Anal. Chem. 2005, 77, 6092-6095
Characterization of Enterobacteria Using MALDI-TOF Mass Spectrometry Patrick Pribil and Catherine Fenselau*
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742
A method is proposed for the rapid classification of Gramnegative Enterobacteria using on-slide solubilization and trypsin digestion of proteins, followed by MALDI-TOF MS analysis. Peptides were identified from tryptic digests using microsequencing by tandem mass spectrometry and database searches. Proteins from the outer membrane family (OMP) were consistently identified in the Enterobacteria Escherichia coli, Enterobacter cloacae, Erwinia herbicola, and Salmonella typhimurium. Database searches indicate that these OMP peptides observed are unique to the Enterobacteria order. Recently, this laboratory has proposed that rapid and highly reliable characterization of bacteria can be achieved by brief proteolytic digestion in situ, MALDI desorption of the resulting peptides, and subsequent analysis by tandem mass spectrometry and bioinformatics.1-3 This approach has been demonstrated in the identification of Gram-positive Bacillus spores, where the success of the method is due in part to the fact that Bacillus spores contain a subset of proteins that are both highly abundant and selectively solubilized by treatment with acid (the small, acidsoluble proteins or SASPs). This strategy has also been extended recently to the analysis of Gram-positive Bacillus bacteria in their vegetative state,4 in which case species identifications were made possible by the identification of peptides from a limited number of proteins solubilized during sample preparation. Working with a small number of proteins limits the density of peptides detected in the spectra and facilitates observation of multiple peptides from each protein and, thus, more reliable identifications of proteins and, thereby, species. Gram-negative bacteria contain many species that are of clinical and microbiological importance. For example, members of the order Enterobacteriaceae (also referred to as Enterobacteria) include Escherichia coli and Salmonella typhimurium, each comprising nonpathogenic laboratory strains as well as those that are etiological causes of gastric infections. In this study, the strategy of selective protein solubilization and trypsin digestion, MALDITOF tandem mass spectrometry, and bioinformatic analysis is applied to Gram-negative Enterobacteria. The prevalent proteins * Corresponding author. Phone: 301-405-8616. Fax: 301-405-8615. E-mail:
[email protected]. (1) Warscheid, B.; Fenselau, C. Anal. Chem 2003, 75, 5618-5627. (2) Warscheid, B.; Jackson, K.; Sutton, C.; Fenselau, C. Anal. Chem 2003, 75, 5608-5617. (3) Pribil, P. A.; Patton, E.; Black, G.; Doroshenko, V.; Fenselau, C. J. Mass Spectrom. 2005, 40, 464-474. (4) Warscheid, B.; Fenselau, C. Proteomics 2004, 4, 2877-2892.
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identified in each of the four species studied were members of the family of outer membrane proteins (OMPs). OMP peptides were consistently observed in spectra, regardless of bacterial growth conditions or sample preparation procedure. The OMP peptides observed appear to be unique to Enterobacteria and could therefore serve as potential biomarkers for rapid characterization of the Enterobacteria order. They do not, however, provide the basis for species differentiation. EXPERIMENTAL SECTION Materials. Luria-Bertani broth (LB) and nutrient broth (NB) were obtained from Fisher Scientific (Fairlawn, NJ). Trifluoroacetic acid (TFA), NH4HCO3, NH4OH, ethanol, methanol, acetonitrile, Triton X-100, and R-cyano-4-hydroxycinnamic acid matrix (CHCA) were purchased from Sigma (St. Louis, MO). Immobilized trypsin was purchased from Pierce (Rockford, IL). Bacterial Cultures. E. coli ATCC 25404 (strain K12), Enterobacter cloacae, Erwinia herbicola (also known as Pantoea agglomerans), and Salmonella typhimurium (strain 98-9176) were obtained from the American Type Culture Collection (Manassas, VA) and grown using standard procedures,5 on either LB or NB medium as indicated. Cells were grown in 3-mL cultures overnight to saturation, washed twice with water, and then either immediately analyzed or lyophilized and stored at -20 °C. The bacteria studied here were handled in a Biosafety level 2 facility. Sample Preparation and Mass Spectrometry. Either freshly harvested or lyophilized cells were resuspended in distilled deionized water, or other solvents, as indicated at a concentration of 2.5 mg/mL. Aliquots (1 µL) were spotted onto a stainless steel MALDI target plate and allowed to air-dry, after which 1 µL of immobilized trypsin (washed in 25 mM NH4HCO3) was added. Digestion reactions were carried out at room temperature in a humidified chamber to prevent spot drying. Reactions were terminated after 5 min by addition of 1 µL pf 0.1% TFA. Following spot drying, 1 µL of CHCA matrix (50 mM in 70% acetonitrile/ 0.1% TFA) was applied. Samples were analyzed on an Axima CFR Plus MALDI-TOF mass spectrometer (Shimadzu Scientific, Columbia, MD). Spectra were obtained from 150 laser shots rastered in a zigzag pattern over the spot and then averaged. The laser power setting was 65 (in arbitrary units, between 0 and 180). For MS/MS microsequencing of peptides by unimolecular decay, precursor ions were selected by setting the ion gate to a window of (5 Da, and acquiring and averaging 150 shots as above, with (5) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning: a laboratory manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. 10.1021/ac050737c CCC: $30.25
© 2005 American Chemical Society Published on Web 08/12/2005
Figure 2. Unimolecular decay analysis of peptide with MH+ ) 1642 from E. coli (see Figure 1).
Figure 1. Partial mass spectra of tryptic digests of four different Enterobacteria. Cells were resuspended in water, spotted onto the MALDI slide, and digested with immobilized trypsin. Due to the occurrence of unidentifiable higher intensity background peaks in the lower mass range, only the mass range between m/z ) 800 and 2500 is shown. Only peptides whose microsequences were determined by unimolecular decay analysis and database searching are labeled. The sequences of identified peptides are listed in Table 1.
the laser power increased by 10%. The entire process could be executed in less than 10 min. Database Searches. Fragment peaks from unimolecular decay analysis were submitted to MASCOT (www. matrixscience.com) with the following search parameters: the database searched was NCBInr; taxonomy was limited to Eubacteria; protein and peptide mass tolerances were set to (1 Da, and the number of missed cleavages was set to 1. Once identified, the peptide sequences were also searched against the sequences of all proteins (all kingdoms) in the SwissProt/TrEMBL databases using BLAST (http://au.expasy.org/tools/blast/). RESULTS AND DISCUSSION Identification of Bacterial Proteins. MALDI mass spectra of peptides prepared in situ from four species of Enterobacteria are shown in Figure 1. Using precursor ion selection and unimolecular decay analysis (postsource decay), peptide microsequencing was undertaken for all peaks detected between m/z ) 800 and 2500 in each spectrum. An example of a unimolecular decay spectrum from which a sequence tag was obtained is shown in Figure 2. The sequence positions of leucine and isoleucine are
assigned from the protein matches made by Mascot, since their masses are not distinguishable in the fragment ions. The peptides identified in this way are listed in Table 1, while the list of proteins subsequently identified from each organism is shown in Table 2. As can be seen in Table 1, some peptides have several possible protein origins within a species, and in some cases, peptides could be assigned to more than one species (for example, LGGMVWR). Limited protein information is available in databases for E. herbicola and E. cloacae. However, three peptides were assigned in the former to a protein in a related organism, and one in the latter (indicated in columns 6 and 7 in Table 1). The search engine also assigned one peptide in S. typhimurium to a protein in a related organism, as indicated in column 8. With the exception of OmpN and acyl carrier protein from E. coli, OmpR from E. cloacae, and yahO and the DNA-binding protein from S. typhimurium, all proteins were identified using at least two peptides (Table 2). The identified proteins include representatives from the interior of the cell (for example, the DNA-binding proteins H-ns and Dps, both of which are highly abundant in bacteria6,7), as well as molecules on the surface of the cell (OMPs). Interestingly, OMPs were identified in all four species.8-10 These are abundant proteins; OMP A is present in E. coli at an estimated 100 000 copies.11 The results indicate that members of this family of proteins are easily solubilized from the outer membrane or that trypsin has access to these proteins while they are still associated with the outer membrane. Homologous sequences were noted among the observed OMP peptides (Table 1), suggesting that certain regions of the OMPs have a higher susceptibility to trypsin digestion, efficient desorption, or both. Sequence searches using BLAST indicated that the OMP proteins identified above m/z ) 800 are found among Enterobacteria in the SwissProt TrEMBL database but not in other bacteria in the database (Table 3). Consistent with this conclusion, no OMP peptides were detected in recent proteomic analyses of peptides from vegetative Gram-positive Bacilli.4,12 It should be noted that current databases contain protein sequences from only ∼200 bacterial species. It can be seen in Table 3 that sequences of two (6) Tendeng, C.; Bertin, P. N. Trends Microbiol. 2003, 11, 511-518. (7) Andrews, S. C.; Robinson, A. K.; Rodriguez-Quinones, F. FEMS Microbiol. Rev. 2003, 27, 215-237. (8) Beher, M. G.; Schnaitman, C. A.; Pugsley, A. P. J. Bacteriol. 1980, 143, 906-913. (9) Nikaido, H. Microbiol. Mol. Biol. Rev. 2003, 67, 593-656. (10) Wang, Y. Biochem. Biophys. Res. Commun. 2002, 292, 396-401. (11) Koebnik, R.; Locher, K. P.; Van Gelder, P. Mol. Microbiol. 2000, 37, 239253. (12) Francis, A. W.; Ruggiero, C. E.; Koppisch, A. T.; Dong, J.; Song, J.; Brettin, T.; Iyer, S. Biochim. Biophys. Acta 2005, 1748, 191-200.
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Table 1. Peptides (m/z ) 800-2500) Identified in Analysis of Four Enterobacteria MHobs+ a 805 818
peptide sequence TWTGQGR LGGMVWR
MHcot+ a
protein
E. colib
E. herbicolab
805.9 819.0
H-ns (DNA-binding protein) outer membrane protein A,
gi|43078 gi|7188818,
gi|50120684 (E. carotovora)
gi|12514142, gi|26246978, gi|37624560 gi|384221 gi|43078
943 957
QAHWNMR LEVVVNER
943.1 958.1
outer membrane protein 3a, outer membrane protein A, Outer membrane protein II Dps protein H-ns (DNA-binding protein)
1157
AALIDCLAPDR
1158,4
outer membrane protein A
-
1264
DGSVVVLGFTDR
1265.4
outer membrane protein A
-
1281
DGSVVVLGYTDR
1281.4
1410 1429 1485 1576 1626
IGSDAYNQGLSER TNDQVNHTAAGGDK TALIDHLDTMAER REEESAAAAEVEER LGYPVTDDLDVYTR
1410.5 1428.5 1486.7 1576.6 1627.8
outer membrane protein A, outer membrane protein 3a, outer membrane protein A, outer membrane protein II outer membrane protein 3a outer membrane protein N Dps protein H-ns (DNA-binding protein) outer membrane protein II
gi|7188818, gi|12514142, gi|26246978, gi|37624560 gi|12514142 gi|3273514 gi|384221 gi|43078 -
-
1642 1649 1655
LGYPITDDLDVYTR IGTISTTGEMSPLDAR LGYPITDDLDIYTR
1641.8 1649.9 1655.8
1679 1700 1715
SYPLDIHNVQDHLK EYSAMEASIDVQISR ITTVQAAIDYINGHQA
1679.9 1699.9 1715.9
outer membrane protein A yahO outer membrane protein 3a outer membrane protein A Dps protein outer membrane protein r acyl carrier protein
gi|7188818 gi|12514142, gi|26246978 gi|384221 gi|24112500
?? -
E. cloacaeb gi|3764542
styphimurium gi|758341, gi|7188820
-
-
gi|2914209 (S. enterica)
gi|50120684 (E. carotovora) gi|50120684 (E. carotovora) -
gi|37624542
gi|758341,
-
-
?? gi|148368 (E. aerogenes)
?? ?? -
gi|2613086 -
gi|7188820 gi|1069714 -
a Masses indicated are in daltons. b Where applicable, accession numbers are given for the appropriate proteins. If peptides were identified from a protein from a related species, the species name is indicated in brackets; ??, indicates that the peptide was observed but does not originate from any unkown protein in the given species; -, indicated that the peptide was not observed in the mass spectrum of the given species.
Table 2. Proteins Identified in Analysis of Four Enterobacteria species E. coli
E. herbicola
ACa
protein
gi|43078 gi|7188818 gi|26246978 gi|37624560 gi|384221 gi|12514142 gi|24112500 gi|3273514 gi|50120684
H-ns (DNA-binding protein) outer membrane protein A outer membrane protein A outer membrane protein II Dps protein outer membrane protein 3a acyl carrier protein outer membrane protein N outer membrane protein A (E. carotovora)d outer membrane protein II outer membrane protein R outer membrane protein II (E. aerogenes)d outer membrane protein A outer membrane protein A yahO DNA-binding protein (S. enterica)d
E. cloacae
gi|37624542 gi|2613086 gi|148368
S. typhimurium
gi|758341 gi|7188820 gi|10697141 gi|29142097
size (aa)
Seq Cov (%)b
scorec
135 346 379 157 167 346 78 377 366
21.5 9.5 8.7 11.5 20.4 13.3 20.5 3.7 8.2
142 131 135 105 120 187 115 75 122
158 192 228
12.0 7.8 6.1
94 83 92
350 249 67 137
5.4 8.4 23.9 5.8
102 112 87 78
a Accession number. b Percentage of protein sequence coverage. c See Experimental Section for MASCOT search parameters. Scores higher than 72 indicate matches at the 95% significance level. d Evidence for a homologue of the given protein from the indicated species present in that organism.
of the smaller peptides (m/z ) 805 and 943) were found in both Enterobacteria and non-Enterobacteria protein sequences, reflecting the random match probability of exact matches for small peptides (e-values 57 and 5.4, respectively). In fact, the lack of non-Enterobacteria protein sequences containing the peptide sequence for m/z ) 818 is remarkable, since its exact match 6094 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005
e-value is 24. These search results confirm the distinctiveness of the observed Enterobacteria peptide sequences. Recognition of OMP proteins should characterize unknown bacteria as members of the Enterobacteria order. Species assignment across characterized Enterobacteria, based on the OMP peptides alone, is complicated by the occurrence of identical peptides in multiple
Table 3. Numbers of Exact Matches from Peptide Sequence Searches MHobs+ a
peptide sequence
protein
total matchesb
Enterobacteria matchesc
1715 1700 1679 1655 1649 1642 1626 1576 1485 1429 1410 1281 1264 1157 957 943 818 805
ITTVQAAIDYINGHQA EYSAMEASIDVQISR SYPLDIHNVQDHLK LGYPITDDLDIYTR IGTISTTGEMSPLDAR LGYPITDDLDVYTR LGYPVTDDLDVYTR REEESAAAAEVEER TALIDHLDTMAER TNDQVNHTAAGGDK IGSDAYNQGLSER DGSVVVLGYTDR DGSVVVLGFTDR AALIDCLAPDR LEVVVNER QAHWNMR LGGMVWR TWTGQGR
acyl carrier protein outer membrane protein R Dps protein outer membrane protein 3a yahO outer membrane protein A outer membrane protein II H-ns (DNA-binding protein) Dps protein outer membrane protein N outer membrane protein 3a outer membrane protein II outer membrane protein A outer membrane protein A H-ns (DNA-binding protein) Dps protein outer membrane protein A H-ns (DNA-binding protein)
2 1 8 13 4 7 1 5 4 6 7 20 23 21 15 15 131 45
2 1 8 13 4 7 1 5 4 6 7 20 23 21 15 14 131 32
a Masses indicated are in daltons. b Results of BLAST searches of peptide sequences against all protein sequences in the SwissProt/TrEMBL databases (not limited to any taxonomy). c Results of BLAST searches of peptide sequences against Enterobacterial protein sequences in the SwissProt/TrEMBL databases.
species. The identification may be narrowed by incorporating observed H-ns and Dps peptides into the analyses, basing the species assignment on multiple peptides from multiple proteins. In addition to identical regions, the OMP proteins have regions of variable sequence across the species, and comparison of OMPs among all Enterobacteria may allow a targeted approach using species-specific OMP peptides, as was carried out recently for Bacillus spores using peptides from the readily accessible SASP protein family3. Constancy of Peptides Observed. Factors such as bacterial growth conditions, cellular harvesting methods, and sample preparation procedures have been reported to affect the mass spectra of proteins desorbed from microorganisms.13-15 Consequently, experimental parameters for the peptide analyses reported here, including bacterial growth medium (LB or NB), bacterial growth time (8, 16, or 24 h), bacterial harvest methods (analysis of freshly grown bacteria or harvested and lyophilized cultures, or picking colonies directly off culture plates), bacterial suspension solvents (water, 10% TFA, 25 mM NH4HCO3 pH 10, 0.5 M NH4(13) Wang, Z.; Russon, L.; Li, L.; Roser, D. C.; Long, S. R. Rapid Commun. Mass Spectrom. 1998, 12, 456-464. (14) Saenz, A. J.; Petersen, C. E.; Valentine, N. B.; Gantt, S. L.; Jarman, K. H.; Kingsley, M. T.; Wahl, K. L. Rapid Commun. Mass Spectrom. 1999, 13, 1580-1585. (15) Demirev, P. A.; Ho, Y. P.; Ryzhov, V.; Fenselau, C. Anal. Chem. 1999, 71, 2732-2738.
OH, 50% ethanol, 50% methanol, 50% acetonitrile, 0.05% Triton X-100), and cell suspension treatment (vortexing, boiling, or sonicating for 5 min) were evaluated. Peptide spectra from proteins selectively solubilized in water remained consistent in the m/z range 800-2500, under a variety of experimental conditions (data not shown). CONCLUSIONS The strategy of on-slide solubilization and trypsin digestion was extended to the analysis of examples of Gram-negative Enterobacteria. Peptides from the same class of proteins (OMPs) were observed in the four species studied, under a variety of experimental parameters, indicating that these proteins are easily accessible and abundant in Enterobacteria. These OMP peptides and proteins were found in current databases to be Enterobacteriaspecific, and their use is suggested as biomarkers for rapid identification and characterization of members of this order of bacteria. ACKNOWLEDGMENT We thank Dr. Nathan Edwards for helpful discussions regarding bioinformatics. Received for review April 29, 2005. Accepted July 14, 2005. AC050737C
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