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Sensitive Detection of Bacillus anthracis Spores by Immunocapture and Liquid Chromatography Tandem Mass Spectrometry Jer^ome Chenau,† Franc-ois Fenaille,† Eric Ezan,† Nathalie Morel,† Patricia Lamourette,† Pierre L. Goossens,‡,§ and Franc-ois Becher*,† †
Service de Pharmacologie et d’Immunoanalyse, iBiTec-S, CEA, 91191 Gif-sur-Yvette Cedex, France Pathogenie des Toxi-Infections Bacteriennes, Institut Pasteur, 75724 Paris Cedex 15, France § CNRS URA 2172, Paris, France ‡
bS Supporting Information ABSTRACT: Bacillus anthracis is one of the most dangerous agents of the bioterrorism threat. We present here a sensitive immuno liquid chromatography tandem mass spectrometry (immuno LC MS/MS) approach to spore detection in complex environmental samples. It is based on the combined specificity and sensitivity of two techniques: immunocapture and targeted mass spectrometry. The immunocapture step, realized directly on the intact spores, is essential for their selective isolation and concentration from complex environmental samples. After parallel trypsin and Glu-C digestions, proteotypic peptides corresponding to small acidsoluble spore protein-B (SASP-B) are specifically monitored in the multiple reaction monitoring (MRM) mass spectrometry mode. Peptide ratio is carefully monitored and provides an additional level of specificity, which is shown to be highly useful for distinguishing closely related samples and avoiding false-positive/negative results. Sensitivity at the level of the infectious dose is demonstrated, with limits of detection of 7 103 spores/mL of milk or 10 mg of soil. This mass spectrometry approach is thus complementary to polymerase chain reaction (PCR) techniques.
B
acillus anthracis is a Gram-positive bacterium that causes the acute infectious disease anthrax.1 According to the Centers for Disease Control (CDC), B. anthracis is classified as category A agent, the highest rank of potential bioterrorism agents. The dissemination form of anthrax is a resistant structure called a spore.2 It was used as a bioterrorism weapon in 2001 in the United States when mailed letters containing B. anthracis spores caused 22 cases of inhalational and/or cutaneous anthrax infection, five of which were lethal.3 It is therefore essential to be able to reliably detect the presence of the spores. The prerequisites for an efficient method of detection of B. anthracis are sensitivity, specificity, and speed. The minimal infectious dose (ID50) is estimated to be about 103 to 5 104 spores inhaled, and the corresponding mean lethal dose (LD50) is about 8000 colony-forming units.4,5 A detection method of at least this level of sensitivity is therefore required. Detection specificity is challenged by the high genetic similarity within the B. cereus group (B. anthracis, B. cereus, and B. thuringiensis),6 and so a robust method must be able to unambiguously discriminate B. anthracis from closely related strains. Several approaches to detection of B. anthracis spores have been developed7 and can be classified into five different categories: (i) culture-based methods, (ii) immunological detection, (iii) nucleic acid based assays, (iv) ligand-based (aptamers and peptides) detection, and (v) biosensors. Herzog et al. summarized these different approaches and their corresponding limit of r 2011 American Chemical Society
detection (LOD) in environmental matrixes (soil, air, fomites, water).8 Nucleic acid based assays may be regarded as the “gold standard” and are the most sensitive techniques. For instance, real-time polymerase chain reaction (PCR), targeting regions on virulence plasmids (pXO1 and pXO2) and a segment of the B. anthracis chromosome, has been reported to detect as little as 1 pg of DNA.9 However, a frequently encountered limitation inherent to PCR analysis of environmental samples is the inhibition of the enzymatic reaction or a bias in primer reactivity due to interfering compounds (salts, inhibitors).10 Furthermore, in the context of a bioterrorism event, the combination of complementary methods is necessary to achieve reliable detection.7 In recent years, advances have been made in the field of biodefense in proteomics and mass spectrometry, which can now be considered as particularly useful tools for the detection of pathogenic microorganisms (such as B. anthracis, Yersinia pestis, and Francisella tularensis).11 For B. anthracis spores, these approaches have been used mainly for biomarker discovery. Direct profiling of spores by intact cell mass spectrometry (ICMS) highlights peaks able to discriminate B. anthracis from similar bacteria.12 Several studies have characterized peaks corresponding to small acid-soluble spore proteins (SASPs) as potential Received: August 12, 2011 Accepted: September 30, 2011 Published: September 30, 2011 8675
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Analytical Chemistry biomarkers of B. anthracis spores.13 17 SASPs are DNA-binding proteins located in the core region of Bacillus spores that protect spores from a number of external factors such as chemical and enzymatic cleavages and UV light. They are synthesized exclusively during sporulation and are degraded in the first minutes of spore germination. The concentration of SASPs in spores is substantial and varies between 8% and 20% of the total spore protein content.18 Among SASPs, SASP-B isoforms differ clearly among the species composing the B. cereus group and can thus be regarded as particularly relevant biomarkers of B. anthracis spores.13 17 This assumption was further reinforced by the recent study of Lasch et al., performed on a large cohort of samples.13 The authors analyzed 374 strains from Bacillus and related genera by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (102 strains of B. anthracis and 121 strains of B. cereus) and showed that the SASP-B peaks were among the most discriminating ones even in vegetative cells. They therefore concluded that even small amounts of residual spores in a large surplus of vegetative cells give rise to intense SASP-B signals, and so SASP-B can be regarded as a more widespread and sensitive marker of B. anthracis spores. However, up to now and to the best of our knowledge, SASP-B detection has only been performed through untargeted MS-based approaches, with a rather low sensitivity not in the range of the expected infectious dose (i.e., ∼3 105 spores detected in the most sensitive study).19 Recent examples demonstrate that proteomic approaches can be used for the sensitive and accurate detection and quantification of specific proteins in complex matrixes, especially when mass spectrometry is used in the multiple reaction monitoring mode (MRM).20,21 This mode relies on the ability of triplequadrupole mass spectrometers to detect suitable pairs of precursor and fragment ion masses (transitions) for a given peptide and by inference the corresponding protein. Several studies have already proven the potential of MRM approaches for sensitive and specific protein detection in complex matrixes, such as plasma.22,23 Regarding the detection of bioterrorism agents in complex environmental matrixes, prior isolation of the targeted component could significantly enhance the sensitivity and specificity of the mass spectrometry detection. In this aim, immunocapture could represent an efficient approach, applicable to various components such as vegetative cells,24 toxins,25 or spores.26 In this study, we report for the first time an immuno liquid chromatography tandem mass spectrometry (immuno LC MS/MS) approach for the sensitive and specific detection of B. anthracis spores in complex environmental samples through SASP-B quantification. Our process relies on immunocapture of the intact spores, followed by acid extraction and the subsequent proteolysis of proteins, and finally targeted MRM detection of proteotypic peptides corresponding to the SASP-B isoform specific to B. anthracis. This process allows detection of SASP-B and in extenso of B. anthracis spores in complex mixtures at levels compatible with the infectious dose (i.e., ∼103 104 spores).
’ EXPERIMENTAL SECTION Chemicals and Reagents. Brain heart infusion (BHI) agar was from Difco (Difco, Detroit, MI, U.S.A.), and radioselectan was from Schering (Renografin 76%; Schering, Lys Lez-Lannoy, France). Sequencing-grade modified trypsin was from Promega (Promega, Madison, WI, U.S.A.), and endoproteinase Glu-C was
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from Sigma (Sigma Chemical Co., St. Louis, MO, U.S.A.). Synthetic peptides of SASP-B protein were synthesized by Bachem (Burgdorf, Switzerland). Mouse monoclonal antibody 77A44 directed against formalin-inactivated B. anthracis 7702 spores was provided by the Laboratoire d’Etudes et de Recherches en Immunoanalyse (CEA, Saclay). The monoclonal antibody was purified using protein A and its characterization showed that it was an IgG-γ-2B isotype directed against B. anthracis major spore surface protein BclA (Morel et al., manuscript in preparation). Sodium acetate, Hepes, and bovine serum albumin (BSA) were from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, U.S.A.). Ultrapure water was from a Milli-Qplus 185 purifier (Millipore, Bedford, MA), HPLC-grade acetonitrile (ACN) was from SDS (Peypin, France), and analytical grade formic acid and trifluoroacetic acid (TFA) were from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, U.S.A.). Commercial milk powder was prepared at 5% v/w in ultrapure water. Soil was sampled in a field around the laboratory. Bacterial Strains Studied and Preparation of Spores. The bacterial strains used in this study originated from the collection of the Toxines et Pathogenie Bacterienne Unit and Pathogenie des Toxi-Infections Bacteriennes laboratory at the Institut Pasteur de Paris. Fourteen strains from the B. cereus group were selected: five B. anthracis (Sterne 7702, 9602R, RA3R, 7611R, and 6183R), six B. cereus (ATCC 569, ATCC 10987, ATCC 14579, AH259, AH819, and 97 25) and three B. thuringiensis (97 27, 97 11, and kur 97 15). B. anthracis 9602R, RA3R, 7611R, and 6183R are derivatives of the highly virulent natural isolates 9602, RA3, 7611, and 6183, respectively, from which plasmid pX02, required for human infection, has been deleted, and therefore they do not produce a capsule but still produce toxins (i.e., phenotypic equivalent of the Sterne strain) and could be handled in BSL-2 laboratory.27 Spores were produced and then purified on radioselectan (Renografin 76%) using previously described methods and were stored at 4 °C in sterile deionized water until use.28 Sample Preparation. Magnetic beads with captured IgG (beads-IgG) were prepared as described in the Supporting Information (p S-2). Twenty microliters of these beads-IgG was then added to 200 μL of spores diluted in Hepes/BSA solution (ratio 1:1 v/v). Samples were incubated for 45 min at room temperature with gentle shaking and then washed three times with Hepes buffer, pH 7.4, to remove weak nonspecific binding. For soil analysis, 10 mg of soil was mixed with 100 μL of spore preparation in water. Proteins were extracted from the spores directly on magnetic beads by using the TFA protocol for highly pathogenic microorganisms described by Lasch et al.29 Briefly, 100 μL of 80% TFA solution was added to complex beads IgG spores and vortexed for 10 min. Beads were then retained on the magnetic support, and supernatant was centrifuged at 14 000g, 4 °C for 15 min. Finally, the supernatant was transferred into Millipore’s Ultrafree MC filter tubes of 0.22 μm pore size (Millipore, Billerica, MA, U.S.A.) and spun at 10 000g for 5 min. The supernatant containing the total protein extract was stored at 20 °C until use. The total inactivation and elimination of spores were routinely checked by neutralization of TFA in the extracts and verification of the absence of colonies after culture on BHI agar, as described previously.29 Each extract was divided into two aliquots (∼2 45 μL) and dried by vacuum centrifugation to eliminate TFA. Proteins were resuspended in 20 μL of 50 mM ammonium bicarbonate 8676
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Table 1. Mass Spectrometer Settings for MRM Detection of SASP-B Isoforms theoretical
precursor
product
collision
tube
average m/z
ion (m/z)
ion (m/z)
energy (eV)
lens (V)
438.8 (3+)
438.7 (3+)
f
583.9 (b122+)
12
60
438.8 (3+)
438.7 (3+)
f
471.5 (b92+)
12
60
441.1 (3+)
441.2 (3+)
f
587.9 (b122+)
12
50
441.1 (3+)
441.2 (3+)
f
475.3 (b92+)
12
50
SRSTNKLAVPGAE
444.2 (3+)
444.2 (3+)
f
592.0 (b122+)
12
60
LVSLAEQQLGGFQK
444.2 (3+) 759.9 (2+)
444.2 (3+) 759.7 (2+)
f f
479.4 (b92+) 536.2 (y51+)
12 24
60 80
759.9 (2+)
759.7 (2+)
f
653.6 (y122+)
22
80
763.4 (2+)
763.3 (2+)
f
536.3 (y51+)
24
80
763.4 (2+)
763.3 (2+)
f
653.6 (y122+)
22
80
767.9 (2+)
767.7 (2+)
f
552.4 (y51+)
25
80
767.9 (2+)
767.7 (2+)
f
661.4 (y122+)
22
80
peptidea N-terminus peptide of Ba (Glu-C) N-terminus peptide of Ba isotopically
sequenceb ARSTNKLAVPGAE ARSTNK[13C615N]LAVPGAE
labeled (Glu-C) N-terminus peptide of Bc (Glu-C) C-terminus peptide of Ba (trypsin) C-terminus peptide of Ba isotopically
[
13
C615N]LVSLAEQQLGGFQK
labeled (trypsin) C-terminus peptide of Bc (trypsin) a
LVSLAEQQLGGYQK
Ba, B. anthracis; Bc, B. cereus. b For each peptide, the mutated amino acid is underlined.
(NH4HCO3) buffer, pH 8.0. Each sample was then reduced by the addition of 5 μL of 45 mM dithiothreitol (DTT) solution (in 50 mM NH4HCO3 buffer) at 55 °C for 25 min and alkylated by the addition of 5 μL of 100 mM iodoacetamide (IAA) solution (in 50 mM NH4HCO3 buffer) at room temperature for 35 min. Finally, enzymatic digestion was performed by the addition of 1.5 μL of 1 μg/μL trypsin or Glu-C (in 50 mM NH4HCO3 buffer) with incubation overnight at 37 °C. Each digest (30 μL) was mixed with 30 μL of internal standard (IS) solution (see the Supporting Information for preparation of the IS). Chromatographic and Mass Spectrometric Conditions. LC MS/MS experiments were performed using an HP 1100 HPLC system from Agilent (Palo Alto, CA, U.S.A.) coupled to a triple-quadrupole TSQ Quantum Ultra mass spectrometer (Thermo Scientific, San Jose, CA, U.S.A.). Chromatographic separation was performed on a Zorbax SB-C18 column (150 mm 2.1 mm i.d., 5 μm particle size, 300 Å porosity) from Agilent Technology (Palo Alto, CA, U.S.A.) as described in the Supporting Information. The column effluent was directly introduced into the electrospray source of the mass spectrometer. Analyses were performed in the positive ion mode (see the Supporting Information for MS parameters). Two MRM transitions were monitored for each peptide to improve detection specificity. Table 1 reports the MRM transitions monitored and their optimized tube lens and collision energy values.
’ RESULTS AND DISCUSSION Analytical Strategy. We have developed a targeted immuno LC MS/MS (MRM) process for sensitive detection of B. anthracis spores and their discrimination from other closely related species of the B. cereus group (B. cereus and B. thuringiensis) (Supporting Information Figure S-1). The first step of the assay is a specific capture of B. anthracis spores, using magnetic beads coated with antibodies directed against B. anthracis major spore surface protein BclA. This immunocapture step is essential to isolate specifically and purify spores from complex mixtures and to concentrate them for further improved sensitivity. Proteins are then directly extracted on the beads using a 80% TFA solution. Such strong acid solutions have been reported to selectively
solubilize SASPs, promoting their extraction from spores.13,18,29 After acid neutralization, protein extracts are digested with two endoproteases (trypsin and Glu-C) in parallel and the resulting SASP-B proteotypic peptides are monitored by mass spectrometry operating in the MRM mode. Development of the LC MRM Method. The amino acid sequences of many SASPs are species-specific, and several studies have underlined a characteristic SASP-B isoform in B. anthracis spores.13 16,19 Moreover, since B. anthracis is a highly conserved species,30 this particular SASP-B isoform should be retrieved in all strains of B. anthracis. So we chose to target the SASP-B isoform specific to B. anthracis and also the corresponding isoforms of other closely related bacteria (B. cereus and B. thuringiensis). SASP-B is a 65 amino acid protein present exclusively in the spores where it is expressed at a high level. The mature form has undergone a methionine cleavage at the N-terminus. As previously described,14,31 the B. anthracis isoform is characterized by the presence of two particular amino acids in the sequence, one near the N-terminus (alanine in position 2) and the other near the C-terminus (phenylalanine in position 63) (Figure 1). Only B. anthracis spores have a SASP-B carrying simultaneously these two particular amino acids (isoform of 6674.45 Da); other B. cereus group bacteria have only one (isoform of 6690.45 Da) or none (isoform of 6706.44 Da) of them (serine in position 2 and/ or tyrosine in position 63). With the aim of specifically detecting SASP-B isoforms, Swatkoski et al. have established the benefit of observing both the precursor proteins and their corresponding hydrolyzed peptides.17 We first directly monitored the intact SASP-B proteins using a high-resolution LTQ Orbitrap mass spectrometer operating in the MS mode. The sensitivity achieved was ∼105 spores, which is similar to the best results obtained by MALDI-TOF analysis17 but insufficient to be at the level of infectious dose (103 104 spores) (data not shown). Switching to the MRM acquisition mode using a triple-quadrupole mass spectrometer did not improve the sensitivity because the low fragmentation yield did not produce intense and specific product ions. Moreover the chromatographic behavior was poor (data not shown). We therefore decided to detect only specific SASP-B proteotypic peptides, but with improved sample preparation and MS 8677
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Figure 1. Amino acid sequences of the four different SASP-B isoforms found in the B. cereus group.
analysis to encompass the absence of intact protein detection. A first optimization was the use of a specific anti-B. anthracis spore antibody for the immunocapture. The second was to target both N- and C-terminus SASP-B peptides and carefully monitor the ratio of these two peptides for a highly specific detection (see below the section on detection selectivity). Particular attention was paid to the choice of targeted peptides. The analysis of amino acid sequences revealed that the use of only one endoprotease is not sufficient to obtain suitable proteotypic peptides, i.e., specific peptides with optimal LC separation and MS fragmentation characteristics (Figure 1). Trypsin was employed to obtain a C-terminus signature peptide since it cleaves after the arginine in position 51 to generate a 14-residue proteotypic peptide that carries the specific amino acid in position 63 (phenylalanine or tyrosine). However, the arginine in position 3 at the N-terminus would generate a peptide of two amino acids in length and so preclude the use of trypsin to produce an adequate N-terminus peptide. Thus, endoprotease Glu-C was employed to obtain the N-terminus signature peptide. It cleaves after the glutamic acid in position 14 to generate a 13-residue proteotypic peptide that carries the mutated amino acid in position 2 (alanine or serine). Glu-C also produces a peptide at the C-terminus by cleaving after glutamic acid in position 57, but this peptide exhibited poor stability since the N-terminus glutamine partially appeared under a pyro-glutamate form that precludes its use for the MRM analysis. Finally, both these endoproteases were required to obtain suitable specific peptides for monitoring the specific B. anthracis SASP-B isoform during the MRM analysis and so to discriminate B. anthracis spores from other B. cereus species. Moreover, a BLAST similarity search was performed using the whole Uniprot database to confirm peptide specificity for SASPB proteins from the B. cereus group bacteria (data not shown). Peptides corresponding to the different N- and C-terminus proteotypic peptides retrieved for SASP-B of the B. cereus group bacteria were synthesized (Table 1). The N- and C-terminus peptides of the B. anthracis isoform were also produced in an isotopically labeled form to be used as internal standard. Each peptide was injected into the mass spectrometer and analyzed by MS and MS/MS to further optimize analytical conditions. The electrospray ionization mass spectrometry (ESI-MS) (positive ion mode) analysis gave prominent triply charged ions for the N-terminus peptides and doubly charged ions for the C-terminus peptides. The measured masses were in agreement with the expected ones (Table 1). These precursor ions were then submitted to collision-induced dissociation (CID) fragmentation (Supporting Information Figure S-2). The analysis of MS/MS spectra showed preferential cleavages after proline and acidic amino acids in accordance with classic fragmentation rules (e.g., see fragment ions b9 and b12 in Supporting Information
Figure S-2A). Among the most intense product ions in the MS/MS spectra, two carrying the specific mutation of each isoform were selected for development of the MRM method. For each peptide, two MRM transitions were monitored for improved detection specificity. Moreover, when possible, product ions were selected with an m/z ratio higher than that of the parent ion (see Supporting Information Figure S-2A). For a given peptide, the retention time observed for the two MRM transitions monitored and the ratio between these two transition areas must match those obtained when analyzing corresponding IS peptides under the same analytical conditions. Table 1 reports the MRM transitions designed and monitored for each peptide and the optimized analytical parameters (tube lens and collision energy values). Detection Selectivity. As previously mentioned, selectivity is crucial when B. anthracis spore detection is envisioned. In order to establish the selectivity of the whole analytical process, purified spores from 14 different strains were analyzed (Table 2). Strains were chosen to cover previously described phylogenetic distribution in the B. cereus group.6 For example, Bt 97 27 is very close to B. anthracis genetically, whereas Bc 14579 and Bc 569 are more distant. A first level of selectivity was achieved by the specific antibody capture. The efficiency of spore capture for each strain was calculated as the percentage of the normalized MRM areas observed with and without immunocapture, i.e., immunocapture yield (Table 2). The antibody is directed against B. anthracis spores and so greatly promotes the specific extraction of these spores (Morel et al., manuscript in preparation). Immunocapture yield was between 49% and 99% for the different B. anthracis spores, but was below ∼1% for other B. cereus group strains. It should be noted that efficient capture (87%) was observed for Bt 97 11 spores. However, mass spectrometry would easily discriminate this strain. The second level of selectivity was provided by the MRM analysis itself. Of the 14 strains screened, the five B. anthracis showed the two SASP-B peptides of the B. anthracis species. Of the 10 strains of the other members of the B. cereus group, five had the two variant peptides, i.e., A f S and F f Y (isoform of 6706.44 Da), whereas four had only one peptide affected (isoforms of 6690.45 Da) (Figure 1). Typical LC MS/MS chromatograms obtained for B. anthracis spores (i.e., Ba RA3R) and for three spores from other strains of the B. cereus group (i.e., Bc ATCC 10987, Bc AH259, and Bc AH819) are shown in Supporting Information Figure S-3. Moreover, for the five B. anthracis strains analyzed, the ratio of the normalized signals detected for the SASP-B N- and C-terminus peptides was calculated and used as confirmation (Table 2). This ratio was rather homogeneous (between 8.9 and 13.3 for all B. anthracis strains and 10.9 ( 1.6 on average), which proves that parallel 8678
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Table 2. SASP-B Isoforms Detected in 14 Strains of the B. cereus Group N-Ter Peptide strainsa
C-Ter Peptide
SASP-B MW (Da)b
Ba (A)c
Bc (S)c
Ba (F)c
Bc (Y)c
ratiod
IC yield (%)e
Ba 7702
6674.45
59.1
ND
3.84
ND
15.4
57
Ba 9602R
6674.45
1027
ND
67.1
ND
15.3
55
Ba RA3R
6674.45
156.1
ND
10.9
ND
14.3
87
Ba 7611R
6674.45
131.1
ND
9.75
ND
13.4
99
Ba 6183R
6674.45
55.3
ND
4.85
ND
11.4
49
Bc AH819
6690.45
5.59
ND
ND
0.16
ND
2
Bc 10987
6690.45
ND
0.12
0.01
ND
ND
1
Bc 97 25 Bc 569
6690.45 6706.44
12.3 ND
ND 2.58
ND ND
0.33 0.09
ND ND
3 1
Bc 14579
6706.44
ND
1.15
ND
0.06
ND
1
Bc AH259
6706.44
ND
0.71
ND
0.04
ND
2
Bt 97 27
6690.45
18.4
ND
ND
0.65
ND
4
Bt 97 11
6706.44
ND
89.1
ND
3.80
ND
83
Bt Kur
6706.44
ND
0.67
ND
0.04
ND
1
a
Ba, B. anthracis; Bc, B. cereus; Bt, B. thuringiensis. b MW: molecular weight of the detected SASP-B protein isoform. c Means of the normalized peak area detected. ND: not determined. d Ratio between N- and C-terminus peptide normalized areas calculated for SASP-B of B. anthracis spores. e IC yield: immunocapture yield calculated as the percentage (%) of normalized peak area of SASP-B peptides with and without spore immunocapture.
Table 3. Selectivity of SASP-B Detection in Spore Mixtures mixturea strain 1 (SASP-B MW)
IC yield (%)d
SASP-B Ba peptides
strain 2 (SASP-B MW)
spores proportionb
N-Terc
C-Terc
ratioc
6.90 6.44
12.0 12.5
strain 1
strain 2
74 80
ND 3.6
Ba RA3R A2F63
Bc 14579 S2Y63
10:0 10:1
82.96 80.59
(6674.45 Da)
(6706.44 Da)
10:10
82.75
5.47
15.1
83
2.7
1:10
7.11
0.58
12.3
87
4.6
0:10
0.00
0.00
ND
ND
2.3
Bc AH819
Bc 10987
10:0
20.59
0.00
ND
8.1
ND
A2Y63
S2F63
10:1
24.14
0.0007
35914
6.9
0.25
(6690.45 Da)
(6690.45 Da)
10:10 1:10
18.93 1.41
0.0078 0.0070
2429 201
8.0 4.8
0.47 0.41
0:10
0.00
0.0076
ND
ND
0.34
a
Spores from four strains were used: B. anthracis RA3R (SASP-B of 6674.45 Da with the A2F63 combination, i.e., alanine in the N-terminus and phenylalanine in the C-terminus peptide); B. cereus 14579 (SASP-B of 6706.44 Da with the S2Y63 combination); B. cereus AH819 (SASP-B of 6690.45 Da with the A2Y63 combination); B. cereus 10987 (SASP-B of 6690.45 Da with the S2F63 combination). b In each case, spores from two strains were mixed in five different ratios: 10:0; 10:1; 10:10; 1:10; 0:10. c Means of the normalized N- and C-terminus peptide peak areas of SASP-B from B. anthracis and ratio between them. ND: not determined d IC yield: immunocapture yield calculated as the percentage (%) of normalized peak area SASP-B peptides with and without spore immunocapture.
proteolysis by trypsin and Glu-C constitutes a reproducible process. This ratio represents a simple and robust way to ensure that the two signature peptides specifically indicate the presence of B. anthracis, which is crucial to avoid false-positive results. The capacity to specifically point out the presence of B. anthracis spores in mixtures of different Bacillus species is critical. Therefore, we designed two different test mixtures (with varying concentrations of two distinct species) that could lead to ambiguous results with MRM signals for the four SASP-B peptides monitored. A first binary mixture of B. anthracis (strain RA3R) and B. cereus (strain 14579) spores was prepared to reflect potential artifacts linked to B. cereus, and a second one contained spores from two B. cereus strains (AH819 and 10987) that can potentially generate false-
positive results (Table 3). In the former case, and whatever the concentrations of the two strains, the peptide ratios were in good agreement with the values observed for pure species (variation ∼16% around average values), thus confirming the presence of B. anthracis. In contrast, the peptide ratios obtained for the second test mixture involving only B. cereus species were considerably modified, with a 20- to 200-fold increase, which unambiguously reflects the absence of B. anthracis. Altogether these data confirm that this peptide ratio combined with the specificity of the immunocapture step (immunocapture yield ∼80% for B. anthracis spores vs 0.2 8% for B. cereus spores) is a particularly efficient and reliable way of confirming the presence/absence of B. anthracis strains in suspect samples. 8679
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Figure 2. Assay sensitivity in water. A 100 μL sample of B. anthracis 7702 spores prepared in water was analyzed. (A) Regression curves expressed as the number of initial spores. (B) LC MS/MS chromatograms for each transition at the instrumental LOD, i.e., 7 103 spores.
Our experimental data and previous published findings on a large number of strains (Lasch et al., 102 Ba and 121 Bc strains13) underline the value of the SASP-B protein in specifically detecting B. anthracis spores. However, it should be noted that exceptions may be expected for a few B. cereus spores as deduced from an in silico study of publicly available databases (Uniprot and EMBLEBI databases), for example, with the virulent natural African chimpanzee isolate B. cereus CI (carrying pXO1 and pXO2 plasmids of B. anthracis) that is predicted to have exactly the same SASP-B isoform as B. anthracis strains (data not shown). Nevertheless, one should bear in mind that this particular strain corresponds to a virulent B. cereus that it would also be useful to detect. Studies with such specific strains are under way. Last but not least, it should be emphasized that all the B. anthracis spores described up to now shared the same SASP-B isoform, and above all no false-negative results were obtained in the present study. Assay Sensitivity. To determine the sensitivity of our method, we assessed its linear range and LOD with purified B. anthracis 7702 spores diluted in water (Figure 2A). LOD is an important measure of performance as it defines the lowest analyte quantity (i.e., B. anthracis spores) that can be accurately measured. The coefficient of determination (r2) indicated a good linearity of the quantification in the range of 7 103 to 7 107 spores. MRM signals for each transition of both the N- and C-terminus
peptides were still differentiable from background noise at the dilution of 7 103 initial spores, corresponding to the instrumental LOD (Figure 2B). This LOD was in excellent agreement with the required level of sensitivity (i.e., ID50 ∼ 103 to 5 104 spores). Application to Environmental Samples. In the case of a bioterrorism event, it is essential to ascertain whether suspicious samples are actually contaminated by B. anthracis spores. B. anthracis spores are the most stable category A agent overall,32 so traces of past contamination should remain detectable. Contamination by B. anthracis could be achieved by different methods of exposure, such as infected food ingestion (e.g., milk) or aerosol inhalation (e.g., powder). It is crucial to validate the applicability of our newly developed immuno LC MS/MS method to such complex environmental samples and to evaluate the corresponding sensitivity. We investigated detection in two different complex matrixes (milk and soil). It should be emphasized that it was not possible to detect spores in these samples without any prior spore immunocapture, which is in line with previously reported data26 and illustrates the method’s usefulness. To determine the sensitivity of the method, different quantities of B. anthracis 7702 spores were spiked into milk and soil samples. For milk samples, analyses were performed with a sample volume of 10 mL. The immunocapture step allowed the spores to be easily extracted/concentrated from such a high 8680
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Analytical Chemistry sample volume, thus yielding improved detection sensitivity (data not shown). The determined LOD was 7 103 spores/mL of milk or and 7 103 spores in 10 mg of soil (Supporting Information Figure S-4). Moreover, the regression curves were linear for the studied range, demonstrating that matrix components do not significantly impact the method’s robustness and sensitivity. Negative controls (i.e., milk or soil blank samples) did not show any interfering signal, thus underlining the efficiency of the immunocapture and the specificity of the LC MS/MS detection. No MRM signal was observed in soil for signature peptides corresponding to B. cereus spores, which indicates that these strains, usually found in natural soils, were either not present in our sample or not recognized by the antibodies. The reproducibility of the assay was also evaluated by independently preparing and analyzing four different milk samples spiked with the same amount of B. anthracis spores (7 106 spores spiked into 10 mL of milk). Coefficients of variation (CV) of 5% and 3% were obtained for the N- and C-terminus peptides, respectively. These values showed that the method has good precision and reproducibility despite the multiple steps involved in the sample preparation (i.e., immunocapture of spores, 80% TFA extraction, enzymatic digestion, and LC MS/MS analysis). These results constitute a relevant proof of concept for the application of our immuno LC MS/MS process to the sensitive and specific detection of B. anthracis spores in environmental samples.
’ CONCLUSION We have developed a mass spectrometry approach to the detection of B. anthracis spores. It combines the specificity and sensitivity of two complementary methods, immunocapture and targeted mass spectrometry, allowing detection of spores at the level of the inhalation dose, i.e., 7 103 spores. To our knowledge, this is the first time that such sensitivity has been achieved in the detection of B. anthracis spores by mass spectrometry. The method is intended to be implemented in regulatory laboratories for confirmation, within 24 h, of results obtained with rapid field detection assays. Further improvements could be made for highthroughput use. For instance, sample throughput could be increased via automation of the immunocapture step. In addition, proteolysis times could be significantly reduced using rapid digestion protocols (from overnight to a few minutes) such as microwave-assisted digestion33 or aqueous organic conditions.34 The final short-/medium-term objective is to reduce the time to results to a few hours, which is far below the time needed for culture-based methods. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: 33-1-69-08-13-15. Fax: 33-1-69-08-59-07. E-mail:
[email protected].
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’ ACKNOWLEDGMENT We wish to warmly thank Christophe Brezillon for assuming the co-responsibility for the National Reference Center for Anthrax, and for his expert help in discussing and providing the bacterial strains needed for this study. We are deeply indebted to both Christophe Brezillon and Michel TCM Haustant for their expert assistance. ’ REFERENCES (1) Mock, M.; Fouet, A. Annu. Rev. Microbiol. 2001, 55, 647–671. (2) Driks, A. Mol. Aspects Med. 2009, 30 (6), 368–73. (3) Bartlett, J. G.; Inglesby, T. V.; Borio, L. Clin. Infect. Dis. 2002, 35 (7), 851–858. (4) Franz, D. R.; Jahrling, P. B.; Friedlander, A. M.; McClain, D. J.; Hoover, D. L.; Bryne, W. R.; Pavlin, J. A.; Christopher, G. W.; Eitzen, E. M. JAMA, J. Am. Med. Assoc. 1997, 278 (5), 399–411. (5) Peters, C. J.; Hartley, D. M. Lancet 2002, 359 (9307), 710–711. (6) Kolstø, A. B.; Tourasse, N. J.; Økstad, O. A. Annu. Rev. Microbiol. 2009, 63, 451–476. (7) Rao, S. S.; Mohan, K. V.; Atreya, C. D. J. Microbiol. Methods 2010, 82 (1), 1–10. (8) Herzog, A. B.; McLennan, S. D.; Pandey, A. K.; Gerba, C. P.; Haas, C. N.; Rose, J. B.; Hashsham, S. A. Appl. Environ. Microbiol. 2009, 75 (19), 6331–6339. (9) Hoffmaster, A. R.; Meyer, R. F.; Bowen, M. D.; Marston, C. K.; Weyant, R. S.; Thurman, K.; Messenger, S. L.; Minor, E. E.; Winchell, J. M.; Rassmussen, M. V.; Newton, B. R.; Parker, J. T.; Morrill, W. E.; McKinney, N.; Barnett, G. A.; Sejvar, J. J.; Jernigan, J. A.; Perkins, B. A.; Popovic, T. Emerging Infect. Dis. 2002, 8 (10), 1178–1182. (10) Arbeli, Z.; Fuentes, C. L. FEMS Microbiol. Lett. 2007, 272 (2), 269–275. (11) Demirev, P. A.; Fenselau, C. J. Mass Spectrom. 2008, 43 (11), 1441–1457. (12) Hathout, Y.; Demirev, P. A.; Ho, Y. P.; Bundy, J. L.; Ryzhov, V.; Sapp, L.; Stutler, J.; Jackman, J.; Fenselau, C. Appl. Environ. Microbiol. 1999, 65 (10), 4313–4319. (13) Lasch, P.; Beyer, W.; Nattermann, H.; St€ammler, M.; Siegbrecht, E.; Grunow, R.; Naumann, D. Appl. Environ. Microbiol. 2009, 75 (22), 7229–7242. (14) Castanha, E. R.; Fox, A.; Fox, K. F. J. Microbiol. Methods 2006, 67 (2), 230–240. (15) Castanha, E. R.; Vestal, M.; Hattan, S.; Fox, A.; Fox, K. F.; Dickinson, D. Mol. Cell. Probes 2007, 21 (3), 190–201. (16) Hathout, Y.; Setlow, B.; Cabrera-Martinez, R. M.; Fenselau, C.; Setlow, P. Appl. Environ. Microbiol. 2003, 69 (2), 1100–1107. (17) Swatkoski, S.; Russell, S. C.; Edwards, N.; Fenselau, C. Anal. Chem. 2006, 78 (1), 181–188. (18) Setlow, P. Annu. Rev. Microbiol. 1988, 42, 319–338. (19) Demirev, P. A.; Feldman, A. B.; Kowalski, P.; Lin, J. S. Anal. Chem. 2005, 77 (22), 7455–7461. (20) Picotti, P.; Rinner, O.; Stallmach, R.; Dautel, F.; Farrah, T.; Domon, B.; Wenschuh, H.; Aebersold, R. Nat. Methods 2010, 7 (1), 43–46. (21) Makawita, S.; Diamandis, E. P. Clin. Chem. 2010, 56 (2), 212–222. (22) Kuzyk, M. A.; Smith, D.; Yang, J.; Cross, T. J.; Jackson, A. M.; Hardie, D. B.; Anderson, N. L.; Borchers, C. H. Mol. Cell. Proteomics 2009, 8 (8), 1860–1877. (23) Anderson, L.; Hunter, C. L. Mol. Cell. Proteomics 2006, 5 (4), 573–588. (24) Madonna, A. J.; Basile, F.; Furlong, E.; Voorhees, K. J. Rapid Commun. Mass Spectrom. 2001, 15 (13), 1068–1074. (25) Boyer, A. E.; Quinn, C. P.; Woolfitt, A. R.; Pirkle, J. L.; McWilliams, L. G.; Stamey, K. L.; Bagarozzi, D. A.; Hart, J. C.; Barr, J. R. Anal. Chem. 2007, 79 (22), 8463–8470. (26) Whiteaker, J.; Karns, J.; Fenselau, C.; Perdue, M. L. Foodborne Pathog. Dis. 2004, 1 (3), 185–194. 8681
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