Anal. Chem. 2006, 78, 5124-5133
Using Capillary Electrophoresis-Selective Tandem Mass Spectrometry To Identify Pathogens in Clinical Samples Anren Hu,†,‡ Cheng-Tung Chen,† Pei-Jen Tsai,‡ and Yen-Peng Ho*,†
Department of Chemistry, National Dong Hwa University, Hualien, Taiwan, and Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien, Taiwan
Analysis of microbial mixtures in complex systems, such as clinical samples, using mass spectrometry can be challenging because the specimens may contain mixtures of several pathogens or both pathogens and nonpathogens. We have successfully applied capillary electrophoresis-selective MS/MS of unique peptide marker ions to the identification of common pathogens in clinical diagnosis. We searched the CE-MS/MS spectra acquired from the proteolytic digests of pure bacterial cell extracts against protein databases. The identified peptides that matched a protein associated with a particular pathogen were selected as marker ions to identify that bacterium in clinical specimens. Thirty-four clinical specimens, obtained from pus, wound, sputum, and urine samples, were analyzed using both biochemical and selective MS/ MS methods. The bacteria in these clinical samples were cultivated directly, without prior isolation of a pure colony, before performing the selective MS/MS analyses. The bacteria analyzed included both Gram-positive and -negative strains. The match with respect to the pathogens identified was good between the biochemical and the selective MS/MS methods; the matching rate was 91%. The rate was as high as 97% when not considering two specimens for which the bacteria were not grown successfully. Two of the specimens that we identified using the biochemical method as containing two bacterial species were confirmed also through selective tandem MS analysis. Characterization of microorganisms is critical for the diagnosis and subsequent treatment of infectious diseases, as well as for the detection of biohazards in the environment.1,2 Various culture methods, serologic diagnostic tests, and molecular techniques are used presently for the identification of pathogenic bacteria. These methods can be classified into phenotyping and genotyping methods.3-5 Genotypic techniques are based on the genetic * Corresponding author. Phone: 886-3-8633591. E-mail: ypho@ mail.ndhu.edu.tw. † National Dong Hwa University. ‡ Tzu Chi University. (1) Wilson, M. Microbial Inhabitants of Humans: Their Ecology and Role in Health and Disease; Cambridge University Press: New York, 2005. (2) Fenselau, C., Ed. Mass Spectrometry for the Characterization of Microorganisms; American Chemical Society: Washington, DC, 1994.
5124 Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
conservation of a species and the genetic variability among different species. Genotypic methods, such as the polymerase chain reaction, employ nucleic acid amplification to detect speciesspecific sequences. Phenotypic testing, which relies upon physiological or biochemical characteristics, can provide information about specific metabolic pathways or antibiotic resistance. Most currently employed traditional methods are labor-intensive and time-consuming. Mass spectrometry provides an alternative means for identifying microorganisms. The use of protein biomarkers to identify microorganisms is a very successful approach. Both matrixassisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS) have been used to analyze microorganisms.6-8 There are a number of advantages to using MALDI-MS for the direct identification of bacteria, including the high speed and sensitivity of the technique, the simplified mass spectra obtained (predominantly singly charged ions), and the approach tolerance to contaminants. When a large set of digested peptides obtained from a complex microorganism is analyzed, however, MALDI-MS provides spectra that are too complicated for interpretation and with a low sensitivity for peptide sequencing. ESI-MS allows on-line detection to be combined with sample purification, concentration, and separation techniques, such as microdialysis, solid-phase extraction, liquid chromatography, and capillary electrophoresis (CE).9-13 Thus, ESI-MS has become a powerful tool for the analysis of complex systems. Although ESIMS has been used less frequently for microbial identification, several groups have characterized microorganisms by performing (3) Ammor, S.; Rachman, C.; Chaillou, S.; Pervost, H.; Dousset, X.; Zagorec, M.; Dufour, E.; Chevallier, I. Food Microbiol. 2005, 22, 373-382. (4) Yeung, P. S. M.; Kitts, C. L.; Cano, R.; Tong, P. S.; Sanders, M. E. J. Appl. Microbiol. 2004, 97, 1095-1104. (5) Wagner, R. D.; Paine, D. D.; Cerniglia, C. E. J. Appl. Microbiol. 2003, 94, 1098-1107. (6) van Baar, B. L. M. FEMS Microbiol. Rev. 2000, 24, 193-219. (7) Lay, J. O., Jr. Mass Spectrom. Rev. 2001, 20, 172-194. (8) Fenselau, C.; Demirev, P. A. Mass Spectrom. Rev. 2001, 20, 157-171. (9) Liu, C. L.; Hofstadler, S. A.; Bresson, J. A.; Udseth, H. R.; Tsukuda, T.; Smith, R. D.; Snyder, A. P. Anal. Chem. 1998, 70, 1797-1801. (10) Estrela, R. D. C. E.; Salvadori, M. C.; Suarez-Kurtz, G. Rapid Commun. Mass Spectrom. 2004, 18, 1147-1155. (11) Pruvost, A.; Ragueneau, I.; Ferry, A.; Jaillon, P.; Grognet, J. M.; Benech, H. J. Mass Spectrom. 2000, 35, 625-633. (12) Zamfir, A.; Seidler, D. G.; Schonherr, E.; Kresse, H.; Peter-Katalinic, J. Electrophoresis 2004, 25, 2010-2016. (13) Janini, G. M.; Conrads, T. P.; Wilkens, K. L.; Issaq, H. J.; Veenstra, T. D. Anal. Chem. 2003, 75, 1615-1619. 10.1021/ac060513+ CCC: $33.50
© 2006 American Chemical Society Published on Web 06/03/2006
ESI-MS analyses of the protein profiles of whole bacterial cells14-20 and ESI-MS/MS analyses of cell lysates,21,22 with or without prior separation. The challenges encountered when attempting microbial identification through protein profiling are the need for mass spectral reproducibility and the difficulty in identifying the components of mixtures. An approach based on proteome database searches to overcome the poor spectral reproducibility has been described.23,24 Proteins extracted from microbial samples can be digested by enzymes; the sequence information of the peptidess obtained using tandem mass spectrometryscan then be used to identify the proteins and deduce the microorganism’s source through a proteome database search.24,25 Yao et al. demonstrated a method for virus identification based on the MS/MS analysis of tryptic peptides and the construction of databases of virusspecific tryptic peptide masses.9 Warscheid et al. proposed a strategy to identify Bacillus spores through a combination of peptide sequencing of tryptic digests of specific spore proteins and database searching.10,11 Li’s group employed a constructed database of tryptic peptides generated from specific biomarkers to analyze Bacillus spores.22,26 Recently, two groups described the application of top-down proteomic approaches toward microbial identification.27,28 Most of these approaches involving database searching have employed MALDI-MS to analyze the samples. Recently, a method employing the selective MS/MS analysis of proteotypic peptides that had been separated through CE was reported by our group for the accurate identification of bacterial mixtures.29 Proteotypic peptides refer to those experimentally observable peptides that identify specific proteins.30,31 The selective MS/MS approach is similar to that of selected reaction monitoring (SRM),32-36 with the major difference being that the allowable (14) Krishnamurthy, T.; Davis, M. T.; Stahl, D. C.; Lee, T. D. Rapid Commun. Mass Spectrom. 1999, 13, 39-49. (15) Chong, B. E.; Kim, J.; Lubman, D. M.; Tiedje, J. M.; Kathariou, S. J. Chromatogr., B 2000, 748, 167-177. (16) Vaidyanathan, S.; Rowland, J. J.; Kell, D. B.; Goodacre, R. Anal. Chem. 2001, 73, 4134-4144. (17) Vaidyanathan, S.; Kell, D. B.; Goodacre, R. J. Am. Soc. Mass Spectrom. 2002, 13, 118-128. (18) Williams, T. L.; Leopold, P.; Musser, S. Anal. Chem. 2002, 74, 5807-5813. (19) Ho, Y. P.; Hsu, P. H. J. Chromatogr., A 2002, 976, 103-111. (20) Zheng, S.; Schneider, K. A.; Barder, T. J.; Lubman, D. M. BioTechniques 2003, 35, 1202-1212. (21) Xiang, F.; Anderson, G. A.; Veenstra, T. D.; Lipton, M. S.; Smith, R. D. Anal. Chem. 2000, 72, 2475-2481. (22) Dworzanski, J. P.; Snyder, A. P.; Chen, R.; Zhang, H. Y.; Wishart, D.; Li, L. Anal. Chem. 2004, 76, 2355-2366. (23) Demirev, P. A.; Ho, Y. P.; Ryzhov, V.; Fenselau, C. Anal. Chem. 1999, 71, 2732-2738. (24) Warscheid, B.; Jackson, K.; Sutton, C.; Fenselau, C. Anal. Chem. 2003, 75, 5608-5617. (25) Warscheid, B.; Fenselau, C. Anal. Chem. 2003, 75, 5618-5627. (26) Dworzanski, J. P.; Deshpande, S. V.; Chen, R.; Jabbour, R. E.; Snyder, A. P.; Wick, C. H.; Li, L. J. Proteome Res. 2006, 5, 76-87. (27) Demirev, P. A.; Feldman, A. B.; Kowalski, P.; Lin, J. S. Anal. Chem. 2005, 77, 7455-7461. (28) Williams, T. L.; Monday, S. R.; Edelson-Mammel, S.; Buchanan, R.; Musser, S. M. Proteomics 2005, 5, 4161-4169. (29) Hu, A.; Tsai, P. J.; Ho, Y. P. Anal. Chem. 2005, 77, 1488-1495. (30) Craig, R.; Cortens, J. P.; Beavis, R. C. Rapid Commun. Mass Spectrom. 2005, 19, 1844-1850. (31) Kuster, B.; Schirle, M.; Mallick, P.; Aebersold, R. Nat. Rev. Mol. Cell. Bio. 2005, 6, 577-583. (32) King, R. C.; Gundersdorf, R.; Fernandez-Metzler, C. L. Rapid Commun. Mass Spectrom. 2003, 17, 2413-2422. (33) Barbarin, N.; Mawhinney, D. B.; Black, R.; Henion, J. J. Chromatogr., B 2003, 783, 73-83. (34) Zweigenbaum, J.; Henion, J. Anal. Chem. 2000, 72, 2446-2454.
maximum mass range is monitored to cover all of the product ions. The coupling of chromatographic tools, such as CE and LC, to MS provides a highly selective method for characterizing the analytes present in complex mixtures. The proteolytic digests of cell extracts obtained from pure bacteria of interest can be subjected to CE-MS/MS analysis with subsequent database searching. The identified peptides that match the protein associated with the corresponding bacterial species are selected as marker ions for the identification of bacteria within the mixtures. Preliminary analysis of pure bacteria through database searching can be used to determine whether the peptides lead to multiple or false microbial identification; these peptides can then be excluded for selective MS/MS. To identify a bacterial species in a sample, the authors of that study performed CE-MS analysis of the proteolytic digest of the cell extract and monitored only the selected marker peptide masses using MS/MS. Thus, the corresponding bacterial species could be identified if the selected peptides were identified correctly from the database searches. The objective of this study was to evaluate the use of selective MS/MS for the direct identification of bacteria in clinical specimens, which is a challenge, given their complexity. In this study, we cultured the bacteria in clinical samples without prior isolation of a pure colony. Generally, traditional bacteriological methods require the isolation of a pure colony to perform microbial identification. Thirty-four clinical samples, whose pathogens were identified using a biochemical method, were analyzed using the selective MS/MS method. Bacterial biomarkers corresponding to as many as eight bacteria were monitored during a single run of the CE-MS experiment. The results were compared with those obtained from the biochemical identification method. EXPERIMENTAL SECTION Materials. Acetonitrile and methyl alcohol were obtained from J. T. Baker (Phillipsburg, NJ). R-Cyano-4-hydroxycinnamic acid was obtained from Aldrich (Milwaukee, WI). Acetic acid, ammonium acetate, ammonia solution, formic acid, and trifluoroacetic acid were purchased from Reidel-de Hae¨n (Seelze, Germany). Ammonium bicarbonate and ferulic acid (FA) were purchased from the Sigma Chemical Co. (St. Louis, MO). Porcine trypsin (sequence grade) was obtained from Promega (Madison, WI). Water was purified using a Milli-Q system (Millipore, Bedford, MA). Luria broth (LB) was obtained from Alpha Biosciences, Inc. (Baltimore, MD). Tryptic soy broth was purchased from Becton Dickinson Co. (Franklin Lakes, NJ). Yeast extract was obtained from Sigma. Bacterial Sample Preparations. All the bacterial cells were prepared in a BLS2 facility and killed by heating at 100 °C for 10 min before transferring to the MS laboratory. Pure bacterial samples were provided by Dr. Tsai’s lab. Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, and Vibrio parahaemolyticus were grown in LB broth at 37 °C for 24 h. Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus epidermidis, and Streptococcus pyogenes were grown in tryptic soy broth supplemented with 0.5% yeast extract (TSBY) at 37 °C for 24 h. The optical density of the cell cultures was measured at 600 nm using a Biowave CO8000 cell density meter (WPA, Cambridge, UK). The cells were harvested, washed three times (35) Onorato, J. M.; Henion, J. D.; Lefebvre, P. M.; Kiplinger, J. P. Anal. Chem. 2001, 73, 119-125. (36) Zhang, H. W.; Henion, J. J. Chromatogr., B 2001, 757, 151-159.
Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
5125
with water, placed in boiling water for 10 min, lyophilized to dryness, and then stored at -20 °C. The lyophilized bacteria were suspended in water at a concentration of 5 mg/mL. Thirty-four clinical specimens were obtained from the Division of Bacteriology at Tzu-Chi General Hospital, Hualien, Taiwan, during the years 2004 and 2005. These clinical samples were from pus, wound, sputum, and urine sources; they were previously identified using a biochemical method. All clinical specimens, containing Grampositive and -negative strains, were cultured directly in TSBY broth at 37 °C for 6 h. The cells were harvested, washed three times with water, placed in boiling water for 10 min, and then centrifuged. Water was used for extraction of proteins from the cells of the pure and clinical samples. The cell suspension was centrifuged, and a sample (1 mL) of the supernatant was collected and filtered through Microcon-YM3 filters (Millipore, Mississauga, ON, Canada). For proteolytic digestion, the protein extract was dissolved in ammonium bicarbonate buffer (55 mM, pH 8.5; 5 µL) and treated with trypsin solution (0.1 mM in 55 mM ammonium bicarbonate, pH 8.5; 0.5 µL). The digestion was performed for 15 min in a domestic microwave system having a maximum output power of 600 W (MOB-0705, Sampo, Taiwan).37 The peptide digest was dried using a SpeedVac vacuum concentrator and then redissolved in water (1 µL). Biochemical Identification. All clinical isolates were initially differentiated into Gram-positive and -negative strains, on the basis of the results of Gram staining. Both the Gram-positive and -negative strains were subsequently identified through biochemical means according to routine clinical laboratory procedures.38 CE Analysis. The experiments were performed using a homebuilt CE system equipped with a model PS/EH30R03.0 power supply (Glassman High Voltage, Inc., Whitehouse Station, NJ). A poly(vinyl alcohol)-coated capillary (100 cm × 75 µm i.d. × 380 µm o.d.) was obtained from Agilent Technologies (Palo Alto, CA). Pressure injection at 100 mBar was applied for 5 s. The temperature was maintained at 25 °C, and the applied potential was 20 kV. The running buffer was a mixture of 10 mM ammonium acetate (70%) and 10 mM acetic acid (30%) at pH 4.8. The capillary was conditioned with water and running buffer for 30 min each prior to the first run and for 5 min between runs. The CE separation time was 30 min for data-dependent experiments and reduced to 20 min for selective MS/MS experiments by increasing the sheath gas flow. MS Analysis. An ion trap mass spectrometer (LCQ Duo, Finnigan, San Jose, CA) equipped with an electrospray ionization source was used for all MS analyses. The CE apparatus was coupled to the spectrometer through a Finnigan coaxial sheathflow interface. The spray voltage was maintained at 4 kV. The capillary voltage and temperature were maintained at 27 V and 200 °C, respectively. The sheath gas flow was set at 20 (datadependent experiments) or 40 (selective MS/MS experiments) arbitrary units. The sheath liquid, 50% MeOH containing 0.5% acetic acid, entered the system at a flow rate of 4 µL/min. The mass spectrometer was operated under the control of the Xcaliber program. Spectra were collected in the positive-ion mode. The automatic gain control was maintained at 5 × 108, the microscan (37) Lin, S. S.; Wu, C. H.; Sun, M. C.; Sun, C. M.; Ho, Y. P. J. Am. Soc. Mass Spectrom. 2005, 16, 581-588. (38) Murray, P. R., Baron, E. J., Jorgensen, J. H., Pfaller, M. A., Yolken, R. H., Eds. Manual of Clinical Microbiology, 8th ed.; American Society for Microbiology: Washington, D.C., 2003.
5126
Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
count was 3, and the maximum injection time was 200 ms. When the spectra were acquired in a data-dependent mode, a full MS scan was acquired from m/z 400 to 2000, followed by three MS/ MS scans between m/z 400 and 2000 of the most-, second-most-, and third-most-intense ions of the full MS scan. The wideband excitation for the MS/MS scan was set at 42% of the normalized collision energy for 30 ms. In the selective MS/MS experiments, the spectra were acquired in the SRM mode while the mass range of the monitored product ion was set to the allowable maximum value to cover the dissociation fragments. This experiment differs from that of normal SRM, which only monitors the abundant and structurally unique transitions (from molecular ion to specific product ions). Several selected ions were scanned sequentially and repeatedly when monitoring multiple ions. For instance, eight ions were scanned sequentially in the selective MS/MS mode; the scan cycle was then repeated during the whole acquisition period. For most selective MS/MS analyses of clinical specimens, marker ions associated with E. faecalis, P. aeruginosa, S. typhimurium, S. aureus, S. agalactiae, S. epidermidis, S. pyogenes, and V. parahaemolyticus were simultaneously monitored. When the clinical sample containing E. coli was analyzed, marker ions associated with E. coli, P. aeruginosa, S. typhimurium, S. aureus, S. agalactiae, S. epidermidis, S. pyogenes, and V. parahaemolyticus were simultaneously monitored. MALDI spectra were acquired using an Autoflex time-of-flight mass spectrometer (Bruker Daltonic, Germany) equipped with a 337-nm nitrogen laser (10 Hz; 3-ns pulse width). FA was used as a MALDI matrix, which was prepared by dissolving FA in a mixture of acetonitrile, water, and formic acid (33:50:17) at a concentration of ∼50 mM. Database Searches. The MS/MS spectra were searched against the NCBInr database using SEQUEST (ThermoFinnigan Inc.). The protein database comprising all kingdoms was downloaded from the web site of the National Center for Biotechnology Information (NCBI; ftp://www.ncbi.nlm.nih.gov/blast/db). In the data-dependent experiments, the mass tolerances of the precursor and fragment ions were set to 2.5. Only those peptides that gave cross-correlation scores (Xcorr) above 1.8 for singly charged peptides, above 2.2 for doubly charged peptides, and above 3.3 for triply charged peptides [each with delta-correlation scores (DelCn) > 0.1] were considered as legitimate identifications. In addition, there must be some continuity to the b or y ion series, and the y ions corresponding to a proline residue should be intense ions. Each identified peptide leads to a matched protein and organism source associated with the protein. RESULTS AND DISCUSSION The bacteria present in clinical samples were directly cultured prior to selective MS/MS analyses. We first investigated the minimum culturing time required to correctly identify a bacterium. Figure 1 displays the optical densities (OD) of cell cultures of S. epidermidis, E. faecalis, P. aeruginosa, and S. pyogenes at growth stages ranging from 0.5 to 12 h. We attribute the smaller OD values for S. pyogenes, relative to those for other bacteria, to the difficulty in cultivating the former organism. The cell extracts of S. epidermidis and S. pyogenes harvested after several periods of time were subjected to selective tandem mass analysis after digestion. Both bacteria were correctly identified after culturing for 4 h. The success of each identification was based on the use of qualified Sequest search scores (Xcorr scores). The scores for S. epidermidis and S. pyogenes were 2.7 and 2.0, respectively. For
Figure 1. Optical densities at 600 nm of cell cultures of S. epidermidis, E. faecalis, P. aeruginosa, and S. pyogenes at different stages of growth ranging from 0.5 to 12 h.
S. epidermidis, the species could be identified with a score of 1.6 after culturing for 2 h. Because S. pyogenes is relatively difficult to culture among all of the pathogens we investigated, it is reasonable to presume that a culturing time of 4 h is the maximum time required for bacterial amplification from a pure colony to identify all of the pathogens investigated. Determining the minimum culturing time for real clinical samples is not straightforward because the amount of bacteria present in the sample can vary tremendously. When all of the clinical samples were cultured for 6 h, the number of harvested cells was more than sufficient for a single analysis. This culturing time is much lower than that required when using the conventional method, which usually requires 1-2 days to culture the bacteria, including colony isolation and cultivation. The MS/MS spectra of proteolytic digests of cell extracts obtained from nine pure bacterial samples were recorded in a datadependent mode prior to selective MS/MS analysis. The peptides were separated through CE and then subjected to MS/MS analyses. Figure 2 illustrates the data-dependent total ion chromatogram for E. faecalis. All of the tandem mass spectra of the peptides were searched against the NCBInr protein database using the SEQUEST application. Proteotypic peptides that led to the correct identification of microorganisms were chosen as speciesspecific biomarkers according to the rules described previously.29 One major consideration when selecting suitable biomarkers is that the peptides corresponding to several microbial sources must be excluded because some proteins may be shared in common by different bacteria, or the same peptide sequence may be associated with several different proteins; if so, database searches against the MS/MS data of the corresponding peptides will lead to multiple identifications of microorganisms. We found many unique and qualified marker ions for each species. Table 1 lists two selected peptides for each pathogen that we chose from the data-dependent experiments; these selected peptides were used for subsequent bacterial identification. The identified proteins
listed in Table 1 are most likely the abundant ones, so that we can observe them. The genomes of more than a hundred pathogenic bacteria are currently available in databases. Many more will be sequenced soon. If the microorganism’s protein database is not available, we may create an experimental protein database in which the proteotypic peptide markers can identify these proteins and, therefore, the microorganism associated with them. Figures 3a displays a selected CE-MS/MS ion chromatogram for the tryptic digest of the cell extract obtained from a clinical urine sample, in which E. faecalis was previously identified using a biochemical method. The sample was cultured directly prior to MS analysis. The ions at m/z 955.9 (2+) and 1935.9 (1+) that are listed in Table 1 were monitored in the SRM mode, whereas the product ion mass window was set to the allowable maximum mass range to cover the dissociation fragments. The product ion mass spectra of the selected precursor ions were searched against the protein database using SEQUEST (data not shown). The identified peptides, which had Xcorr values of 4.7 and 3.9, gave the correct source of the proteins: E. faecalis. Several bacterial species could be monitored in a single CE/ MS analysis through sequential detection of the biomarkers corresponding to each species. Figure 3b displays a selective ion chromatogram of eight ions corresponding to eight species, including E. faecalis. The sample was the same one used previously (Figure 3a). The predominant peaks were derived from MS/MS of the selected doubly charged ion at m/z 955.9 for E. faecalis. The lack of ion abundances of the other seven ions gave rise to the peak spacing. Furthermore, if these seven ions were present in the sample, peaks should appear somewhere during the elution period. The product ions observed in the tandem mass spectrum (Figure 3c) of the doubly charged ions at m/z 955.9 identify the peptide sequence TLEEGQAVTFDVEDSDR. A database search led to the identification of a cold-shock protein associated with the corresponding bacterium (E. faecalis). The Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
5127
Figure 2. Data-dependent total ion chromatogram obtained from the CE-MS/MS analysis of E. faecalis. Table 1. Selected Peptides Obtained from Data-Dependent Experiments of Each Pathogen bacterium S. aureus S. epidermidis P. aeruginosa S. pyogenes S. typhimurium S. pneumoniae E. coli E. faecalis S. agalactiae
isolated ion
sequence
protein
MW
peptide position
1076.52 (1+) 1262.60 (1+) 1040.49 (2+) 949.99 (2+) 1638.79 (1+) 1036.04 (2+) 1087.56 (1+) 1374.69 (1+) 1968.99 (1+) 1272.67 (2+) 1451.74 (1+) 1521.80 (1+) 1833.97 (1+) 1681.78 (1+) 955.93 (2+) 1935.93 (1+) 1308.67 (1+) 1073.55 (1+)
-ETVGNVTDNK-SGEESEVLVADK-YGPVDGDPITSTEEIPFDK-AHLVDLAQHNPEELNAK-IEDTDFAAETANLTK-TVIHTDNAPAAIGTYSQAIK-EAVEGAVDAVK-HGELLSEYDALK-AQPDLAEAAATTTENPLQK-TLHLADSELSEEALIQALVEHPK-DFHVVAETGIHAR-TVGDLVAYVEEQAK-EAAIQVSNVAIFNATTGK-EAIGYADSVHDYVSR.-TLEEGQAVTFDVEDSDR-TLEEGQAVTFEIEEGQR-VTVEVTYPDGTK-DAVEGAVDAVK-
hypothetical protein hypothetical protein accumulation-associated protein immunodominant antigen A flagellin conserved hypothetical protein hypothetical protein SPy2005 M protein phase 1 flagellin putative arsenate reductase phosphocarrier protein of the PTS acyl carrier protein 50S ribosomal protein L24 bacterioferritin cold-shock domain family protein cold-shock domain family protein alphalike protein 4 hypothetical protein
6 996 21 243 157 025 24 514 34 341 13 583 6 939 11 914 41 275 13 351 8 916 8 245 11 321 18 495 4 949 7 163 38 597 6 999
16-25 92-103 1086-1104 37-53 307-321 4-23 52-62 54-65 340-358 70-92 5-17 61-74 62-79 103-117 29-45 40-56 286-297 52-62
data presented in Figure 3c were derived from a single scan spectrum. Although the spectra associated with the selected precursor ion may, in principle, be summed and averaged, the software we used did not allow averaging over discontinued spectra that were acquired in the multiple selective MS/MS mode. Nonetheless, the quality of the spectrum was sufficiently high to allow positive identification of the peptide with an Xcorr score of 3.9. Although disease-related pathogens may dominate after direct cultivation of clinical specimens, it would not be a surprise if there were other bacterial species present in the clinical samples, considering the complexity of the sample. Figure 4 displays MALDI mass spectra of S. agalactiae obtained from a pure bacterial culture and a clinical specimen (wound). Although most of the ions observed in the pure sample were also observed in the clinical sample, the spectra clearly differ, such that identifica5128
Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
tion of the bacterium in a clinical sample could be difficult. The selective CE-MS/MS analyses target only bacteria-specific marker peptides. Therefore, any potential interference from other bacterial species present in the complex system will be eliminated. Figure 5a illustrates a selected CE-MS/MS ion chromatogram for the tryptic digest of the cell extract obtained from the clinical specimen containing S. agalactiae. Eight bacteria-specific ions, including that of S. agalactiae, were monitored. The marker ion at m/z 1308.7, corresponding to S. agalactiae, was detected in the equally spaced peaks of the spectrum. The identified sequence VTVEVTYPDGTK has a qualified Xcorr score of 2.5 (Figure 5b). A pus sample, which was identified through the biochemical method to contain both S. aureus and P. aeruginosa, was subjected to selective MS/MS analysis. Eight ions corresponding to eight species, including these two pathogens, were monitored in the selective MS/MS mode. The peaks within the range of retention
Figure 3. (a) Selected CE-MS/MS ion chromatogram of two ions at m/z 955.9 (2+) and 1935.9 (1+). The analytes were in the tryptic digest of the cell extract obtained from a clinical urine sample, in which E. faecalis was previously identified biochemically. (b) Selective ion chromatogram of eight ions corresponding to eight pathogen species, including E. faecalis. Only the doubly charged ion at m/z 955.9 for E. faecalis was detected. (c) Tandem mass spectrum of the doubly charged ions at m/z 955.9, which identified the peptide sequence TLEEGQAVTFDVEDSDR.
Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
5129
Figure 4. MALDI mass spectra of S. agalactiae obtained from (a) a pure bacterial culture and (b) a clinical wound specimen.
times from 11.0 to 11.6 min correspond to the S. aureus-specific ion at m/z 1076.5. The peaks at retention times ranging from 11.6 to 12.2 min relate to the P. aeruginosa-specific ion at m/z 1638.8. The ion chromatogram (Figure 6a) appears more complicated than those presented in Figures 3b and 5a. Although some peaks were detected during the retention period from 10 to 11 min, no peptides were identified with qualified scores. The selective MS/ MS method correctly identified the two species despite the complex appearance of the chromatogram. Figure 6b displays a spectrum of the selected ion at m/z 1076.5. The peptide sequence ETVGNVTDNK associated with S. aureus was correctly identified with an Xcorr value of 2.3. The peptide IEDTDFAAETANLTK, which suggests the microorganism source as P. aeruginosa, was identified with an Xcorr score of 3.1 (Figure 6c). Thirty-four clinical samples that had been identified previously through biochemical methods were analyzed using the selective MS/MS method. These samples were obtained from sputum, swab, and urine sources. Table 2 summarizes the results. One marker peptide was monitored for each species, and the masses of marker ions are listed in Table 2. The bacteria present in 31 of the 34 samples were identified in accordance with those identified biochemically. None of the expected bacteria were identified in the other three specimens. One of these specimens was a urine sample containing E. coli. The failure in identifying the other two specimens was a result of the bacteria’s not being grown successfully, possibly because of the death of the bacteria in the samples prior to their handling in our laboratory. We note that sample no. 11 was identified only to the genus level using the biochemical method. (Salmonella spp.) This specimen was identified to contain S. typhimurium using selective MS/MS of the biomarker of S. typhimurium, which is most often identified at Tsu-Chi General Hospital. The overall degree of successful 5130 Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
identification and matching between the biochemical and present methods was 97% without taking into account the samples that did not grow. Since the microorganisms in clinical specimens were identified to the species level, we did not attempt to differentiate the strains. In general, there is little difference in the proteomes among strains; however, if we can experimentally observe strainspecific biomarkers, we should be able to identify bacteria to the strain level. The presence of bacterial mixtures (S. aureus and P. aeruginosa; E. coli and S. agalactiae) in two samples that had been detected using the biochemical method were confirmed when using the selective MS/MS method. Many MS-based microbial detection methods rely on the spectral patterns of specific species; these patterns contain information relating to the masses of protein or other markers. Such spectral patterns become complicated when microbial mixtures are present in the sample. Because the masses may not be unique or may overlap with those of other species present in the sample, the detection may be rendered inaccurate. In contrast, the selective MS/MS method analyzes bacteria-specific marker peptides for each species present in the mixture. Not only are the marker masses analyzed but also their sequences. Multiple species may be identified simultaneously as long as the markers are recognized; therefore, this method provides the advantages of excellent selectivity and high accuracy in the identification of bacterial species present in complex systems. The CE separation was not ideal. The purpose of using CE was to provide a simple method to reduce sample complexity. If the digest of the cell extract was analyzed using the selective MS/ MS method without performing any separation, the tandem MS spectra were so complicated that they failed to identify any expected peptides (data not shown). The CE separation voltage
Figure 5. (a) Selected CE-MS/MS ion chromatogram of eight ions corresponding to eight pathogen species, including S. agalactiae, obtained from a wound sample. (b) MS/MS spectrum of the ions at m/z 1308.7, corresponding to S. agalactiae, obtained from one of the equally spaced peaks of the spectrum presented in (a).
could be lowered to broaden the CE peak width and to extend the MS/MS acquisition time. However, the peak width was sufficiently broad when using the present separation setup without lowering the voltage. If the elution period of each ion is too short, the ion might be lost during the sequential MS/MS interrogation period. Therefore, a narrow elution period for each ion will make it impossible to perform multiple monitoring of as many as eight ions. Bacterial biomarkers corresponding to as many as eight bacteria were monitored during a single run of the CE-MS
experiment. Although the instrumental setup allows monitoring of 12 ions, the computer tended to stop running when more than eight ions were monitored; we had to run a second CE-MS/MS analysis of the second set of ions when we required the monitoring of more than eight bacteria. The selective MS/MS method limits the analysis of microorganisms to eight species per run. This method is useful for clinical identification of some specific groups of pathogens, such as those obtained from swab samples, for which the pathogens are derived from pus or wound sources. Furthermore, each run of the CE-MS analysis takes ∼30 min, Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
5131
Figure 6. (a) Selected CE-MS/MS ion chromatogram of eight ions, including two markers associated with S. aureus and P. aeruginosa, obtained from a pus sample. The sample was identified biochemically to contain both S. aureus and P. aeruginosa. (b) MS/MS spectrum of the selected ion at m/z 1076.5 was correctly identified to be a peptide associated with S. aureus. (c) MS/MS spectrum of the ion at m/z 1638.8, which was identified to be the peptide IEDTDFAAETANLTK, suggesting that the microorganism source was P. aeruginosa. 5132 Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
Table 2. Search Results Generated from the Selective MS/MS Analysis of Each Clinical Specimen no.
bacterium
Gram stain
specimen
marker ion (m/z)
Xcorr score
identificationb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
E. faecalis E. coli E. coli E. coli E. coli P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa Salmonella spp.a S.s aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. epidermidis S. epidermidis S. epidermidis S. agalactiae S. agalactiae S. agalactiae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pneumoniae S. pyogenes S. pyogenes S. pyogenes S. aureus + P. aeruginosa E. coli + P. aeruginosa
positive negative negative negative negative negative negative negative negative negative negative positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive positive pos + neg neg + pos
pus pus pus urine urine sputum sputum sputum sputum sputum pus sputum sputum sputum sputum sputum sputum sputum wound wound wound wound pus urine sputum sputum sputum sputum sputum pus pus pus pus urine
955.93 1833.97 1833.97 1833.97 1833.97 1638.79 1638.79 1638.79 1638.79 1638.79 1968.99 1076.52 1076.52 1076.52 1076.52 1076.52 1076.52 1076.52 1040.49 1040.49 1040.49 1308.67 1308.67 1308.67 1451.74 1451.74 1451.74 1451.74 1451.74 1087.56 1087.56 1087.56 1076.52, 1638.79 1833.97, 1638.79
3.9255 2.7110 2.1852 2.0233 1.0822 3.0127 2.3362 2.4052 2.7347 2.9643 2.1326 1.9927 1.8878 3.2274 3.0217 2.5541 2.2627 2.4334 2.5592 2.5143 2.4174 2.0023 3.6754 2.0167 3.0872 3.0996 3.0574 2.4158 1.8617 2.0330 2.3385, 3.0979 2.5079, 3.0397
+ + + + + + + + + + + + + + + + no growth no growth + + + + + + + + + + + + + + +
a
Identifed as S. typhimurium using the selective MS/MS method. b +, species identified; -, species not identified.
including between-run preparation; this length of time is acceptable when performing several analyses. To monitor more than eight marker ions, we may also employ the elution times of specific marker peptides as an extra parameter, in addition to the ion masses, for the identification of bacteria. For instance, every eight marker ions can be blocked into a group according to their retention times. The application of using the retention time as a constraint to the selective MS/MS analysis of bacteria will be published elsewhere. The total analysis time, including the time required for direct culturing, protein digestion, and data analysis, was ∼8 h. The most time-consuming step was the cultivation process (6 h). The conventionally time-consuming digestion step was improved to less than 15 min through the application of microwave-assisted proteolysis.37 Although the present method is not as rapid as other MS-based methods, the salient features of this approach are its accuracy in bacterial identification and the ability to analyze bacterial mixtures. These favorable features arise from the selection of unique marker ions and the separation of complex peptides. Generally, traditional bacteriological methods require the isolation of a pure colony prior to microbial identification. With the present method, it is not necessary to use pure samples of bacteria. The main disadvantage of this method is that it is relatively expensive, considering the need to use mass spectrometers. In addition, the analysis time is longer than that required for direct MALDI-MS analysis because of the use of a coupled separation system.
CONCLUSIONS Our proposed selective CE-MS/MS method allows the identification of specific pathogens in clinical specimens. These clinical specimens were subjected to CE/MS analysis after direct cultivation without the need for prior isolation of the colony, which is required when using most bacterial identification methods. The direct culturing method greatly simplified the sample preparation process. The selective CE-MS/MS analyses of bacteria-specific marker peptides provide the advantages of excellent selectivity and high confidence in the identification of the bacterial species present in potentially complex systems, such as clinical samples. The overall degree of identification and matching between the biochemical and present methods was 97% from 34 clinical samples when not considering two no-growth specimens. Our results suggest that this method will have great potential for use in the identification of pathogens in clinical specimens. We are currently exploring further application of the selective MS/MS approach to the differentiation of bacterial strains ACKNOWLEDGMENT We thank the National Science Council of Republic of China for supporting this research financially.
Received for review March 21, 2006. Accepted May 5, 2006. AC060513+
Analytical Chemistry, Vol. 78, No. 14, July 15, 2006
5133
