC MS analysis to create density maps of the ion current from the identified peptides. This information can then be used for relative quantification of the proteins of interest. Moreover, this approach allows identification of a number of outer membrane proteins with similar molecular weight and Journal of Proteome Research • Vol. 5, No. 11, 2006 3201
technical notes
Outer Membrane Protein Profile of Helicobacter pylori Isolates
Figure 4. Proteins identified by nano-LC FT-ICR MS analysis of a single 1D-GE band (**) from the clinical H. pylori isolate Can13. Seventeen unique proteins were detected in the sample (**) of the outer membrane fraction from a H. pylori strain (Can 13) isolated from a patient with gastric cancer. The great majority of the identified proteins are known to be expressed on the bacterial surface. Protein identity is significant at the 99.9% level. The correspondig nano-LC chromatogram is shown in Figure 3. The RMS error shown in the table is the mean mass error for the matched peptides.
Figure 5. Reproducibility test of the nano-LC FT-ICR MS and MS/MS analysis. A tryptic digest of a protein band from an OM preparation was analyzed at two different time points. (A) The LC chromatogram illustrates the high reproducibility of both the retention time and the peak distribution in the LC separation. (B) The mass measurement of the doubly protonated peptide at m/z 836.94 eluted at 21.32 and 21.25 min, respectively, differ by only 0.1 ppm. (C) The CID spectrum of the doubly protonated peptide at m/z 836.94. The assigned amino acid sequence corresponds to the H. pylori protein Omp6.
isoelectric point (Table 1). For example, AlpA, AlpB, SabA, and a putative outer membrane protein (JHP0725) differ only by 1 kDa and 0.3 pH unit, respectively. Even if these membrane proteins could be analyzed by 2D-GE, their resolution in the gel would be very difficult to achieve and that would preclude unambiguous identification and quantification. 3202
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Conclusion We have demonstrated that subcellular fractionation combined with nano-LC FT-ICR MS analysis enables identification of the majority of the theoretically predicted OMPs including the complete Hop family in clinical H. pylori isolates. We therefore conclude that this rapid subproteomic approach is
technical notes
Figure 6. SDS-PAGE analysis of the outer membrane fractions from selected H. pylori strains. Lane 1: Hel 524, lane 2: Hel 516; isolated from duodenal ulcer patients. Lane 3: Can 51, lane 4: Can 12; isolated from cancer patients. Lane 5: Hel 231, lane 6: Hel 386; isolated from asymtomatic carriers. Lane 7: Molecular weight marker. The major component of the OM fractions is the flagellar proteins flagellins A and B.
suitable for the characterization of H. pylori outer membrane profiles in the search for novel markers for clinical outcome and new potential target proteins, for example vaccine development, and that use of this approach will help to provide new insight to the membrane biology of other Gram-negative bacteria.
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PR060181P
