MS Proteomic Approach for Bacterial

Virginie Ruelle, Nandini Falisse-Poirrier, Benaissa ElMoualij, Danièle Zorzi, Olivier Pierard, Ernst Heinen, Edwin De Pauw, and Willy Zorzi*. Center ...
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An Immuno-PF2D-MS/MS Proteomic Approach for Bacterial Antigenic Characterization: To Bacillus and Beyond Virginie Ruelle,†,‡,§ Nandini Falisse-Poirrier,†,§ Benaissa ElMoualij,† Danie` le Zorzi,† Olivier Pierard,† Ernst Heinen,† Edwin De Pauw,‡ and Willy Zorzi*,† Center of Research on Prion Proteins, University of Lie`ge, B-4000 Lie`ge, Belgium, and Mass Spectrometry Laboratory, University of Lie`ge, B-4000 Lie`ge, Belgium Received December 11, 2006

We are confronted daily to unknown microorganisms that have yet to be characterized, detected, and/ or analyzed. We propose, in this study, a multidimensional strategy using polyclonal antibodies, consisting of a novel proteomic tool, the ProteomeLab PF2D, coupled to immunological techniques and mass spectrometry (i-PF2D-MS/MS). To evaluate this strategy, we have applied it to Bacillus subtilis, considered here as our unknown bacterial model. Keywords: Bacillus subtilis • flagellin • in-gel digest • mass spectrometry • PF2D

Introduction It is commonly admitted that the microbial world is everevolving and is represented by several million species out of which, in January 2006, only 235 bacterial genomes have been sequenced and published.1 In recent times, screening for the presence of microorganisms and accurately identifying them during epidemiological surveys, food processing, medical diagnosis, public health, and biosafety assessments has gained unprecedented attention. Monitoring their presence has been possible using traditional methods such as culture on selective media, enrichment and isolation techniques, biochemical activity tests, and polymerase chain reaction (genotyping techniques).2-4 Another technique for the detection, analysis, and characterization of microorganisms in different biomatrices is the use of antibodies directed against specific immunogens.5-7 This approach necessitates a good understanding of the nature of the immunogen against which an antibody needs to be developed. The production of monoclonal antibodies is well established in the biotechnological domain consisting in developing, screening, and subsequently cloning hybridomas. Previous work done in this domain8 shows this approach to be particularly laborious and time-consuming, often resulting in monoclonal antibodies that present a low specificity toward the organism of interest. Alternatively, if one chooses to produce a monoclonal antibody using a synthetic antigen formulated after a genetic sequence comparison, it is necessary to question the host-specific, epitope-specific, and immunogenic properties of the chosen antigen. Moreover, inspite of * To whom correspondence should be addressed. Willy Zorzi, University of Lie`ge, Center of Research on Prion Proteins (CRPP), 1 avenue de l’hoˆpital B36 Sart Tilman, B-4000 Lie`ge, Belgium. Tel: +32-4-366.43.27. Fax: +324-366.43.21. E-mail: [email protected]. † Center of Research on Prion Proteins. ‡ Mass Spectrometry Laboratory. § These authors have equally contributed to the work.

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specificity toward the organism of interest, it is known that the choice of the synthetic antigen formulated using bioinformatic tools could be biased because of its potential similarity with other non-identified (unsequenced) components. This can lead to problems of cross-reactivity in immunodiagnostic assays.9 Finally and most importantly, for unknown bacteria, the strategies described above, for monoclonal antibody generation, are risky and not very appropriate. The use of polyclonal antibodies could therefore provide an alternative solution. Polyclonal antibodies have the advantage of being of reasonable cost and relatively simple to produce besides presenting a higher avidity than monoclonal antibodies. This makes them robust and tolerant of small changes in the nature of the antigen(s) they recognize. They could hence facilitate the choice of one, or more, antigenic targets at a low risk-to-cost ratio. Polyclonal antibody characterization has previously been used to detect the immunogen(s) of pathogenic microorganisms and hence identify potential biomarkers and novel vaccine candidates.10 These techniques have mostly relied on the traditional 2-DE combined with western-blotting to achieve this. In our study, a multidimensional strategy was developed to provide an approach for characterizing the immunogens of unknown and/or poorly characterized bacteria, using a polyclonal antibody. This strategy, termed “i-PF2D-MS/MS”,11 integrates for the first time an analytical 2DLC based protein fractionating technique (ProteomeLab PF2D) coupled to immuno-blotting and mass spectrometry. The immunoproteomic approach presented here is proposed as a determining step to identify the unknown target immunogens. Being well characterized and especially nonpathogenic to humans, Bacillus subtilis was chosen here as a model to evaluate the “i-PF2DMS/MS” strategy. This bacterium is also known to be a key producer of industrialized enzymes (amylases) and antibiotics such as proteases or bacitracine. However, as it also produces toxins, it is of great interest to detect its presence in several environmental, food, and clinical samples. Moreover, using it 10.1021/pr060661g CCC: $37.00

 2007 American Chemical Society

Serological Characterization of Bacillus subtilis

as a model, the approach developed here could be extended to characterize the immunogens of unknown bacteria, using a polyclonal antibody.

Experimental Section Reagents. Chemicals were purchased from Sigma-Aldrich Inc. (St. Louis, MO) and used without further purification. Water (18.2 MΩ.cm at 25 °C) was obtained from a Milli-Q water filtration system (Millipore Co., U.S.A.). Biological Material. The bacterial strains Bacillus cereus AH183, Bacillus pumilus P059, and Bacillus laterosporus LAT011 were obtained from the Laboratory of Microbial Genetics (GEMI, Catholic University of Leuven, Belgium); Escherichia coli ATCC11775 and Salmonella enteritidis ATCC13076 were obtained from the American Type Culture Collection (ATCC, U.S.A.); Bacillus megaterium LMG7127 and Enterococcus faecium LMG15877 were obtained from the Laboratorium voor Microbiologie (LMG, University of Gent, Belgium); Bacillus subtilis 1A1 was obtained from the Bacillus Genetic Stock Center (BGSC, Ohio State University, OH); and Enterobacter cloacae 4C2 was obtained from a clinical isolate characterized by Api Gallery 50 CHB and API 20E. Cell Culture. Bacteria (106 cells/mL) were inoculated in liquid Luria-Bertani culture medium (Difco, U.S.A.) and grown 24 h at 37 °C. Bacterial density was measured spectrophotometrically at 600 nm. Bacteria were pelletted by centrifugation (10 min, 20 000g, 4 °C) and washed thrice in sterile PBS (Na2HPO4 0.01 M; NaCl 0.15 M; 0.05% Tween 20 pH 7.4). Preparation of Anti-Bacillus Serum. The rabbit serum used in this study had previously been developed against inactivated bacterial whole cell preparations from Bacillus cereus, pumilus, laterosporus, megaterium, and subtilis. It had been produced according to the standard IgG purification and immunization protocol from Eurogentec (Eurogentec S.A., Seraing, Belgium). The pre-serum derived from rabbit before immunization was used as a control in our study. Preparation of “Biotinylated Anti-Bacillus Serum”. The anti-Bacillus serum and pre-serum had been biotinylated using the “Biotin Labeling Kit” (Roche, Germany) and adjusted to 1.5 mg/mL. Preparation of “Depleted Serum”. The biotinylated antiBacillus serum was depleted against a non-exhaustive list, as is usually the case, of gram positive and gram negative bacteria that included Escherichia coli, Salmonella enteritidis, Enterobacter cloacae, Enterococcus faecium, and Bacillus megaterium. Briefly, a mixture of these bacterial strains (1010 cells/strain) was suspended in 5 mL of biotinylated anti-Bacillus serum (3.75 ng/mL in PBS) and incubated, under agitation, 1 h at 37 °C. The solution was then centrifuged (20 min, 20 000g, 4 °C), and the recovered supernatant was used as the “depleted serum”. The efficacy of the serum against Bacillus subtilis was confirmed by ELISA and western-blot. Bacterial Cell Lysis. After cell culture, bacterial pellets were lysed for 1 h in the lysis buffer (saccharose 0.78 M, Tris-HCl 30 mM pH 8, EDTA 5 mM, and lysozyme 100 µg/mL). Flow Cytometry (FACS). Bacillus subtilis cells were fixed in 2% paraformaldehyde and labeled with anti-Bacillus serum or pre-serum as primary antibody (incubation 45 min at 4 °C, dilution 1/50). The swine anti-rabbit-FITC (DakoCytomation, Denmark) was used as secondary antibody (incubation 45 min at 4 °C, dilution 1/100). The Bacillus subtilis cells (106 cells/ mL) were resuspended in PBS and then analyzed by flow cytometry using a FACS Calibur (Becton-Dickinson, U.S.A.). The

research articles excitation of the FITC was realized at 488 nm, and the green fluorescence emitted was collected between 500 and 540 nm. Electron Microscopy. The Bacillus subtilis cells were fixed in paraformaldehyde and labeled with the primary antibody (serum diluted at 1/50) and a secondary antibody (anti-rabbit coupled to a gold particle, AuroProbe EM-Labeled (Amersham Auroprobe, U.K.). After labeling, the cells were recovered and successively fixed in 2% glutaraldehyde and 3% potassium ferrocyanide/2% osmium tetraoxide (Agar Scientific, U.K.) diluted in 0.1 M cacodylate buffer. The sample was then dehydrated using ethanol and enchased in epoxy resin. Sections of 100 nm were realized and observed on a transmission electron microscope Jeol 100 SX (Jeol, Japan) magnified 15 000×. Western-Blot. Bacterial lysates (5 × 107 cells/mL) were diluted in Bio-Rad Laemmli Sample Buffer and denatured 5 min at 100 °C. Samples and molecular mass markers (Precision Plus Protein Dual Color Standards, Bio-Rad Laboratories, USA) were loaded on a SDS-PAGE mini-gel (separating gel 12%, stacking gel 4%). The electrophoresis was realized at a constant voltage of 120 V. After electrophoresis, the proteins were blotted in a Tris-glycine buffer containing 5% methanol onto Hybond-P PolyVinylidene DiFluoride membranes. The membranes were incubated in blocking buffer overnight at 4 °C. The proteins were detected by a biotinylated anti-Bacillus serum (3.75 ng/ mL), and the reaction was visualized by “ECL Advance Western Blotting Detection Kit” (Amersham-Pharmacia, U.K.). Dot-Blot. The fractions obtained after proteins purification were directly blotted (15 µL/spot) on a nitrocellulose membrane. The membrane was blocked and revealed using the same protocol as western-blot. Protein Fractionation. The protocol used was that recommended by the “ProteomeLab PF2D” manufacturer (Beckman Coulter, Fullerton, CA). Briefly, a bacterial lysate (10 × 1010 cells/mL) was centrifuged (20 min, 20 000g, 4 °C) to eliminate cellular debris. This was followed by an exchange step, on a Sephadex PD-10 column (Amersham-Pharmacia, UK), that involved the replacement of the lysis buffer by the PF2D first dimension start buffer (pH 8.5). The protein concentration of the solution obtained after elution was determined using the “DC protein assay” (Bio-Rad, U.S.A.) and was adjusted to 2.5 mg/mL of protein. A total of 5 mg of Bacillus subtilis protein lysate was first injected onto an HPCF-1D column (250 mm × 2.1 mm, Eprogen, Darien, IL), previously equilibrated with start buffer for 210 min. Twenty minutes after sample injection, the PF2D eluting buffer (pH 4) was passed through the column. A pH gradient (pH 8.5-4) was, thus, gradually established, and the proteins were eluted according to their respective isoelectric points at a flow rate of 0.2 mL/min. The protein fractions were collected at intervals of every 0.3 pH unit monitored by a pH meter placed at the outlet of the column. All proteins that had a pI beyond the gradient were also eluted and collected but on the basis of time (every 5 min). A UV detector, set at 280 nm, allowed the detection of the eluted proteins and subsequent dosage of each 1-D protein fraction. At the end of the gradient, the column was washed with NaCl (1 M) HPLC grade water (J.T. Baker, U.S.A.). The 1-D separated fractions were further separated according to relative hydrophobicity on an RP column (33 mm × 4.6 mm, 1.5 µm nonporous C18 silica beads, Eprogen), maintained at 50 °C and eluted at a flow rate of 0.75 mL/min with a 0-100% linear gradient of solvent A (0.1% v/v TFA) and solvent B (0.08% v/v TFA in acetonitrile) for 35 min. Eluted proteins were detected online by an UV detector at 214 nm and protein dosage was determined using Journal of Proteome Research • Vol. 6, No. 6, 2007 2169

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a standard BSA curve. A fraction collector, placed outside the second UV detector, allowed the recuperation of protein fractions for subsequent analyses by immuno-blot and mass spectrometry. Determination of Protein Concentration. Protein concentrations were estimated online using the PF2D platform. Indeed, for the first dimension fractions, the separated proteins were quantified using the UV absorbance at 280 nm integrated in the platform according to the eq 1: C ) A280/l

(1)

where “C” is the concentration (mg/mL), “A280” is the absorbance at UV 280 nm, and “l” is the path length ()1 cm).12 For the second dimension, we estimated the protein concentration of the eluted fractions using an online BSA standard curve. This was established by relating the area under the absorbance peaks in function of known quantities of BSA injected on the column. Micro-Sequencing by Mass Spectrometry. After a separation by SDS-PAGE, gels were stained with a MS-compatible silverstain protocol.13 Protein(s) of interest were characterized by ingel digestion and analyzed by MS/MS as reported by Douette et al.14 Search in the Swiss-Prot Database. This search was realized on the Swiss-Prot database (Expasy, Swiss Bioinformatics Institute) using the research module “TagIdent” (http://us. expasy.org/tools/tagident.html). The used parameters were the choice of the organism (Bacillus subtilis), the mass range (32 ( 3 kDa) and the pI (7 ( 10).

Results The strategy used for characterizing an anti-Bacillus polyclonal antibody consisted in using a rabbit serum, originally directed against whole bacilli cells, in improving its specificity vis-a`-vis the Bacillus subtilis species and in identifying its target immunogen(s). Characterization of an Anti-Bacillus Serum on Whole Bacillus subtilis Cells. Previously we had developed an antiBacillus polyclonal antibody by immunizing rabbits with whole inactivated bacilli cells and collecting the resulting serum. The efficacy of this immunization, and consequently the production of antibodies directed against bacilli, had been confirmed by ELISA at the time of the anti-Bacillus serum production (data not shown). The antigenic reactivity of the serum against whole Bacillus subtilis cells was studied using flow cytometry and electron microscopy. Flow Cytometry. Bacillus subtilis cells were fixed in paraformaldehyde and incubated with either rabbit pre-serum or antiBacillus serum as primary antibody. Immuno-detection was realized using an anti-rabbit antibody coupled to FITC. Figure 1 shows the reactivity of the serum before (trace B) and after immunization (trace A) vis-a`-vis Bacillus subtilis. As cells used for this study were fixed in paraformaldehyde, known to preserve super-structures, the results in Figure 1 show that the anti-Bacillus serum presents a potential reactivity toward surface antigens of Bacillus subtilis. Electron Microscopy. After having characterized the antiBacillus serum avidity and reactivity toward Bacillus subtilis, a transmission electron microscopy was used to confirm the cell surface localization of the target immunogen(s). The cells, fixed in paraformaldehyde, were incubated with the anti-Bacillus serum (as primary antibody) and then labeled using a second2170

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Figure 1. Histogram showing the number of events versus the logarithm of the intensity of emitted fluorescence. FACScan flow cytometer analysis of Bacillus subtilis cells labeled with (A) rabbit anti-Bacillus serum and (B) pre-serum as primary antibody. The swine anti-rabbit-FITC was used as secondary antibody. (C) A control was realized without primary antibody.

ary antibody coupled to a gold particle. In Figure 2B, gold particles (black dots) were detected on the surface of the bacterial cells, implying that the primary antibody had again been fixed on the Bacillus subtilis surface. Labeling is indicated by black arrows. The results obtained by electron microscopy were in agreement with those obtained previously by flow cytometry and show that the anti-Bacillus rabbit serum presented a response toward at least one (or several) cell surface antigen(s) of Bacillus subtilis. Immunoproteomic Characterization of the Anti-Bacillus Serum. The biochemical characterization and identification of the antigenic target(s) of the serum was realized using an immunoproteomic approach consisting of combining western/ dot-blotting techniques to a 2DLC protein fractionating tool and mass spectrometry. Western-blotting was used to analyze the reactivity of the serum toward the whole antigenic content of the bacterial cell, the specificity of the serum vis-a`-vis Bacillus subtilis and the apparent molecular mass of its specific target antigen(s). Characterization by Western-Blot. The present step consisted in checking the antigenic response of the serum toward the denatured proteins of Bacillus subtilis. Figure 3A shows that the biotinylated anti-Bacillus serum produces several reaction bands, one of which presents a higher signal than the others (see black arrow: 32-35 kDa). The specificity of the serum visa`-vis Bacillus subtilis was tested by western-blot on protein lysates of a non-exhaustive list of other bacterial species. As the serum cross-reacted with several antigens (Figure 3A: lanes 1, 3-6), the specificity of the biotinylated anti-Bacillus serum toward Bacillus subtilis had to be improved. Improvement of Serum Specificity toward Bacillus subtilis. To raise the specificity of the anti-Bacillus serum toward Bacillus subtilis, a “serum depletion” step was carried out (see Experimental Section: Preparation of “Depleted Serum”). This serum preparation was tested for its specificity against Bacillus subtilis 1A1. The western-blot results, presented in Figure 3B, show that the specificity of the biotinylated serum toward Bacillus subtilis was enhanced by this depletion step and the reactivity toward the antigenic target at 32-35 kDa was maintained. Further depletion was not pursued so as to conserve sufficient anti-Bacillus antibody titer. To further investigate the non-ambiguous identification of the antigenic targets, a fractionation step, in this case by 2-D chromatography, was performed on the complex bacterial cell lysate.

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Figure 2. Electron microscopy (magnification 15 000×) illustrating the fixing of the antibodies on Bacillus subtilis cells. The black arrows indicate the position of some fixed antibodies (B). A control, realized without the primary antibody, did not fix any gold particle (A).

Figure 3. Western-blot analysis realized with (A) biotinylated anti-Bacillus serum and (B) exhausted serum as detection antibody on bacterial protein extracts (5 µg/wells) of Bacillus megaterium (1) and subtilis (2) as well as Enterobacter (3), Enterococcus (4), Escherichia coli (5), and Salmonella (6). PM ) molecular mass marker (Precision Plus Protein Dual Color Standards, Bio-Rad). This marker, not biotinylated, was colored with Coomasie Blue.

First Dimension Protein Fractionation of Bacillus subtilis Protein Lysate According to Isoelectric Point. The protein lysate was fractionated by coupling 2-DLC to immunological techniques. 2-DLC was performed on the ProteomeLab PF2D platform. The protein lysate, prepurified by molecular sieve chromatography over a PD-10 column, was injected onto an anion exchanger previously equilibrated at pH 8.5. A pH gradient ranging from pH 8.5-4 was established during the elution run, and the protein fractions were eluted by intervals of 0.3 pH units according to their isoelectric points (Figure 4). In total, 30 fractions (eluted within and on either side of the gradient) were chosen for analysis (Figure 4, fractions contained between the two black bars). These fractions were screened, in relatively native conditions, using the dot-blot technique. They were spotted onto a nitrocellulose membrane and revealed with the depleted serum, used as detection antibody. Out of the six fractions (21-24 and 28-29) that reacted with the detection antibody (Figure 5A), two were discarded (28 and 29), as they were collected beyond the pH gradient and during the column washing step (Table 1). On the basis of these results, about 2.7 µg of each positive fraction (21-24) was analyzed by western-blot. Only one protein, contained in fraction 22, reacted with the depleted serum (Figure 5B). This protein of

relative mass 32-35 kDa, as shown previously, had a pI ranging from 4.72 to 5.02. We verified whether its identification was possible at this stage. For this, we analyzed the protein band of interest excised from a silver-stained SDS-PAGE gel. The large number of protein bands made it difficult and ambiguous to identify the band of interest in spite of an additional separation brought about on the gel (Figure 5C). Second Dimension Protein Fractionation According to the Relative Hydrophobicity. An additional fractionation of fraction 22 was considered necessary and was performed on the basis of relative hydrophobicity. This was accomplished on a nonporous reverse phase column, and the resulting liquid fractions were systematically examined by dot-blot to identify the reactive fraction(s) (Figure 6A). Some fractions presented a positive response toward the serum (fractions 41 to 62). As shown in Figure 7, these corresponded to the region collected under the highlighted peak in the 2-D chromatogram and were thus characterized by a relative hydrophobicity (retention time of peak 18.5 min corresponding to about 50% in acetonitrile). By western-blot, the 32-35 kDa protein was most intensely detected in fraction 41 (Figure 6B). The band of interest containing the target antigen was thus excised from a silverstained SDS-PAGE gel (Figure 6C) for non-ambiguous peptide Journal of Proteome Research • Vol. 6, No. 6, 2007 2171

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Figure 4. First dimension chromatogram obtained for the Bacillus subtilis lysate along a pH gradient (4-8.5). The pH trace (left ordinate) and the absorbance, monitored at 280 nm, (right ordinate) are expressed in function of elution time (abscissa). Out of a total of 42 fractions, 30 (contained within the two black bars) were analyzed.

the reactive protein band by precise excision allowing a MS/ MS sequencing. After micro-sequencing by mass spectrometry (Figure 6D), an identification by Mascot confirmed flagellin to be the target antigen of the anti-Bacillus subtilis serum. The Mowse Score was of 643 for the 19 matched digested peptides, which covered 22% of the total protein sequence. Flagellin is a protein of 304 amino acids with a molecular mass of 32 607 Da and an isoelectric point of 4.97. This identification corresponds perfectly with our earlier results obtained by purification on a pH gradient by ProteomeLab PF2D and by western-blot. Figure 5. Immunoproteomic characterization of the target antigen after 1-D fractionation of Bacillus subtilis lysate. Table 1. pH Range and Protein Concentration of the Fractions Having a Positive Response toward the Anti-Bacillus subtilis Serum fraction number

pH range

21 22 23 24 28 29

5.32-5.02 5.02-4.72 4.72-4.42 4.42-4.12 end of gradient end of gradient

protein concentration (mg/mL)

0.070 0.100 0.130 0.160

sequencing by mass spectrometry. The additional separation of the 2-D antigenic fraction on the silver nitrate gel (along relative mass) had the consequence of enhancing the fractionation of the antigenic fraction. This facilitated the isolation of 2172

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Discussion Out of the several million species of microorganisms that exist in the nature, only a few hundred bacterial genomes have been sequenced and published. Currently, to detect the presence of the poorly characterized microorganisms in several matrices, the use of antibodies directed against specific immunogens is helpful. In the case of monoclonal antibodies, the development strategy often necessitates a good characterization of the target microorganism concerning protein expression pattern and/or genetic sequence. Bioinformatic tools are thus of precious help for comparing the information contained in the several databases and for finding a specific sequence to synthesize an antigen allowing the production of the corresponding antibody. However, for all bacteria having not yet been characterized, this strategy is not directly applicable. For this reason, we have developed an original immunoproteomic approach, using a

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Figure 6. Immunoproteomic characterization of the target antigen after 2-D separation of fraction 22. The bold amino acids correspond to the peptide matched in the sequencing realized after 2-D fractionation.

In our work, the results obtained from FACS and electronic microscopy showed that the reactivity of the serum was directed toward at least some surface immunogen(s). It is important to note that membrane localization of an immunogen can hence be confirmed by FACS and electronic microscopy. Next, the specificity of the serum was studied by Westernblotting and showed that the serum reacted against others bacterial species. A serum depletion step was undertaken against a non-exhaustive list of bacterial species, taken as example. In this context, the depletion enhanced the specificity of the serum toward Bacillus subtilis without significant loss of reactivity. Indeed, the notion of specificity of an antibody is relative and closely depends on the domain of its application. Although the development of antibodies is often accompanied by specificity tests on a large number of organisms, this does not exclude eventual cross-reactions possible in the presence of unknown or uncharacterized microorganisms or biomatrices. The reactivity of the depleted serum was shown to be directed toward a 32-35 kDa protein band, but due to the complexity of the protein lysate, the identification of the protein band was not possible and fractionation was a required step.

Figure 7. Second dimension chromatogram obtained for fraction 22 (F) along relative hydrophobicity. UV detection was set at 214 nm. The absorbance (ordinate) is expressed in function of the retention time (abscissa).

polyclonal antibody, which can be applied to characterize the target antigens of unknown bacteria. This approach was evaluated using the well-characterized Bacillus subtilis 1A1 model, for which no monoclonal antibody is commercially available. The preliminary identification of an antigen specifically expressed by the target microorganism and presenting a high immunogenicity presents a big advantage for monoclonal antibody production. Indeed, the cost and difficulty of generating a monoclonal antibody often restricts its production to specific requirements,15 and the candidates produced can present cross-reactivity if the properties of the target antigen are not well studied.

To clearly identify the antigenic target of the depleted serum, an immunoproteomic strategy, i-PF2D-MSMS11 was chosen for the final fractionation of a Bacillus subtilis protein lysate. Traditionally, 2-DE has played a strategic role in several applications in combination with western-blot.10,16-20 The development of liquid chromatography-based protein fractionating systems gives way to alternative proteomic techniques, potentially providing the same advantages as 2-DE.21 Moreover, owing to their gel-free nature, PF2D systems offer the possibility of being completely automated and being interfaced to several other analytical and biochemical methods, indicative of their high versatility and user-friendly work procedures.22-25 It is possible, by PF2D, to detect up to 100 ng of purified protein mix (data not shown). The method, however, requires relatively high amounts of total protein lysate (1-5 mg) for integral proteome analysis. For the most complete visualization of the proteome that would allow the detection of low abundant proteins, it is almost a necessity to work with such relatively high amounts to generate sufficient analytical material in each chromatographic step. As in every purification method, it is necessary to take into account the relative loss of analytical material at each step. In our case, 5 mg of a complex Bacillus subtilis protein lysate was separated according to Journal of Proteome Research • Vol. 6, No. 6, 2007 2173

research articles isoelectric point and relative hydrophobicity on the ProteomeLab PF2D platform. After each fractionation, the collected fractions were rapidly screened by an immunoassay (dot-blot, Western-blot) and the antigenic fraction(s) were consequently highlighted. This strategy greatly improved the separation resolution subsequently brought about by SDS-PAGE and facilitated a nonambiguous excision of the target antigen for mass spectrometry-based identification. The target antigen was hence identified as being flagellin, a bacterial surface protein of theoretical pI 4.97 and molecular mass 32.6 kDa. The external location of this target antigen as well as its relative molecular mass, determined in the first part of our work, was hence completed by the immuno-characterization and peptide sequencing by i-PF2D-MS/MS. The identified flagellin, located on the external membrane of Bacillus subtilis,26 constitutes the subunit of the bacterial flagellar filament, the largest portion of the flagellum. The arrangement and composition of flagella varies greatly between microorganisms. Flagellin possesses two highly conserved regions (N- and C-terminal) and one central domain varying considerably in both amino acid sequence and size.27 This central domain appears to be responsible for flagellar antigenic variability28 and, consequently, for the virulence of pathogenic bacteria.29 Moreover, surface proteins having a higher variability than cytoplasmic ones,30 they could be useful as biomarkers for bacterial identification.31 Flagellar serotype variation is already one of the basic criteria used in the classification of Enterobacteriaceae,32 especially for Human pathogens as Escherichia coli O157:H7.33 Furthermore, Asano et al. have proposed the flagellin of Bacillus subtilis DB9011 as a biomarker for its detection and discrimination against others bacterial strains.34 This highly antigenic protein is known to be a pathogen-associated molecular pattern constituting the dominant target in diseases such as Crohn disease.35 It is also the only known ligand for TLR5, an innate immunity receptor, and could trigger autoimmune tissue injury.36 The characterized flagellin in our study is proposed here as an appropriate external marker that can be used to identify the Bacillus subtilis 1A1 strain, originally called Bacillus subtilis 168 in classical nomenclature.37 In this way, on the basis of the high variability of the central domain reported between different Bacillus subtilis strains34,38 and its high immunogenicity, as seen in this study, flagellin can be proposed as a potential marker candidate for the development of monoclonal antibodies for the identification and rapid detection, as well as on-line monitoring of other whole Bacillus subtilis strains in complex biological mixtures. Indeed, as of now, commercially available anti-Bacillus subtilis monoclonal antibodies are absent. In conclusion, the i-PF2D-MS/MS methodology is a multidimensional approach that can be used to characterize immunogens of microorganisms from a polyclonal serum. Produced at low cost, this polyclonal antibody-based approach, permitting the identification of immunogenic candidates, will facilitate the choice of a specific epitope target for ulterior monoclonal antibody development. Abbreviations: PF2D, protein fractionating two dimensional system; 2-DLC, two-dimensional liquid chromatography; i-PF2DMS/MS, immuno-PF2D-tandem mass spectrometry.

Acknowledgment. The work has been funded by the “Region Wallonne” (Grants RW 981/3799, RW 14531-iPCRq, RW 2174

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114713-i-M@ldi, RW 114747-P3 prion, and FSE W2002134). V.R. is supported by a fellowship from ARC 99/04-245.

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