Anal. Chem. 1987, 59, 2806-2809
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Profiling of Bacteria by Fast Atom Bombardment Mass Spectrometry D. N. Heller,* R. J. Cotter, a n d Catherine Fenselau' Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
0 . M. Uy The Johns Hopkins University Applied Physics Laboratory, Laurel, Maryland 20707
Positive and negative ion fast atom bombardment mass spectra have been obtained from lysed bacteria introduced without exlractlon Into the mass spectrometer. Phospho#pids and other polar llplds are found to be selectively desorbed to provlde molecular ions consistent with those observed In spectra of pure lipid samples. Three Grampodtlve and three Gram-negathre bacteria were studled. The anion spectra from these six bacteria are presented and are found to reflect qualttatively their dbtindhre phospholipid content. Fast atom bombardment mass spectra may serve as an aid In differentiating species.
Extensive chemotaxonomic studies through the last 25 years have shown that lipids and other classes of biochemicals extracted from bacteria can serve as fingerprints for taxonomic classification (1-3). Currently two approaches are widely used for the chemical characterization of bacteria. In one protocol, pyrolysis of intact bacteria is followed by gas chromatographic or mass spectral analysis ( 4 ) . In the second protocol, total fatty acids are extracted and derivatized for gas chromatographic analysis (5). In this laboratory mass spectrometric approaches are being developed and evaluated with the twin objectives of speed and reliability, by minimizing pretreatment of the sample, including culturing, and directly analyzing intact phospholipids and glycolipids already known to be chemotaxonomic biomarkers (6). Earlier observations had suggested that intact biochemicals might be preferentially desorbed from bacteria by some or all of the newer desorption techniques. These observations included the variability of fast atom bombardment (FAB), laser and plasma desorption efficiencies, which favor some compounds over others in mixtures (7-9), and the enhanced desorption of some compounds when these are present in a solid matrix such as lactalbumin or glutathione (9). In a preliminary study (6),FAB,laser, and plasma desorption were all found to desorb phospholipids selectively from lysed E. coli. In the present study the capabilities of fast atom bombardment mass spectrometry are evaluated to distinguish Gram-negative from Gram-positive bacteria and to characterize different species. Toward these objectives, three Gram-negative and three Gram-positive species of bacteria have been cultured or procured, four protocols have been evaluated for preparing cells for FAB analysis, and both cation and anion spectra have been measured under fast atom bombardment. EXPERIMENTAL SECTION Bacteria. Bacillus subtilis, var. niger (ATCC 9372), was grown in tryptic soy broth (Sigma, 30 g/L in H20)with aeration for 15
Current address: Department of Chemistry, University of Maryland, Baltimore, MD 21228.
h at 37 "C. Staphylococcus aureus (ATCC 6538) and Proteus vulgaris (ATCC 842'7) were purchased as lyophilized tablets (BBL Microbiology Systems), and cultures from these sources were grown in tryptic soy broth at 37 "C with agitation for 16 and 20 h, respectively. Escherichia coli strain C-600 was grown in a complex LB broth with agitation for 15 h at 37 "C. Cell cultures were harvested by chilling in an ice water bath followed by centrifugation. After the supernatant was removed, the pelleted cells were washed three times with distilled water, lyophilized, and stored at -80 OC. Micrococcus luteus (or, M. lysadeikticus, ATCC 4698) and Pseudomonas fluorescem (ATCC 13430) were purchased as lyophilized cells from the Sigma Chemical Co. These samples were suspended in H20, relyophilized, and stored a t 4 0 "C. Sample Preparation. Samples of lyophilized cells were suspended in 1mL of MeOH-CHC13 (21) for 30 min. The solvent was blown off with N2,and distilled water was added to make suspensions of 20 pg/pL of H20. Samples were stored at -80 "C prior to analysis. In studies of the efficacy of different chaotropic agents, 0.1 N HC1 and 0.1 N NaOH replaced the organic solvent, and aqueous suspensions were agitated with equal volumes of glass microbeads (Du Pont Clinical Systems) for 5 min. Total lipid extracts were prepared by a modified Bligh-Dyer procedure (10). Mass Spectrometry. Positive and negative ion fast atom bombardment mass spectra were obtained on a Kratos ( b e y , NJ) MS-50 double-focusing mass spectrometer. The magnet was scanned at 30 s/decade, and spectra were acquired and processed with the Kratos DS-90 data system. Sample suspensions were vortexed briefly before being added to the liquid matrices on the FAB probe: diethanolamine for anion spectra and thioglycerol for cation spectra. An amount of sample equivalent to 30 pg dry weight of cells was applied to the probe. B/E linked scanning was carried out with both oscillographic and computer-supported data acquisition. RESULTS In contrast to the case with E. coli (6),biomarkers were not reproducibly desorbed from lyophilized intact Gram-positive species under fast atom bombardment. Therefore, four minimal protocols were evaluated for breaking down the cell wall to facilitate desorption of lipids. Exposure to acid and base was found not to be effective; however both agitation with glass beads and short exposures to methanol/chloroform solutions did lead to biomarker ions in the FAB spectra. When relative efficiencies were evaluated, based on intensities of the molecular anions of the diacylphosphatidylethanolamine of mass 702 desorbed from E. coli, solvent lysing was found to provide about 50% more ion current than mechanical agitation. Cation and anion spectra from the three Gram-negative and three Gram-positive species were found to be of good quality and were judged suitable for interpretation. The anion spectra are presented in Figures 1 and 2. The major ions in the cation and anion spectra of the six species were attributed to polar lipids already shown to be components of the bacteria by chemotaxonomic studies (11-16). Structures and monoisotopic molecular weights of these phospho- and glycolipids are
0003-2700/87/0359-2806$01.50/00 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
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values are reported. Table I. Monoisotopic Mass of Molecular Anions of Polar Lipids in Figures 1 and 2 fatty acids
(R’+ R”) total
carbons:unsaturations 29:O 30:O 31:O 32:O 33:l 33:O 34:l 34:O 35:1 35:O
PE
PG
679.5 693.5 707.5 721.5 733.5 735.5 116.5 747.5 749.5 730.5 761.5 763.5
662.5 676.5 690.5 702.5
(M - H)LPG PI 767.5 821.6 781.5 835.6 795.5 849.6 809.5
DGDG 863.6 877.6 891.6
863.6 877.6 891.7
shown in Figure 3 and in Table I, respectively. Note that monoisotopic molecular weights are presented in the table while nominal masses are assigned on the spectra. Further, these nominal masses reflect ion formation by loss of a proton. The correlation between biomarkers in the mass spectra and lipid components in the chemotaxonomy literature was additionally supported by examining the desorption spectra of the total lipid fractions obtained by the Bligh-Dyer procedure (10). Anion spectra were similar to those of the bacteria, while the cation spectra showed higher signal to noise ratios. In addition, B/E linked scans of selected molecular cation species provided daughter ion spectra consistent with FAB spectra of reference samples. Examination of the spectra in Figures 1and 2 indicates that the various phospholipid families are selectively desorbed from
the complex mixtures under analysis. Some of these desorbed biomarkers are indicated on the spectra and are summarized by lipid family in Table 11. This table also indicates relative abundances of the various polar lipid families as reported in the literature (11-16). Relative abundances of phospholipid anions desorbed from the complex matrix of lysed E. coli remain relatively constant through more than 5 min in contrast, for example, to anions from a synthetic mixture of (16:0,18:l) phosphatidylethanolamine and (180, 180) phosphatidylglycerol. The cation spectra obtained from this set of samples contained fewer discernible biomarkers and more chemical noise than the anion spectra.
DISCUSSION The detection of membrane lipids from intact, lyophilized bacteria cells via FAB-MS appears to be dependent on cell envelope structure. Gram-positive species have a relatively thick, lipid-free wall, also called the peptidoglycan layer, surrounding the cytoplasmic membrane. When unlysed B. subtilis cells are analyzed by anion FAB, the early spectra do not show strong lipid ions. However, as particle bombardment continues over several minutes, lipid signals appear and grow more intense, presumably as more of the peptidoglycan layer is destroyed, releasing membrane lipids into the matrix. Gram-negative species on the other hand have an additional lipid-containing membrane outside of a relatively thin peptidoglycan layer. This outer membrane apparently disperses into the liquid matrix, allowing ready detection of lipids from intact cells. For the general case, a means of lysing cells prior to FAB analysis is important to obtain consistent mass spectra.
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
l l
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Figwe 2. Partial anion spectra from (a) 8.subtllis , (b) M . luteus, and (c) S . aweus. Abbrevlatlons are deflned in Figure 3. Nominal mass values are reported.
Table 11. Relative Abundance8 of Molecular Anions of Polar Lipids Desorbed from Lysed Bacteria
bacteria type lipid family
PE PG LPG DPG
E. coli
Gram negative P. fluorescens
100 (100)O 41 (19) (7)
Gram positive P. vulgaris
B. subtilis
100 (100) 10 (11)
100 (100)
(21)
(23)
26 (83) 100 (100) 13 (61) tr (33)
(9)
PI 3 (NRIb
DGDG
U1'
M . luteus
S. aureus
100 (71)
100 (100) 25 (15)
20 (100) 19 (18) 6 (14)
2
U2'
20
OValues in parentheses are approximate molar ratios, from literature sources (ref 11-16, respectively). bNR,not reported. 'U1 and U2, unknown h i d % Desorption spectra obtained in this study permit the Gram-positive and Gram-negative species to be readily grouped according to whether phosphatidylethanolamines or phosphatidylglycerols are the dominant phospholipid family detected in the FAB spectra. The high abundance of the phosphatidylethanolamine family in the walls of Gram-negative species and its reduced abundance relative to that of the phosphatidylglycerol family in Gram-positive species are well documented in the literature (1-3). The distribution of fatty acid combinations within each polar head group family may also be discerned from the distribution of molecular anions in Figures 1 and 2, with qualifications for positional isomerism. Assignment of the fatty acid combinations which make up these distributions
within each type of bacteria is made with reference to independent chemotaxonomic studies (1-3): phospholipids in Gram-negative species typically contain 16:O and 18:l fatty acids along with 17cp (cyclopropane) and 19cp fatty acids. Ions corresponding to these predominant carboxylate anions appear in the anion FAB spectra at m / z 255, 281,267, and 295, respectively. On the basis of published chemotaxonomic studies and also the fatty acids detected in the lower mass range of the anion spectra, the phosphatidylethanolamine ions of masses 690,702,716, and 730 in Figure 1are presumed to carry two palmitoyl moieties (160, 16:0), a (16:0, 17cp) combination, a (16:0, 18:l) combination, and a (16:0, 19cp) combination, respectively, as major isobars. On the other hand, phospholipids in Gram-positive species carry mainly saturated
ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
$ 1
R'- C-0-CH,
R'L c-0- CH
8 1
&*R', R "'
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i
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VARIABLE FATTY ACIDS
I
8
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We thank David White and David Hedrick (University of Tennessee) and Timothy Cooley (Johns Hopkins) for helpful discussions.
LITERATURE CITED
cnon-cn,-o-~-o-cn,
OH
other. However PG/PE ratios and unassigned lipid ions reproducibly detected above mass 800 permit their distinction. These unknown ions show heterogeneity typical of diacyl lipids (i.e., 14 mass unit separation), and their identity is under study. Comparison (Table 11) of the relative intensities of molecular species in the desorption spectra and relative molalities in the chemotaxonomic literature shows only a qualitative correspondence. This could reflect the use of different cultures in the different studies, however, the major factor is the difference in ease of desorption of the various polar lipid families under the fast atom beam as discussed in the introduction. Relative ion abundances in the spectra of E. coli samples prepared in the same way are reproducible. Twenty-one spectra from four E. coli experiments carried out in the same way were correlated with the average of the whole to yield coefficients ranging from 0.9536 to 0.9967. In contrast to some other studies (7,17) and in contrast to the behavior of a mixture of pure phospholipids, time after insertion has been found not to be a major experimental variable affecting relative abundances of ions desorbed from solvent-lysed bacteria.
ACKNOWLEDGMENT
OH
- o on - ' - o ~ o H
9 - 0-7-0 -CH,-
2809
CH-0-CO-A' CH,-0-CO-R"
-HEXOSE
DPG
DGDG
Flgure 3. Structures of representative polar membrane IlpMs: E, phosphatidylethanolamine; PG, phosphatidylglycerol; Lffi, lysylphosphatldylglycerol; PI, phosphatidylinositol; Dffi, dlphosphatidylglycerol or cardiolipin; DODO, dlglycosyldlglyceride.
fatty acids (1-3). These differences are reflected in the mass spectra. However, fatty acid distribution in the cells varies quantitatively and qualitatively according to the composition and temperature of the culture medium and also the growth phase and health of the bacteria. It is important to note that this characterization of bacteria rests on polar head groups rather than fatty acids. Within the Gram stain groups each species studied can be qualitatively differentiated by the anion spectra presented. Different combinations of biomarkers readily distinguish the three Gram-positive examples studied (Figure 1 and Table I) and qualitatively reflect literature values (Table 11). The detection of phosphatidylinoaitol in the spedrum of M. Zuteus, for example, is consistent with the observations of classical as is the observation of some chemotaxonomic studies (14), phosphatidylethanolamine in the spectrum from B. subtilis (13). The patterns of phospholipids desorbed (Figure 2) from the three Gram-negative species are more similar to one an-
Kates, M. I n Advances in Lipid Research; Paoletti, R., Ed.; Academic: New York. 1964; Vol. 2, pp 17-90. Shaw. N. Adv. Appl. Mlcroblol. 1074, 63-108. Lechevaller, M. P. CRC Crit. Rev. Micr&i. 1977, 5 , 109-210. Wieten, G.; Meuzelaar, H. L. C.; Haverkamp, J. I n GClMS Applicetlons in Microbiology; Odham, G., Larsson, L.. Mardh, P. A., Eds.; Pla num: London, 1983; pp 335-380. Moss, C. W. J. Chromatogr. 1981, 203, 337-347. Heller, D. N.; Fenselau, C.; Cotter, R. J.; Demirev, P.; Olthotf, J. K.; Honovich, J.; Uy, M.; Tanaka, T.; Klshlmoto, Y. Blachem. Biophys. Res. Commun. 1987, 142, 194-199. Ho, B. C.; Fenselau. C.; Hansen, 0.;Larsen, J.; Daniel, A. Clin. Chem. (Winston-Salem, N . C . ) 1983, 2 9 , 1349-1353. Fenselau, C.; Cotter, R. J. Chem. Rev. 1987, 8 7 , 501-512. Alai, M.; Demirev. P.; Fenselau, C.; Cotter, R. J. Anal. Chem. 1986, 58, 1303-1307. Kates, M. Techniques of Lipldology; American Elsevier: New York, 1972; p 351. Cronan. J. E., Jr.; Vagelos, P. R. Biochim. Siophys. Acta 1071, 265, 25-60. Cuilen, J.; Phillips, M. C.; Shipley, G. G. Biochem. J . 1071, 125, 733-742. Randle, C. L.; Albro, P. W.; Dittmer, J. C. Biochim. Siophys. Acta 1989, 187, 214-220. Op den Kamp, J. A. F.; Redai, I.; van Deenen, L. L. M. J . Becterlol. 1089, 99, 298-303. Thomas, T. D.; Ellar, D. J. Biochim. Siophys. Acta 1973, 316, 180-195. Kanemasa, Yasuhiro; Yoshoko, Tieko; Hayashi, Hideo Biochim . BioPhyS. Acta 1972, 280, 444-450. Townsend, R. R.; Heiler, D. N.; Fenselau, C.; Lee, Y. C. Biochemistv 1984, 23. 6389-6392.
RECEIVED for review March 16,1987. Accepted August 3,1987. This work was supported by the U.S. Army CRDEC under U.S. Navy Contract N00024-85-C-5301 and by the Applied Physics Laboratory Independent Research and Development Fund. Measurements were made at the Middle Atlantic Mass Spectrometry Facility, an NSF shared instrumentation facility.
