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Anal. Chem. 2003, 75, 5618-5627

Characterization of Bacillus Spore Species and Their Mixtures Using Postsource Decay with a Curved-Field Reflectron Bettina Warscheid* and Catherine Fenselau

Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742

A strategy is proposed for the rapid identification of Bacillus spores, which relies on the selective release of a family of proteins, referred to as small, acid-soluble spore proteins (SASPs). In this work, SASPs were selectively solubilized from Bacillus spores on the MALDI sample plate by using 10% TFA. Proteolytic digests of SASPs generated in situ from spores of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki HD-1, B. cereus T, and the nonpathogenic strain B. anthracis Sterne were prepared in 5-25 min by using trypsin immobilized on Agarose beads and subsequently analyzed by MALDI-TOFMS using a curved-field reflectron. Protein identification was obtained by partial sequencing of distinctive tryptic peptides from Bacillus spores via postsource decay analysis combined with genome-based database searches by Mascot Sequence Query. Various unique SASPs were identified, allowing the characterization of Bacillus species by obtaining sequence-specific information on single peptides. The applicability of this approach for the rapid identification of Bacillus species was further established by analyzing spore mixtures. To date, a number of approaches have been used for the identification of microorganisms, applying the analytic power of mass spectrometry.1 Among these, electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have provided access to cellular proteins as biomarkers. MALDI provides several advantages for the rapid analysis of complex biological samples such as bacteria, e.g., formation primarily of singly charged ions,2 high sensitivity, tolerance to contaminants,3 and alleviation of sample preparation in general. In several previous studies, proteins have first been separated from microbial debris by extraction and subsequently isolated by sophisticated techniques, e.g., two-dimensional gel electrophoresis or high-performance liquid chromatography, for mass spectrometric analysis.4-17 For identification of proteolytic peptides derived from these proteins, partial sequencing has been per* Corresponding author. Phone: (301) 405-8618. Fax: (301) 405-8615. E-mail: [email protected]. (1) Fenselau, C.; Demirev, P. A. Mass Spectrom Rev. 2001, 20, 157-171. (2) Karas, M.; Glu ¨ ckmann, M.; Scha¨fer, J. J. Mass Spectrom. 2000, 35, 1-12. (3) Lay, O. J., Jr. Mass Spectrom Rev. 2001, 20, 172-194. (4) Harris, W. A.; Reilly, J. P. Anal. Chem 2002, 74, 4410-4416. (5) Vaidyanathan, S.; Winder, C. L.; Wade, S.; Kell, D. B.; Goodrace, R. Rapid Commun. Mass Spectrom. 2002, 16, 1276-1286. (6) Cargile, B. J.; McLuckey, S. A.; Stephenson, J. L. Anal. Chem. 2001, 73, 1277-1285.

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formed by collision-induced dissociation (CID) or postsource decay (PSD)18 analysis. Tandem mass spectrometry of intact isolated bacterial proteins of moderately low molecular mass can also provide limited sequence information for characterization by database searches of both the protein and its microbial source.19,20 To avoid time-consuming separation and isolation steps, intact microorganisms have been lysed on the sample holder and released biomarkers directly analyzed by MALDI.21-23 Based on the suite of biomarkers detected, either mass mapping with a carefully created library of microbial mass signatures24,25 or mass matching to protein masses predicted from the genome26-28 enables microorganism identification. More recently, an approach for microbial mixture characterization by MALDI-TOFMS has (7) Zhou, X.; Gonnet, G.; Hallet, M.; Muenchbach, M.; Folkers, G.; James, P. Proteomics 2001, 1, 683-690. (8) Krishnamurthy, T.; Rajamani, U.; Ross, P. L.; Jabbour, R.; Nair, H.; Eng, J.; Yates, J.; Davis, M. T.; Stahl, D. C.; Lee, T. D. J. Toxicol. Toxin Rev. 2000, 19, 95-117. (9) Xiang, F.; Anderson, G. A.; Veenstra, T. D.; Lipton, M. S.; Smith, R. D. Anal. Chem. 2000, 72, 2475-2481. (10) Arnold, R. J.; Reilly, J. P. Anal. Biochem. 1999, 269, 105-112. (11) Holland, R. D.; Duffy, C. R.; Rafii, F.; Sutherland, J. B.; Heinze, T. M.; Holder, C. L.; Voorhees, K. J.; Lay, J. O. Anal. Chem. 1999, 71, 3226-3230. (12) Yates, J. R.; Eng, J. K. U.S. Patent 5,538,879, 1996. (13) Dai, Y.; Liang, L.; Roser, D. C.; Long, S. R. Rapid Commun. Mass Spectrom. 1999, 13, 73-78. (14) Lopaticki, S.; Morrow, C. L.; Gorman, J. J. J. Mass Spectrom. 1998, 33, 950-960. (15) Liu, C.; Hofstadler, S. A.; Bresson, J. A.; Udseth, H. R.; Tsukuda, T.; Smith, R. D.; Snyder, A. P. Anal. Chem. 1998, 70, 1797-1801. (16) Despeyroux, D.; Phillpotts, R.; Watts, P. Rapid Commun. Mass Spectrom. 1996, 10, 937-941. (17) Cain, T. C.; Lubman, D. M.; Weber, W. J., Jr. Rapid Commun. Mass Spectrom. 1994, 8, 1026-1030. (18) Spengler, B. J. Mass Spectrom. 1997, 32, 1019-1036. (19) Demirev, P. A.; Ramirez, J.; Fenselau, C. Anal. Chem. 2001, 73, 57255731. (20) Reid, G. E.; Shang, H.; Hogan, J.; Lee, G. U.; McLuckey, S. A. J. Am. Chem. Soc. 2002, 124, 7353-7362. (21) Claydon, M. A.; Davey, S. N.; Edward-Jones, V.; Gordon, D. B. Nat. Biotechnol. 1996, 14, 1584-1593. (22) Holland, R. D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Voorhees, K. J.; Lay, J. O. Rapid Commun. Mass Spectrom. 1996, 10, 1227-1232. (23) Krishnamurthy, T.; Ross, P. L.; Rajamani, U. Rapid Commun. Mass Spectrom. 1996, 10, 883-888. (24) Conway, G. C.; Smole, S. C.; Sarracino, D. A.; Arbeit, R. D.; Leopold, P. E. J. Mol. Microbiol. Biotechnol. 2001, 3, 103-112. (25) Jarman, K. H.; Cebula, S. T.; Saenz, C.; Peterson, C. E.; Valentine, N. B.; Kingsley, M. T.; Wahl, K. L. Anal. Chem. 2000, 72, 1217-1223. (26) Demirev, P. A.; Lin, Y. S.; Pineda, F. J.; Fenselau, C. Anal. Chem. 2001, 73, 4566-4573. (27) Pineda, F. J.; Lin, J. S.; Fenselau, C.; Demirev, P. A. Anal. Chem. 2000, 72, 3739-3744. (28) Demirev, P. A.; Ho, Y.-P.; Ryzhov, V.; Fenselau, C. Anal. Chem. 1999, 71, 2732-2738. 10.1021/ac034200f CCC: $25.00

© 2003 American Chemical Society Published on Web 09/16/2003

been presented, testing complex search algorithms for fingerprint library matching.29 Drawbacks encountered in MALDI-TOFMS analysis on the protein level include low mass accuracy, limited mass resolution, and poor spectral reproducibility. On-probe enzymatic digestion of proteins obtained from simple viruses without fractionation or isolation has been proposed to overcome these limitations, using partial sequencing of peptides derived from abundant proteins for rapid identification by database searching.30,31 When this strategy is applied to more complex microorganisms, e.g., Clostridium and Bacillus species, however, a large mixture of peptides is obtained, with poor signal-to-noise ratios, low sensitivity for partial sequencing experiments, and limited reproducibility. Among these microorganisms, spores of the genus Bacillus are of particular interest since they are monitored as important targets in battle spaces, immune buildings, counterterrorism activities, and some environmental and medical fields. Bacillus species also require discrimination between pathogenic and nonpathogenic species. The rigidity and chemical resistance of the outer spore coat makes the direct desorption of proteins from Bacillus species in the dormant (spore) state challenging.32 Nonetheless, biomarkers such as the abundant small, acid-soluble protein (SASP) family can be extracted from Bacillus spores by treatment with acids.33,34 Since sequences differ among the SASPs, which taken in total are distinctive for different Bacillus species, identification of Bacillus spores on the species or even strain level is feasible.33,35 In addition, the utility of SASPs of bacterial endospores as tools for investigating the molecular evolution of closely related species from the Bacillus cereus group has been recently explored.36 Several SASPs are constituents of the bacterial endospore cytoplasm protecting the spore chromosome from DNA damage by, for example, heat37 or UV radiation.38,39 The γ-type SASPs play an important role in spore germination functions and processes.40, 41 In this study, we report selective solubilization of SASPs from several spore species of the genus Bacillus on the MALDI sample plate, followed by rapid proteolytic digestion of SASPs. MALDIMS spectra of proteolytic digests generated in situ were carefully evaluated for species-unique tryptic peptides. Partial sequencing of distinctive peptides was performed by PSD analysis, revealing a wealth of sequence-specific information. Peptide and product ion masses were used in database searches to identify Bacillus (29) Wahl, K. L.; Wunschel, S. C.; Jarman, K. H.; Valentine, N. B.; Petersen, C. E.; Kingsley, M. T.; Zartolas, K. A.; Saenz, A. J. Anal. Chem. 2002, 74, 6191-6199. (30) Yao, Z.-P.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2002, 74, 2529-2534. (31) Yao, Z-P.; Afonso, C.; Fenselau, C. Rapid Commun. Mass Spectrom. 2002, 16, 1953-1956. (32) Driks, A. Microbiol. Mol. Biol. Rev. 1999, 63, 1-20. (33) Hathout, Y.; Setlow, B.; Cabrera-Martinez, R. M.; Fenselau, C.; Setlow, P. Appl. Environ. Microbiol. 2003, 1100-1107. (34) Setlow, P. J. Bacteriol. 1978, 136, 331-340. (35) El-Helow, E. R. FEMS Microbiol. Lett. 2001, 196, 119-122. (36) McKinney, N.; Goldman, S.; Hunter-Cevera, J.; Leighton, T.; Long, G.; Nelson, B. Society for Applied Microbiology, 3rd International Anthrax Conference, Plymouth, 1998. (37) Setlow, B.; Setlow, P. Appl. Environ. Microbiol. 1995, 61, 2787-2790. (38) Mason, J. M.; Setlow, P. J. Bacteriol. 1986, 167, 174-178. (39) Setlow, P. Annu. Rev. Microbial. 1995, 49, 29-54. (40) Cucchi, A.; Rivas, C. S. D. Curr. Microbial. 1998, 36, 220-225. (41) Ruzal, S. M.; Alice, A. F.; Sanchez-Rivas, C. Microbiology 1994, 140, 21732177.

spore proteins. This strategy was also applied to rapidly identify Bacillus spores in mixtures. EXPERIMENTAL SECTION Materials and Bacillus Spores. Trifluoroacetic acid (TFA) and R-cyano-4-hydroxycinnamic acid (R-HCHA) were purchased from Sigma Chemical Co. (St. Lous, MO). Trypsin immobilized on Agarose beads was purchased from Pierce Biotechnology (Rockford, IL) and washed with 25 mM NH4HCO3 before use. Bacillus cereus T, Bacillus thuringiensis subs. Kurstaki HD-1, Bacillus subtilis 168, and Bacillus globigii spores were grown by standard techniques described elsewhere.42 Spores of the nonpathogenic strain Bacillus anthracis Sterne were grown on Agar plates containing new sporulation media. Spores were harvested, purified by lysozyme treatment and salt detergent washes,43,44 and stored at -20 °C before use. Sample Preparation. Aliquots of 0.8 µL of aqueous Bacillus spore suspension (2.5 mg/mL) were placed onto a MALDI sample plate and mixed with 1.0 µL of diluted TFA (10% in H2O). Bacillus spore samples treated by acid were allowed to air-dry before adding 1 µL of immobilized trypsin suspended in 25 mM NH4HCO3. Spore samples prepared on the MALDI plate were then covered with a humidified chamber (100% relative humidity) at room temperature to prevent sample drying. Digestion was stopped after 5-25 min by allowing the samples to dry and addition of 0.5 µL of aqueous 0.1% TFA solution. Aliquots of 0.8 µL of R-HCHA (100 mM in 70:30:0.1 ACN/H2O/TFA) were placed on proteolytic digests from Bacillus spores for MALDI analysis. Mass Spectrometry. MALDI spectra were obtained using an Axima-CFR time-of-flight instrument (Kratos Analytical by Shimadzu Biotech, Manchester, U.K.) with delayed extraction operated in the reflectron positive ion mode. Generally, the MALDITOFMS spectra presented are a sum of 100 laser shots rastered across the sample surface with a laser power of 40 arbitrary units (range of laser power 0-180, where 0 represents minimum and 180 maximum transmission). For PSD analysis, protonated peptide ions were isolated with an ion gate set to a 10-15-Da window. Enhanced fragmentation of parent ions was observed by increasing the laser power by 40%. Standard peptides were used for external calibration. Database Search. Protonated peptide and product ion masses were used for database searches in the NCBInr database taxonomically restricted to bacteria (eubacteria). Mascot Sequence Query45 parameters were usually set as follows: missed cleavages, up to 1; protein mass, unrestricted; product ion matches, b- and y-type ions; peptide ion mass tolerance, (0.5 Da; product ion mass tolerance, (1 Da. Since PSD spectra showed intense and wellresolved ion signals, product ions with signal intensities at least 5% above the noise level were included in database searches. Product ions formed by expulsion of small molecules, e.g., H2O, were not removed for database searching. (42) Hathout, Y.; Demirev, P. A.; Ho, Y.-P.; Bundy, J.; Ryzhov, V.; Sapp, L.; Stutler, J.; Jackman, J.; Fenselau, C. Appl. Environ. Microbiol. 1999, 65, 43134319. (43) Nicholson, W. L.; Setlow, P. In Molecular biological methods for Bacillus; Harwood, C. R., Cutting, S. M., Eds.; John Wiley and Sons: Chichester, England, 1990; pp 391-450. (44) Jenkinson, H. F.; Sawyer, W. D.; Mandelstam, J. J. Gen. Microbiol. 1981, 123, 1-16. (45) Available: http://www.matrixscience.com/cgi/index.pl?-page)./home.html.

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Figure 1. MALDI-MS spectra of tryptic digests generated in situ from B. subtilis 168 spores by on-probe solubilization of SASPs using 10% TFA and enzymatic proteolysis for 5 (a), 15 (b), and 25 min (c).

RESULTS Small, acid-soluble proteins have been previously shown to represent reliable biomarkers for the mass spectrometric analysis of Bacillus spores. In one study, extracts of SASP were obtained by treatment of Bacillus spores with 1 N HCl for 90 min and shown to be species-specific.33 In general, little is known about the processes involved in the extraction of SASPs from the spore core region by acids; however, 1 N HCl is reported to cause complete spore disruption.34 In the present work, SASPs were directly solubilized from Bacillus spores on the MALDI sample plate by acid treatment, reducing total sample-processing time to ∼5 min. Among common acids (HCOOH, TFA, HCl, HNO3) tested, an aqueous solution of 10% TFA was shown to be most suitable for on-probe solubilization of SASPs from Bacillus spores combined with MALDI analysis using sinnapinic acid as matrix (data not shown). Proteolytic digestion of SASPs solubilized on-probe was carried out in situ, using trypsin immobilized on Agarose beads. Peptide ion signals with intensities adequate for sequencing on the Axima-CFR could be generated by tryptic digestion for 5 min. As the digestion time was extended, e.g., to 25 min, the extent of proteolysis and the peptide ion currents were increased. This is illustrated in Figure 1 for B. subtilis 168. No sample cleanup was needed, and trypsin autolysis products were not observed in these MALDI-MS spectra. At 25 min, the SASPs are usually completely cleaved to peptides, and signal intensities are not further increased by extending the digestion time, for example, to 45 min. When a faster identification of Bacillus spores is required, the analysis may be made without waiting for complete digestion of SASPs, though with some loss of sensitivity. B. subtilis 168 was chosen first to demonstrate the applicability of our approach for rapid spore identification, since its genome has been completely sequenced. The MALDI-MS spectra of the proteolytic digest generated in situ through various times from B. subtilis 168 spores revealed distinctive tryptic peptides as shown in Figure 1. Molecular masses of SASPs listed in this work 5620 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

correspond to amino acid sequences in which the N-terminal methionine residue (131.2 Da) has been removed posttranslationally.46 All the masses detected for protonated peptides match peptide masses predicted from theoretical digestion of the various SASPs from B. subtilis 168 (Table 1). This observation is consistent with enzymatic digestion only of selectively solubilized SASPs, as proposed in this work. The R- and β-type SASPs exhibit some sequence homologies within and also across Bacillus species. Thus, the protonated tryptic peptides observed at m/z 1880.0 and 1033.1 (Table 1) are identified as digestion products of both the R- and β-type SASPs from B. subtilis 168. Protonated tryptic peptides with unique molecular masses were also detected from the R- and β-type SASPs, e.g., at m/z ratios of 1484.4, 1640.5, 2442.2, 1322.1, and 2285.6, respectively (Figure 1 and Table 1). As indicated in Table 1, some protonated peptides, formed by partial cleavages of the larger γ-SASPs, could be detected. Among these, two partial cleavage products of identical mass but different sequences could contribute to the ion signal at m/z 2911.4 (Figure 1 and Table 1). To move beyond ambiguous peptide mass matching, distinctive tryptic peptides were selected and partially sequenced by PSD analysis. To exemplify the high quality of PSD data obtained, fragmentation spectra of protonated peptides observed at m/z 1880.8 and 2783.4 are presented in Figure 2. Extended fragmentation was observed for both peptides by PSD analysis. Product ions obtained could be matched to b- and y-type ions formed by the tryptic peptides (LEIASEFGVNLGADTTSR) and (QNQQSAAGQGQFGTEFASETNAQQVR), tentatively identified in Table 1. Generally, product ions labeled as y-type ions refer to Y′′-type ions,47 signifying the addition of two hydrogen atoms to the C-terminal (46) Driks, A.; Setlow, P. In Prokaryotic Development; Brun, Y. V., Shimkets, L. J., Eds.; American Society for Microbiology: Washington, DC, 1999; pp 191218.

Table 1. Compilation of Bacterial Peptides Generated in Situ from B. subtilis 168 Spores That Match Peptides from Theoretical Tryptic Digests of Known SASPs (Average Masses Are Shown) [M + H]+ obsd

theor

span

sequence

SASPa

816.9 1033.1

816.9 1033.1 1033.1 1322.5 1484.7 1640.9 1881.1 1881.1 1906.1 2286.5 2442.7 2783.9 2841.9 2912.1 2912.1 2970.1

9-15 43-53 41-51 53-64 55-68 54-68b 25-42 23-40 20-37 1-22 1-24 17-24 44-69 16-42b 17-43b 43-69b

TNAQQVR ANGSVGGEITK ANGSVGGEITK LVSFAQQQMGGR LEIASEFGVNLGADTTSR RLVSFAQQNMGGGQF LEIASEFGVNLGADTTSR LEIASEFGVNLGADTTSR LEVAQEFGVNLGSDTVAR ANQNSSNDLLVPGAAQAIDQMK ANNNSGNSNNLLVPGAAQAIDQMK QNQQSAAGQGQFGTEFASETNAQQVR QNQQSAGQQGQFGTEFASETDAQQVR QNQQSAAGQGQFGTEFASETNAQQVRK KQNQQSAAGQGQFGTEFASETNAQQVR KQNQQSAGQQGQFGTEFASETDAQQVR

SASP-γ (9136.5) SASP-R (6939.6) SASP-β (6848.6) SASP-β (6848.6 ) SASP-R (6939.6) SASP-R (6939.6) SASP-R (6939.6) SASP-β (6848.6) SASP-D (6672.5) SASP-β (6848.6) SASP-R (6939.6) SASP-γ (9136.5) SASP-γ (9136.5) SASP-γ (9136.5) SASP-γ (9136.5) SASP-γ (9136.5)

1322.1 1484.4 1640.5 1880.8 1905.8 2285.6 2442.2 2783.4 2841.4 2911.4 2969.8

a All masses are calculated without the N-terminal methionine residues. Numbers in parentheses are daltons. b Tryptic peptide formed by partial cleavage.

Figure 2. PSD spectra of protonated tryptic peptides of 1880.8 (a) and 2783.4 Da (b) generated by on-probe solubilization and in situ digestion of SASPs from B. subtilis 168 spores.

side after cleavage of the amino bond of the peptide analyzed. Formation of y-type ions was strongly favored by the presence of basic residues on the C-terminal side, and cleavage of amino bonds containing glutamic acid and aspartic acid produced y-ions with high abundances.48,49 In addition, b-type ions were not observed at all from tryptic peptides with the partial sequence -Q-Q-V-R at the C-terminus. (47) Roepstorff, P.; Fohlman, J. J. Biomed. Mass. Spectrom. 1984, 11, 601.

Toward the objective of promptly identifying the protein and, accordingly, the Bacillus spore species, PSD data were evaluated for database searches using Mascot Sequence Query. For this purpose, product ions with signal intensities at least 5% above the noise level were generically employed in sequence queries. To (48) Wattenber, A.; Organ, A. J.; Schneider, K.; Tyldesley, R.; Bordoli, R.; Bateman, R. H. J. Am. Soc. Mass Spectrom. 2002, 13, 772-783. (49) Schilling, B.; Wang, W.; McMurray, J. S.; Medzihradszky, K. Rapid Commun. Mass Spectrom. 1999, 13, 2174-2179.

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Figure 3. MALDI-MS spectrum of tryptic digest generated in situ from B. globigii spores by on-probe solubilization of SASPs using 10% TFA. Table 2. Compilation of Bacterial Peptides Generated in Situ from B. globigii Spores That Match Peptides from Theoretical Tryptic Digests of the SASP-1 (7095.9 Da) and the γ-SASP (8889.3 Da) (Average Masses Are Shown) [M + H]+ obsd

theor

span

sequence

SASPa

816.8 1032.9 1928.9 2784.3 2827.4

816.9 1033.1 1929.1 2784.9 2827.9

7-13 44-54 26-43 42-67 15-40

TNAQQVR ANGSVGGEITK FEIASEFGVNLGAETTSR QNQQSAAGQGQFGTEFASETDAQQVR QNQQSASGQGQFGTEFASETNVQQVR

SASP-γb (8889.3) SASP-1b (7095.9) SASP-1b (7095.9) SASP-γb (8889.3) SASP-γb (8889.3)

a All masses are calculated without the N-terminal methionine residues. Numbers in parentheses are daltons. b SASP from B. stearothermophilus as listed in the Swiss-Prot database.

keep the data processing straightforward, product ions formed by losses of small molecules (e.g., H2O, NH3) were not removed. Based on the usual performance of the instrument, mass errors set in sequence queries were restricted to (0.5 and (1 Da for precursor and product ions, respectively. If no significant database search result could be accomplished by using these parameters, peptide and product ion mass errors were increased stepwise to (1 and (1.5-2 Da, respectively. The protein, and according to its source, the microorganism, was considered as identified if a Mascot sequence search score of g69 was achieved. The confidence level of protein identification improves with increasing search score, and most scores in this study were over 100. An overview of all tryptic peptides analyzed by PSD in this work, corresponding product ion masses, and search results obtained is provided in the supplemental Table Ι. Database searches were performed using PSD data of distinctive tryptic peptide ions with m/z ratios of 1880.8, 2442.2, 2783.4, and 2841.4 generated in situ from B. subtilis 168 spores. The Rand β-type SASP from B. subtilis 168 could be identified with equal scores of 144, based on the peptide (LEIASEFGVNLGADTTSR) with protonated masses of 1880.8 Da. The tryptic peptide (ANNNSGNSNNLLVPGAAQAIDQMK), assigned for precursor ions of 2442.2 Da, was found to be derived from the R-type SASP from B. subtilis 168 with a search score of 171. Based on PSD analysis on protonated peptides of 2783.4 Da, the sequence (QNQQSAAGQGQFGTEFASETNAQQVR) was identified as originating from the γ-type SASP (9136.5 Da) in B. subtilus 168, with a significant score of 233. However, the search revealed that this sequence also occurs in two additional γ-type SASPs (9207.6 and 9235.7 Da), from B. subtilis strain N10 and strain W23, which were assigned with equal scores. The γ-type SASPs from B. subtilis strains N10, W23, and 168 do differ elsewhere their sequences, and B. subtilis strain 168 could be established as the bacterial 5622 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

source via PSD analysis on peptides ions of 2841.4 Da, identified as the tryptic peptide (QNQQSAGQQGQFGTEFASETDAQQV). This result shows that discrimination of B. subtilis strains can be accomplished as previously reported, based on the sequence of the gene (sspE) that encodes for the γ-type SASPs from B. subtilis subspecies W23 and 168.35 In addition to B. subtilis 168, B. globigii was studied here as an unsequenced, and thus unknown sample to test the analytical strategy. B. globigii has been described as a B. subtilis strain that produces a black pigment when grown on tyrosine, and thus, it has sometimes been referred to as B. subtilis variety niger.50 More recently, mass spectrometric analysis of acid extracts from B. globigii spores resulted in the tentative identification of SASP-1 (7335.1 Da) and the γ-type SASP (8889.1 Da) proteins33 based on correspondence in mass to the SASP-1 and the γ-type SASP from B. cereus and B. stearothermophilus, respectively. To gain sequencespecific information in addition to mass-specific information on SASPs from B. globigii spores, tryptic peptides were generated in situ by proteolytic digestion of SASPs that were selectively solubilized on the MALDI plate. Protonated peptides could be detected by MALDI-TOFMS analysis, ranging from about 800 to 3000 Da (Figure 3). The masses of the five peptide ions of highest abundances are listed in Table 2 and match those of tryptic peptides from both the SASP-1 (7095.9 Da) and the γ-type SASP (8889.3 Da) from B. stearothermophilus with an average mass accuracy of 0.3 Da (Table 2). Tryptic peptides corresponding to the SASP-1 from B. cereus were not detected. Partial sequencing of the bacterial peptide ions detected at m/z 1928.9 was performed, which provided a database match to the tryptic peptide (FEIASEFGVNLGAETTSR) of the SASP-1 (7095.9 (50) Priest, F. In Bacillus subtilis and other Gram-positive bacteria; Sonenshein, A. L., Hoch, J. A., Losick, R., Eds.; American Society for Microbiology: Washington, DC, 1993; pp 3-16.

Figure 4. PSD spectra of protonated tryptic peptides of 2784.3 (a) and 2827.4 Da (b) generated by on-probe solubilization and in situ digestion of SASPs from B. globigii spores.

Da) from B. stearothermophilus with a score of 155 (Supporting Information, Table Ι). PSD spectra of protonated peptides of 2784.3 and 2827.4 Da are shown in Figure 4. Sequences of the peptides identified differ only in three amino acids, and the high specificity of sequence information obtained by PSD is demonstrated. As a result, the γ-type SASP (8889.3 Da) from B. stearothermophilus was identified with search scores of e210, and sequence coverage of 62% could be obtained. Additional PSD studies on protonated peptides at m/z 2685.5 and 1741.3 from the B. globigii sample revealed distinctive product ions, but no matches could be obtained by database searches (Supporting Information, Table Ι). To gain more information on the identity of B. globigii, based on SASPs, de novo sequencing of the genome or of the proteins must be performed. Complete sequences of the major SASPs from B. globigii have been recently determined in our laboratory and will be published soon. B. cereus, B. thuringiensis, and B. anthracis are all classified as members of the B. cereus group of bacteria;51 however, they demonstrate widely different toxicological effects on their environment.52-54 The distinction between these species is often based on DNA plasmids. However, these are reported to depend (51) Helgason, E.; Okstad, O. A.; Caugant, D. A.; Johansen, H. A.; Fouet, A.; Mock, M.; Hegna, I.; Kolsto, A.-B. Appl. Environ. Microbiol. 2000, 66, 26272630. (52) Andersson, A.; Ro ¨nner, U.; Granum, P. E. Int. J. Food Microbiol. 1995, 28, 145-155. (53) Jackson, S. G.; Goodbrand, R. B.; Ahmed, R.; Kasatiya, S. Lett. Apll. Microbiol. 1994, 21, 103-105. (54) Mock, M.; Fouet, A. Annu. Rev. Microbol. 2001, 55, 647-671.

on growth media and conditions and the plasmids can be lost naturally.55 As an example, discrimination between B. cereus and B. thuringiensis is only established based on specific genes coding for the insecticidal toxins characteristic for the latter. If the corresponding plasmids are missing, B. thuringiensis can no longer be distinguished from B. cereus.56 Among the B. cereus group, only the genome of one B. anthracis strain (A 2012) is currently available to the public, along with sequences for the major SASPs from B. cereus T and some additional proteins from other species. Biomarkers in the mass range of 2500-3600 Da have been reported to be characteristic of three B. anthracis strains and to be absent in MALDI spectra of spores from other B. cereus group members.57 Although not identified in that report, these low molecular weight biomarkers are likely to represent secondary metabolites such as lipopeptides, rather than proteins translated from the genome, and thus their presence would depend on growth conditions.42,58-60 More recently, the species-specific R/β(55) Turnbull, P. C. B.; Hutson, R. A.; Ward, M. J.; Jones, M. N.; Quinn, C. P. J. Appl. Bacteriol. 1992, 72, 21-28. (56) Thorne, C. B. In Sonenshein, A. L.,Hoch, J. A., Losick. R., Eds.; Bacillus subtilis and other Gram-positive bacteria; American Society for Microbiology: Washington, DC, 1993; pp 113-124. (57) Elhanany, E.; Barak, R.; Fisher, M.; Kobiler, D.; Altboum, Z. Rapid Commun. Mass Spectrom. 2001, 15, 2110-2116. (58) Hathout, Y.; Ho, Y.-P.; Ryzhov, V.; Demirev, P.; Fenselau, C. J. Nat. Prod. 2000, 63, 1492-1496. (59) Peypoux, F.; Bonmatin, J. M.; Wallach, J. Appl. Microbiol. Biotechnol. 1999, 51, 553. (60) Leenders, F.; Stein, T. H.; Kablitz, B.; Franke, P.; Vater, J. Rapid Commun. Mass Spectrom. 1999, 13, 943-949.

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Figure 5. MALDI-MS spectra of tryptic digests generated in situ from B. thuringiensis subs. Kurstaki spores (a) and B. anthracis Sterne spores (b) by on-probe solubilization of SASPs using 10% TFA. Legends on each spectrum provide m/z ratios of low-abundant peptide ions observed in the mass range of 1100-1850 Da.

type SASP (6678.5 Da) has been identified based on mass spectrometric analysis of B. anthracis Sterne spore extracts.33 B. anthracis Sterne is a nonpathogenic strain widely used as vaccine for livestock and wild animals, missing the pXO2 plasmid.54 To gain additional information on this set of closely related Bacillus spore species, proteolytic digestions of SASPs solubilized from B. cereus T, B. thuringiensis subs. Kurstaki, and B. anthracis Sterne spores were performed in situ. The peptides detected in tryptic digests from B. cereus T and B. thuringiensis subs. Kurstaki showed peptides with matching molecular masses and signal intensities (data not shown). In contrast, a small set of unique tryptic peptides was generated from the proteolytic digests from B. thuringiensis subs. Kurstaki (Figure 5a) and B. anthracis Sterne (Figure 5b). There is considerable redundancy between the two spectra. However, unique peptides are present in each. A compilation is provided in Table 3 of the masses of the tryptic peptides observed from the three members of the B. cereus group studied here. The table also lists SASPs from which peptides arise in theoretical tryptic digests with masses that match the experimental observations. SASPs denoted with the letter c are taken from the B. anthracis A 2012 genome, and all other SASPs are from B. cereus T entries in the protein database. The genome of B. thuringiensis subs. Kurstaki has not been sequenced yet, and no SASP sequences are available in public databases. As discussed above, it is assumed that B. cereus T and B. thuringiensis subs. Kurstaki feature identical SASPs. For the first time, the SASP-1 (7335.1 Da) could be tentatively identified in B. cereus group spore species. Generally, each identification was initially based on several peptides (Table 3) whose masses match those predicted. In addition, tryptic peptides most likely derived from the R/β-type SASP (7162.9 Da), hypothetical protein-1 (9207.3 Da), and hypothetical protein-2 (9737.3 5624 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

Da) provide the first (published) experimental evidence for these proteins from B. anthracis Sterne. Some of the peptides listed in Table 3 may be formed by more than one type of SASPs, due to partial sequence homologies. Nonetheless, unique tryptic peptides were predicted for all SASPs sequences reported in this work for the B. cereus group members studied, and matched protonated peptides in mass as revealed by MALDI analysis (Table 3). Several species-specific protonated peptides were observed in tryptic digests from B. anthracis Sterne and were assigned as deriving from two different R/β-type SASPs (7162.9 and 6678.5 Da), hypothetical protein-1 (9207.3 Da), and hypothetical protein-2 (9737.3 Da). MALDI analysis of tryptic digests from the Bacillus species thuringiensis subs. Kurstaki and cereus T revealed tryptic peptides matching in mass, among which protonated peptides at m/z ratios 1534.5, 2728.5 and 2851.7 were specific for the pair (Table 3). These peptides were formed by tryptic cleavage of the R/β-type SASP (6710.5 Da) and the γ-type SASP (9540.1 Da) that were previously reported for both species.33 MALDI spectra of tryptic digests from B. thuringiensis subs. Kurstaki and B. anthracis Sterne showed additional low-abundant peptide ions, e.g., of 1156.2 and 1140.1 Da, respectively (Figure 5a and b). Although these protonated peptides did not match tryptic peptide masses predicted for known SASPs, they could provide additional information for the discrimination of these closely related Bacillus species. To verify the sequences proposed by peptide mass matching, PSD analyses were performed. Generally, tryptic peptides of identical m/z ratios but generated from different B. cereus group members showed an identical fragmentation pattern in their PSD spectra (Supporting Information, Table I). For example, the tryptic peptide (YEIAQEFGVQLGADATAR), observed in the spectra of B. cereus T, B. thuringiensis subs. Kurstaki, and B. anthracis Sterne, could be completely sequenced by PSD analysis on pre-

Table 3. Compilation of Masses of Tryptic Peptides Generated in Situ from B. thuringiensis Subs. Kurstaki, B. cereus T, and B. anthracis Sterne Spores and Matching Peptides from Theoretical Tryptic Digests of Known Bacterial Proteins from B. cereus Group Members As Listed in Common Databasesa

H]+

H]+

[M + obsd

[M + calcd

span

872.8 1189.1

1488.4 1518.4 1528.4 1534.5

873.1 1189.3 1189.3 1189.3 1252.4 1273.5 1296.4 1336.4 1430.7 1430.7 1489.0 1518.8 1528.7 1534.7

55-61 41-53d 39-50d 43-54d 1-10 55-66 25-37 38-49 7-20 7-20 54-67 52-65 55-69 51-64

LLAELWK ANGSVGGEITKR ANGSVGGEITKR ANGSVGGEITKR SVLDNFDQWK LVAMAEQQLGGR GLDGGAVSDMAFR VGDYLANEVEAR LAVPGAESALDQMK LAVPGAESALDQMK LVAMAEQSLGGFHK LVSLAEQQLGGFQK LVSLAEQQLGGGVTR LVSLAEQQLGGYQK

Hypoth. protein-1c (9207.3) SASP-R/βc (6834.6) SASP-2 (6710.5) SASP-R/βc (7162.9) Hypoth. proteinc (9207.3) SASP-1 (7335.1) Hypoth. protein-1c (9207.3) Hypoth. protein-1c (9207.3) SASP-2 (6710.5) SASP-1c (6678.5) SASP-R/βc (6834.6) SASP-1c (6678.5) SASP-R/βc (7162.9) SASP-2 (6710.5)

1594.5 1826.6 1884.7 1939.8

1594.8 1827.0 1885.1 1940.1

1955.9 1971.7 2258.1 2728.5

1956.1 1972.1 2258.5 2728.8

55-69 62-77 5-22 21-38 21-38 21-38 23-40 25-42 1-22 48-74

LVAMAEQQLGGGYTR VADEQEQHTIANLMVK NSNQLASHGAQAALDQMK YEIAQEFGVQLGADATAR YEIAQEFGVQLGADATAR YEIAQEFGVQLGADATAR YEIAQEFGVQLGADSTAR YEIAQEFGVQLGADTSSR ANQNSSNQLVVPGATAAIDQMK AQASGAQSANASYGTEFATETDVHSVK

SASP-R/βc (7080.8) Hypoth. protein-1c (9207.3) SASP-R/βc (7080.8) SASP-2 (6710.5) SASP-1c (6678.5) SASP-R/βc (7162.9) SASP-R/βc (6834.6) SASP-R/βc (7080.8) SASP-R/βc (6834.6) SASP-γ (9540.1)

2834.4 2851.7

2835.0 2852.0

10-37 10-37

ATSGASIQSTNASYGTEFATETNVQAVK ATSGASIQSTNASYGTEFSTETDVQAVK

Hypoth. protein-2c (9737.3) SASP-γ (9540.1)

2942.6

2943.1

48-76

AQASGASIQSTNASYGTEFATETDVHAVK

Hypoth. protein-2c (9737.3)

1251.9 1273.1 1295.9 1336.0 1430.3

SASPb

sequence

detected in spectra of proteolytic digests from B. cereus group members studied B. anthracis Sterne all three all three all three all three B. anthracis Sterne all three all three B. anthracis Sterne B. anthracis Sterne B. cereus T, B.thuringiensis subs. Kurstaki all three B. anthracis Sterne all three all three all three all three all three B. cereus T, B.thuringiensis subs. Kurstaki B. anthracis Sterne B. cereus T, B.thuringiensis subs. Kurstaki B. anthracis Sterne

a Peptide masses in tryptic digests were observed within (0.2-Da mass differences. Average masses are shown b Masses calculated for proteins from which methionine has been porttranslationally removed. Numbers in parentheses are daltons. c Proteins listed for B. anthracis A2012. d Tryptic peptide formed by partial cleavage.

cursor ions of 1939.8 Da (Figure 6a). The SASP-2 (6710.5 Da), the R/β-type SASP (7162.9 Da), and the SASP-1 (6678.5 Da) were identified as potential sources from all three species for this peptide with identical scores of 218, and accordingly, it did not serve to distinguish B. anthracis Sterne from B. cereus T and B. thuringiensis subs. Kurstaki (Supporting Information, Table I). To distinguish between these B. cereus group members, PSD analysis was performed on the protonated peptide, m/z 1534.5, observed in proteolytic digests from B. cereus T and B. thuringiensis subs. Kurstaki (Figures 5a and 6b) but not from B. anthracis Sterne. The tryptic peptide (LVSLAEQQLGGYQK) was assigned to the SASP-2 (6710.5 Da) with a search score of 157, and B. cereus T was identified as biological source (Supporting Information, Table Ι). Postsource decay studies on tryptic peptides of 1594.5, 1488.8, and 1955.9 Da led to identification of two R/β-type SASPs of 7080.8 and 6834.6 Da with high scores. Although B. anthracis 2012 is the only source for these two proteins listed in public databases, our analytical strategy revealed their presence in spores from all the B. cereus group members studied here. PSD analysis on distinctive protonated peptides observed only in digests from B. anthracis Sterne at m/z ratios of 1336.0, 1518.4, 1528.5, 2834.4, and 2942.6 resulted in identification of hypothetical protein-1 (9207.3 Da), two different R/β-type proteins (7162.9 and 6678.5 Da), and hypothetical protein-2 (9737.3 Da) from B. anthracis

A2012 with scores from 85 to 227. None of these bacterial proteins have been experimentally observed for B. anthracis Sterne in mass spectrometric studies, except the R/β-type SASP of 6678.5 Da.33,57 Sequence coverage of 46% was obtained for the R/β-type protein of 7162.9 Da by PSD analysis on protonated peptides of 1528.5 and 1939.8 Da (Supporting Information, Table Ι). The protonated tryptic peptides with masses of 2834.4 and 2942.6 Da were assigned to hypothetical protein-2 from B. anthracis and account for 58% of its sequence. On the basis of sequence homology with the γ-type SASPs from B. cereus T, we propose that hypothetical protein-2 is the γ-type SASP of B. anthracis strain A2012 and Sterne. One area where the strategy of in situ digestion of a limited set of proteins combined with PSD analysis and database searching is anticipated to provide valuable results is the rapid identification of Bacillus spores in mixtures. To demonstrate this, a binary 3:1 spore mixture of B. thuringiensis subs. Kurstaki and B. globigii was analyzed. Tryptic peptides unique to each Bacillus species were detected by MALDI-TOFMS (Figure 7a). Since the instrument provided a resolution, defined as full width at half-maximum (fwhm), of 4050 at 1939.2 Da in this study, tryptic peptides in the mixture were readily resolved throughout the mass range of interest (see, for example, Figure 7a inset). PSD analysis on characteristic tryptic peptides resulted in unambiguous identification of SASPs and, thus, both Bacillus species. As an example, Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

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Figure 6. PSD spectra of protonated tryptic peptides of 1939.8 (a) and 1534.5 Da (b) generated by on-probe solubilization and in situ digestion of SASPs from B. thuringiensis subs. Kurstaki spores.

the PSD spectrum of the protonated tryptic peptide (FEIASEFGVNLLGAETTSR) observed at m/z 1929.2 is shown, identifying the SASP-1 (7095.9 Da), and, accordingly, B. globigii as the bacterial source (Figure 7b). Additional experiments demonstrated that the strategy presented is applicable to the rapid identification of spore species in various mixtures, e.g., B. globigii and B. subtilis 168 (2:1) and B. thuringiensis subs. Kurstaki and B. subtilis 168 (3:1) (data not shown). DISCUSSION A novel approach has been presented, employing in situ proteolytic digestion of a limited set of proteins to identify rapidly Bacillus spore species by MALDI using a curved-field reflectron time-of-flight instrument. SASPs were selectively solubilized from Bacillus spores by on-probe treatment with 10% TFA. Proteolytic peptides of SASPs were generated in situ within 5 min using immobilized trypsin. The results presented here demonstrate the potential of PSD analysis for unambiguous identification of proteins and Bacillus spore species by applying genome-based database searches. Since the bacterial protein and the microorganism source can be promptly identified based on partial sequencing of a single tryptic peptide, this approach was shown to be suitable for the rapid analysis of Bacillus spore mixtures. Mascot sequence scores from 85 to 233 reflect the significant high confidence levels for protein identification achieved by PSD analysis. In addition, the approach presented is still successful when the mass error is expanded to (2 Da, suggesting that data acquired on instruments with moderate mass accuracies can also be used for database 5626 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

retrievals. Thus, a wide range of instruments with CID or PSD capabilities can be used for the approach discussed in this work. In general, tryptic peptides from the γ-type SASP provided most valuable sequence information for discrimination of different Bacillus species. In situ proteolytic digestion of SASP selectively solubilized from Bacillus spores on the MALDI plate provided reproducible results. The number of tryptic peptides analyzed is limited, and theoretically, only a small set of potential proteins had to be considered in bioinformatics. Signal intensities of protonated peptides were observed to be at least 100 times higher than signal intensities obtained from corresponding SASPs, resulting in excellent signal-to-noise ratios and sensitivity. In some cases, peptides were characterized, even though the protein was not detectable. In addition, tryptic peptides from SASPs could be readily analyzed in the reflectron mode, allowing mass resolution and accuracy superior to higher mass protein profiling. To the best of our knowledge, the (putative) SASP-1 of 7095.9 Da has not been observed before by mass spectrometric analysis of B. globigii spores. Mass spectrometric analysis of proteolytic spore digests from B. cereus T and B. thuringiensis subs. Kurstaki resulted in identification of identical sets of SASPs, suggesting that these species might be only distinguishable by variable secondary metabolites, e.g., lipopeptides or protein toxins. The R/β-type SASP (7162.9 Da), hypothetical protein-1 (9207.3 Da), and γ-type SASP (9737.3 Da) were identified by partial sequencing of tryptic peptides generated in situ from B. anthracis Sterne. Though predicted from the genome, these spore proteins have

Figure 7. MALDI-MS spectrum of the tryptic digest generated in situ from a 3:1 mixture of B. thuringiensis subs. Kurstaki and B. globigii spores by on-probe solubilization of SASPs using 10% TFA (a), and PSD spectrum of selected peptide ions of 1928.8 Da (average mass).

not been previously reported as biomarkers from a B. anthracis strain. Since distinctive tryptic peptides of these proteins are readily accessible by our approach, B. anthracis Sterne could be unambiguously distinguished from closely related species of the Bacillus cereus group studied in this work. CONCLUSIONS The approach reported allows on-probe solubilization and in situ enzymatic digestion of SASPs from Bacillus spores. Identification of Bacillus spores can be accomplished via peptide microsequencing combined with bioinformatics in less than 20 min, comprising sample preparation, mass spectrometric analysis, and database searches. The strategy has the capability to provide identification of species from the genus Bacillus in spore mixtures.

ACKNOWLEDGMENT We thank Suzanne R. Kalb, Dr. Dongxia Wang, and Dr. Yetrib Hahout for helpful discussion regarding this work, and Prof. Robert J. Cotter for the use of the Axima-CFR mass spectrometer. Financial support was provided by the German Research Community (Deutsche Forschungsgemeinschaft, DFG) and the U.S. Defence Advanced Research Projects Agency (DARPA). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review February 27, 2003. Accepted August 11, 2003. AC034200F

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