Anal. Chem. 1994,66,4171-4176
Electrospray Tandem Mass Spectrometry for Analysis of Native Muramic Acid in Whole Bacterial Cell Hydrolysates Gavin E. Black, Alvin Fox,* and Karen Fox School of Medicine, University of South Carolina, Columbia, South Carolina 29208 A. Peter Snyder
Development and Engineering Center, U S . Army Chemical Research, Aberdeen Proving Ground, Maryland 210 10-5423 Philip B. W. Smith Gunpowder Branch, Geo-Centers, Inc., P.O. Box 68, Aberdeen Proving Ground, Maryland 21010-0068
Muramic acid is an amino sugar found in eubacterial cell walls and not elsewhere in nature. This study explored the use of electrospray tandem mass spectrometry (ESI MS/MS) in analysis of underivatized muramic acid in bacterial hydrolysates. Fungal hydrolysates were used as negative controls. The only processing used was hydrolysis in sulfuric acid followed by extraction with an organic base (NjV-dioctylmethylamine).toremove the acid prior to ESI MS/MS analysis. Compared with pure muramic acid, bacterial hydrolysates produced more complex ESI mass spectra, such that the protonated molecular ion at m/z 252 was barely detectable. In contrast, product ion spectra of m/z 252 were identical among pure muramic acid, Gram positive bacteria, and Gram negative bacteria. However, no characteristic product ion spectrum was manifested from m/z252 in fungal samples. This allowed ready, visual differentiation of bacteria and fungi. Multiple reaction monitoring (MRM) followingmuramic acid fragmentations (m/z 252 144 and m/z 252 126) increased sensitivity and allowed quantitative differentiation when compared with the MRM of the internal standard N-methyl-D-glucamine(m/z 196 44). ESI MS/MS required minimal sample preparation and allowed rapid sample throughput for analysis of muramic acid in whole bacterial cell hydrolysates.
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Conventionally,microbes are identified by morphological and metabolic characteristics. For example, the Gram stain and other microscopic tests can readily differentiate bacteria and fungi. Bacteria can be classified into two broad categories based on this stain, Gram positive and Gram negative. Staining characteristics are thought to be related to differences in cell wall structure. In recent years, there has been considerable interest in bacterial differentiation based on chemical composition using mass spectrometry.’-3 Bacterial cell wall composition fall into two categories. Gram positive type indicates a thick peptidoglycan (PG) with no outer * Fax number, (803) 733-3192; e-mail address,
[email protected]., (1) Odham, G.; Larsson, L;M&rdh, P.A Gas Chromatography/Mass Spectrometty Applications in Microbiology; Plenum Press: New York, 1984. 0003-2700/94/0366-4171$04.50/0 0 1994 American Chemical Society
membrane, whereas Gram negative type has inner and outer membranes with a thin PG.4 The so-called “Gram type” does not always correlate with classic Gram-staining characteristics. Indeed, it is best to consider the biochemical information inferred by Gram type as distinct from morphologicalinformation provided by the Gram stain. PG is a polymer that has a glycan backbone consisting of repeating units of N-acetylmuramicacid and N-acetylglucosamine, Muramic acid (MA) is an unusual sugar not found elsewhere in n a t ~ r e . ~ Considerable -~ heterogeneity exists in MA content among bacteria. Indeed, Gram positive bacteria can often have similar levels of MA compared to Gram negative bacteria. Due to this heterogeneity, MA levels do not strictly correlate with Gram-staining characteristics. Furthermore, some eubacteria do not contain MA,6 while fungi lack both PG and MA Classical methods for determination of MA are based on colorimetry. These procedures do not readily distinguish muramic acid from other amino sugars and amino acids at the levels found in bacterial cells. Chromatographic methods are certainly superior to colorimetric approaches. However, definite identification of sugars is not possible without the mass spectrometer. For example, using both colorimetric and chromatographic methods, it has been debated for many years whether the Gram negative bacteria Chlamydia contain MA. By use of gas chromatography/ mass spectrometry (GUMS), Chlamydia were shown to lack MA6 In this work, other bacteria were used as positive controls and fungi served as negative controls. Unfortunately, due to the complexity of the hydrolysis/derivatization procedure, it has been difficult to apply GC/MS analysis of MA (or other neutral and a m i n o s u g a r ~ outside ) ~ ~ ~ of a few research centers. (2) Fox, A; Morgan, S. L.; Larsson, L.; Odham, G. Analytical Microbiology Methods: Chromatography and Mass Spectrometv; Plenum Ress: New York, 1990. (3) Fenselau, C. Mass Spectrometv for the Characterization of Microorganisms; American Chemical Society: Washington, DC, 1994. (4) Eudy L.; Walla M.; Morgan S. L.; Fox, A Analyst 1985,110,381-385. (5) Fox, A.; Schwab, J. H.; Cochran, T. Infect. Immun. 1980,29, 526-531. (6)Fox,A.; Rogers, J. C.; Gilbart,J.; Morgan, S.; Davis, C. H.; Knight, S.; Wyrick, P. B.Infect. Immun. 1990,58, 835-837. (7) Findlay, R H.; Moriarty, D. J. W.; White, D. C. Geomicrobiol. 1.1983,3, 135-150.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994 4171
Pyrolysis G U M S is an attractive technique for Gram typing, in terms of both rapidity of analysis and automation. The differentiation of group B streptococci from other streptococcal groups was acheived by the identilication of a characteristic dehydration product (dianhydroglucitol) generated from the simpler sugar glucitol.1° However, MA (like many other compounds) is destroyed during the extremely high temperatures required for on-line depolmerization and volatilization during pyroly~is.~ Electrospray liquid chromatography/mass spectrometry @SI LC/MS) is a possible alternative to GC/MS of MA. Unfortunately, the technical difficulties of routine analysis of native sugars by LC/MS have not yet been fully solved. Excellent chromatography of native sugars can be obtained with anion exchange LC at highly alkaline pH using a pulsed amperometric detector. The high ionic strength solutions often necessary for LC of sugars are not directly suited to MS, and even the use of on-line suppressors which make eluent suitable for MS can cause loss of some charged sugar species.11J2 Decreased sensitivity can also occur due to suppression of ESI by contaminating ions from the LC mobile phase. Excellent sensitivity may be achievable using ESI tandem mass spectrometry (MS/MS) analysis since problems with the chromatography solvent entering the MS can be eliminated.I3 This study attempted to differentiate a group of Gram negative/ Gram positive bacteria from fungi using ESI MS/MS. The only sample preparation used was hydrolysis to release MA from cell wall polymers prior to analysis. Our aim was to develop a method that avoided both the complexities of derivatization/pyrolysis for sample volatilization in GC/MS and the technical problems associated with LC/MS for routine sugar analysis. EXPERIMENTAL SECTION Preliminary experiments were performed on an API I11 (Sciex, Thomhill, Canada) and the remainder on a Quattro (VG BioTech, Cheshire, England) with comparable results. Both instruments were triple-quadrupole mass spectrometers equipped with electrospray ionization sources. Both systems used a 50%acetonitrile/ water mobile phase, the former acidified with 1%acetic acid. The API I11 solvent delivery was maintained at 7 pL/min by a Harvard Apparatus (Harvard Apparatus, MA) syringe pump. Solvent delivery for the Quattro was achieved by a Pharmacia (Uppsala, Sweden) P-500 fast protein liquid chromatography (FPLC) pump operating at 17 pL/min. A Rheodyne (Cotati, CA) Model 7125 sample injector with a 10 pL sample loop was used to introduce samples into the solvent flow. Nitrogen was used as nebulizing and bath gas delivered at flow rates of 15 and 350 L/min, respectively. The collision cell hexapole region of the API 111used a pressure of 3 x 10-5 Torr with argon, whereas the Quattro was maintained at a pressure of 4 x Torr with argon and used a collision energy of 20 eV. Both Quattro quadrupoles were calibrated using a solution of 40 ng/pL poly(ethy1ene glycol) 400 (8) Fox, A,; Morgan, S. L.; Gilbart, J. In Analysis of Carbohydrates by GLC and MS; Biermann, C. J., McGinnis, G. D., Eds.; CRC Press: FL, 1989; Chapter 5, p 87. (9) Fox, A; Black, G. In Mass Spectrometryfor the Characterization of Microorganisms; Fenselau, C., Ed.; American Chemical Society: Washington, DC; 1994 Chapter 8, p 107. (10) Smith, C. S.; Morgan, S. L.; Parks, C. D.; Fox, A,; Pritchard, D. G. Anal. Chem. 1987,59, 1410-1413. (11) Simpson, R C.; Fenselau, C. C.; Hardy, M. R;Townsend, R R.; Lee, Y. C.; Cotter, R. J. Anal. Chem. 1990,62,248-252. (12) Conboy, J. J.; Henion, J. Bid. Muss Spectrom. 1992,21,397-407. (13) Duffin, K. L.; Henion, J. D.; Shieh. J. J. Anal. Chem. 1991,63,1781-1788.
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Analytical Chemistry, Vol. 66, NO. 23, December 1, 1994
252
100,
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A
A oh*
1~
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252
100,
I
I
I1
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Figure 1. ESI mass spectrum of the protonated molecular ion of muramic acid (MH+= m/z 252) (A), and the ESI product ion mass spectrum (6)exhibiting characteristic ions at mlz 126 and 144, in addition to ions at mlz216 and 234 formed by dehydration reactions.
(380-420) in 50%acetonitrile and 1 mM ammonium acetate. In order to prevent carryover, a minimum of three injections of acetonitrile/water/acetic acid (50:50:1) were injected between each sample. Four different data collection functions were used, each set to require 1.0 s of analysis time: function 1, ESI mass spectrum scanning from m l z 100 to 300; function 2, product ion mass spectrum of the protonated molecular ion of MA (MH+ = m / z 252) after collision-induced dissociation (CID) scanning from m/z 50 to 260; function 3, multiple reaction monitoring (MRM) following from m/z 252 144 and m / z 252 126 ion dissociations, which are specific for MA, with a dwell time of 0.5 s per dissociation; function 4, MRM following the transition m / z 196 44,which is specific for N-methybglucamine WeGlN) (MH+ = m / z 196). Quattro resolution was 1.5 Da at half peak height for both quadrupoles, with an acquisition step size of 8 points/ Da. MA was purchased from Sigma (St. Louis, MO); MeGlN was obtained from Aldrich (Milwaukee, WI); glacial acetic acid and sulfuric acid were Ultrex grade a. T. Baker, Phillipsburg, NJ), and glass distilled chloroform and acetonitrile (Burdick &Jackson, Muskegon, MI) were used as supplied from the manufacturer. N,N-Dioctylmethylamine (Fluka, Ronkonkoma, NY) was only available as reagent grade and was preextracted twice with double distilled water to remove contaminants. Representative Gram positive and Gram negative bacteria and fungi included the following (Gram positive bacteria) Staphylococcus epidemzidis,Bacillus subtilis, Colynebacterium haemolyticum, Clostm'diumpeMngens, and Bacillus anthracis; (Gram negative bacteria) Neisseria sicca, Flavobacterium meningosepticum, En-
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Figure 2. Interpretation of ESI MA product ions produced by dehydration and fragmentation reactions, including the characteristic loss of lactic acid.
terobacter cloacae, Shigella sonnei, Escherichia coli, Aeromonas hydrophilia, Klebsiella pneumoniae, and Legionella pneumophila; (fungi) Aspergillus fumigatus, Cladosporium sp., C@tococcus neoformans, Microsporum canis, Nigrospore sp., and Saccharomyces cerevisiae. Microbes were grown in the appropriate liquid media, washed three times in water, and sterilized by autoclaving prior to lyophilization. Samples of 5-20 mg were suspended in 500 pL of 2 N HzS04 and hydrolyzed for 3 h at 100 "C. All samples were analyzed in duplicate. Samples were cooled, and 100pg of MeGlN was added as an internal standard for quantitation. External standards consisted of mixtures of 50 pg of MA and 100pg of MeGlN treated identically to samples, except with no heating. Samples were neutralized by addition of 1.25 mL of a 50%N,N-dioctylmethylamine/chloroform mixture. The aqueous phase was extracted twice with 1.5 mL of chloroform to remove residual N,Ndiocfylmethylamine. A 200 pL aliquot of aqueous phase was added to 200 p L of acetonitrile and acidfied with 4 p L of 10%acetic acid. A standard curve using MA ranging in concentration from 0 to 250 ng/pL and a constant amount of MeGlN (100 ng/pL) was used to test the quantitative nature of the technique. The possible role of sulfate ions (from sulfuric acid for hydrolysis) and residual N,N-dioctylmethylamine (used for neutralization) on ionization efficiency was addressed by comparing MS signal intensities of sets of mixtures of MA and MeGlN: aqueous solutions without acidfication or neutralization, solutions with neutralization, and solutions with neutralization and acidifcation.
To determine MA concentration in the samples, the ratio of MRM signals for MA and MeGlN (function 3 and function 4) in the sample was compared to the same ratio in the external standards. The concentration was divided by the weight of sample analyzed and the resulting weight of muramic acid expressed as percent dry weight of the microbial cells. RESULTS AND DISCUSSION The purpose of this study was to develop a method for rapid quantification of MA in hydrolysates of whole bacterial cells to provide chemotaxonomic information. Analysis of h g a l hydrolysates (which lack MA) served as negative controls. As MA is a component of cell wall polysaccharides, it must first be released by hydrolysis. Concentrated mineral acid, used for hydrolysis, is unsuitable for ESI MUMS analysis and was readily removed with a water-immiscible organic base, N,N-dioctylmethylamine. The ESI mass spectrum of MA contained a prominent proton. ated molecular ion at m/z 252 (see Figure 1) and a small dehydration species ( m / z 234). The m / z 252 product ion mass spectrum is also shown in Figure 1. Fragmentation of the protonated molecular ion includes single or double dehydrations resulting in m/z 234 and 216, in addition to loss of the lactic acid moiety, which result in fragments m / z 144 and 126 (see Figure 2). Dehydration reaction fragments are observed in the product ion mass spectra of many classes of compound and thus are not suitable for ESI MS/MS characterization analyses. Therefore, m/z 144 and 126 were chosen as distinctive product ions of MA, Analytical Chemistty, Vol. 66, No. 23, December 1, 1994
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196
A B
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Table 1. Suppression of Multiple Reaction Monitoring after Acidification and Neutralization
aqueous solution
MeGlN Added before Acidfication or Neutralization muramic acid 3.01 x lo4 9.19 x lo3 2.79 103
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Figure 3. ESI mass spectrum of the protonated molecular ion of methylglucamine (MH+ = m/z196) (A), and its ESI product ion mass spectrum (B), including the a-cleavage reaction which forms the characteristic methyliminium ion at mlz 44.
The MA product ions were identical on both the Sciex and VG Quattro instruments, as well as comparable to fragments generated by others using plasmaspray and thermospray.14 The abundance of the protonated molecular ion in ESI was increased over that of plasmaspray or thermospray. The MS/MS was tuned to maximize the protonated molecular ion of MA in the ESI mass spectrum and mlz 144 and 126 in the product ion spectrum. The ESI mass spectrum of MeGlN had a protonated molecular ion at mlz 196 and generated few prominent fragment ions after CID (see Figure 3). The only prominent fragment was the methyliminium ion (mlz 44). This fragment ion is produced by a-cleavage between C1 and C2 (the most common fragmentation of amines), where C1 bears the protonated amino group. The MA standard curve consisted of seven replicate samples (including a blank) with a concentration range of 7.8-250 n g l pL. All samples contained 100 ng/pL MeGlN as an internal standard. The plot of MA concentration versus W M e G l N MRM signal ratio gave an excellent linear response (12 > 0.99). Although, ESI MS/MS is generally used as a qualitative technique, others have also shown concentration-dependent responses.13 MS and MS/MS responses were reproducible between samples, and over the several days of experiments. As noted above, samples and standards were hydrolyzed in sulfuric acid and neutralized with N,N-dioctylmethylamine. MA and MeGlN in water alone exhibited higher signals when compared to samples that had been acidified and neutralized. To address this, MRM signal intensities of sets of mixtures of MA (14) Elmroth, I.; Larsson, L.; Westerdahl, G.; Odham, G. J. Chromatogr. 1992, 598, 43-50.
Analytical Chemistry, Vol. 66, No. 23, December
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acidification neutralization and neutralization
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and MeGlN were compared, including aqueous solutions without acidification or neutralization, solutions with neutralization alone, and solutions with neutralization and acidification (see Table 1). In one set of experiments MeGlN was added before any treatment, whereas in the second set of experiments MeGlN was added afterward. In both cases, neutralization with N,N-dioctylmethylamine reduced the signal for both MA and MeGlN. Acidifcation, in addition to neutralization, reduced the signal even further. Overall suppression was comparable regardless of when MeGlN was added. This suggested that suppression of signal, rather than physical loss of compounds, had occurred. Others have previously noted the difficulties of ESI MS analysis of carbohydrates due to ion suppression.12 ESI mass spectra of microbial hydrolysates exhibited more complex patterns than did mixtures of authentic MA and MeGlN and did not allow differentiation of bacteria and fungi. Figure 4 compares the ESI mass spectra from a representative Gram positive bacterium, S. epidermidis, a Gram negative bacterium, E. cloacae, and a fungus, A. ficmigatus. The presence of the m / z 252 protonated molecular ion of MA was often difficult to visually ascertain, although the m / z 196 molecular ion of MeGlN was clear in every sample. In bacterial samples containing at least 0.2%dry cell weight MA, product ions of m l z 252 were readily detected in the full-scan product ion mass spectra and were identical to those of pure MA fragments (Figure 5). The product ion mass spectra of the fungal samples analyzed did not contain characteristic MA fragment ions (see Figure 5). Samples below 0.2%MA did not produce visually identifiable mlz 144 or 126 fragment ions. MRM provided considerably better detection of MA and MeGlN than observation of product spectra. Identification of MA from whole cell hydrolysates using MRM was possible at levels of 1 part per 2000. All samples containing the internal standard, MeGlN, showed a clear signal for the transition m l z 196 44. In samples not containing MeGlN, no signal for that transition was observed. MRM signals for the transitions m/z 252 to 144 and 126, characteristic for MA, were identified in every bacterial sample analyzed, and quantitative analyses resulted in values of 0.06-1.04% MA on a dry weight basis. The MRM transitions used for MA were used to scan fungal samples and resulted in a baseline level, identical to the signal observed for solvent alone. This allowed ready differentiation of bacterial and fungal samples (see Figure 6 and Table 2). However, the MA MRM response for one gram negative bacterial sample, L. pneumoniae, a p proached the range found in the fungal samples.
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Figure 4. ESI full-scan mass spectra of an authentic MA and MeGlN mixture (A) and hydrolyzed whole cells of S. epidermidis (a Gram positive bacterium) (B),E. cloacae (Gram negative) ( C ) , and A. fumigarus (fungus) (D).
CONCLUSIONS Use of ESI MS/MS for the quantitation of muramic acid (a marker for cell wall PG) in hydrolysates of whole microbial cells was demonstrated. ESI MS/MS, compared to ESI MS, allowed ready visual discrimination of bacteria and fungi based on the presence of MA in the former. Certain unusual eubacteria (such as chlamydia) do not contain MA, In a wider sense, it is well established that MA is only found in the kingdom Eubacteria, not Archaebacteria. The content of MA provides information on the chemical composition (PG) among microbes that is not readily apparent by using traditional microbiological approaches. MA is present at different levels in most eubacteria, however, other markers are less widely distributed among different microbial species or genera. There is a great diversity of sugars, both common and uncommon, found in bacteria. For example, most members of the Gram negative LRgionellaceae contain the rare aminodideoxyhexosesquinovosamine and f u ~ o s a m i n e . ~Mem~J~ bers of the Gram positive genus Bacillus contain the uncommon aminohexose mann~samine.'~The alditol acetate procedure for wholecell carbohydrate profiling by G U M S was used to identify these sugars. In fact, these compounds, like MA, contain amino functions that would make them amenable to ESI ionization. Whether ESI MS/MS can provide carbohydrate profiles similar (15) FOX, A; Lau, P.; Brown, A; Morgan, S. L.; Zhu, Z,-T.; Lema, M.; Walla, M. D.3. Clin. Microbiol. 1984, 19, 326-332. (16) Fox, A; Rogers, J. C.; Fox, K F.; Schnitzer, G.; Morgan, S. L.; Brown, A; Aono, R /. Clin. Microbiol. 1990, 28, 546-552. (13 Fox, A; Black, G. E.; Fox, K; Rostovtseva, S. 3. Clin. Microbiol. 1993,31, 887-894.
Figure 5. ESI product ion mass spectra of the samples described in Figure 4.
to GC/MS for these and other sugars, remains to be explored in future work. Many sugars found in microorganisms need to be chromatographically resolved from other coexisting isomers to provide chemotaxonomic information. The presence of galactosamine enables differentiation of B. anthracis and Bacillus cereus.17 Without chromatographic resolution it would be extremely difficult to discriminate galactosamine from the mannosamine present in both species. In this instance, chromatographic separation is essential and MS/MS would be inadequate. However, as demonstrated here, ESI MS/MS is more easily applied than GC/MS to analysis of charged species (as is the case for amino or acidic sugars, such as are found in the teichuronic acid of bacilli), MS/ MS would provide profiling of compounds based on separation by molecular weight. Such proiiling could provide complementary information, but would not replace GC/MS. Compared to more conventional GC/MS analysis of bacterial carbohydrates, ESI MS/MS is simpler in terms of both sample preparation and speed of sample analysis. An ESI MS/MS analysis takes a couple of minutes versus 30-50 min for a typical GC/MS run. These considerationswould be extremely important if this approach were to be adapted for rapid or routine bacterial identification. The current work was concerned with identification of a compound found in pure bacterial cultures. Detection of bacteria, or their chemical components, in complex matrices is more difticult since bacterial markers are only present at trace levels. In the current ESI MS/MS work, the concentration of muramic acid was 0.05-1.04% and analysis was of 5-10 mg of sample. GC/ MS studies have detected MA in animal serum after injection with bacterial PG subunits (muramyl dipeptide) at concentrations of Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
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Table 2. Levels of Muramic Acid in Bacteria and Fungi* %drywt
Gram positive S. epidermidis B. subtilis C . haemoliticum C. perfn'ngens B. anthracis mean Gram negative N . sicca F. meningosepticum E. ccloacae S. sonnei E. coli A. hydrophilia K. pneumoniae L. pnuemophila mean fungi A. fumigutus C. neoformans Cladospon'um sp. M . canis Nigrospon'um sp. S. cerevisiae solvent mean
8 124
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.
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\
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1.036 0.806 0.266 0.188 0.135 0.486
0.781 0.347 0.308 0.300 0.188 0.125 0.201 0.063 0.289 0.010 0.024 0.022 0.051 0.013 0.018 0.050
0.027
As determined by multiple reaction monitoring.
analysis,which could provide chemotaxonomic differentiation, will be vital before widespread use of this technique is possible. 100 ng/mL (1part in 10 million) .I* MA from endogenous bacteria in house dust has been found at levels of 4 ng/mg (1 part in 250 000) using GUMS and GC/MS/MS. With current methodology, GC/MS sensitivity for trace detection of compounds from complex matrices is superior by several orders of magnitude over ESI MS/MS.I9 ESI MS/MS is useful, however, for the rapid identiflcation of compounds present at more elevated levels. In summary, the simplicity and rapidity of ESI MS/MS for the discrimination of bacteria from fungi based on MA content suggests the applicability of this approach with other compounds. Further research exploring compounds amenable to ESI MS/MS (18) Fox, A; Fox, K Infect. Immun. 1991,59, 1202-1205. (19) Fox, A; Wright, L.; Fox, K F. J Microbial. Methods, in press.
4176 Analytical Chemistry, Vol. 66, No. 23, December 7, 7994
ACKNOWLEDGMENT Thanks are extended to William E. Cotham and Michael D. Walla in the Mass Spectrometry Facility, Department of Chemistry and Biochemistry, of the University of South Carolina (Columbia, SC 29208) for help and advice. This work was supported by the Army Research Office (Grant DAAL03-92-0255),a training award from the Army Research Office DoD EPSCoR program, and an equipment grant (DAAHO493-G-0506). Received for review May 9, 1994. Accepted August 1994.e
@
Abstract published in Advance ACS Abstracts, October 1, 1994
26,
