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A perspective on the fourth International Symposium on the Interface between Analytical Chemistry and Microbiology (ISIAM 2000)
Journal of Microbiological Methods 48 (2002) 95 – 100 www.elsevier.com/locate/jmicmeth
Preface
A perspective on the fourth International Symposium on the Interface between Analytical Chemistry and Microbiology (ISIAM 2000) ISIAM 2000 is the 4th in the series and we opportunistically look to the future towards ISIAM 2004. Having been one of the few present at the entire series to date, I was given the honor by the French organizers of helping to close the meeting and give an impromptu summary of where we are. Having now had a little more time for thought, I attempt here to provide a little deeper perspective. The First International Symposium on the Interface between Analytical Chemistry and Microbiology: Applications of Chromatography and Mass spectrometry was held in June 1987 at the University of South Carolina, Columbia, SC. The purpose of the ‘‘interface’’ meeting was to forge connections between analytical chemists and microbiologists that are using chromatography and mass spectrometry to solve common problems. The goals were admirably fulfilled. Nearly 100 participants from seven European countries, Japan and the United States participated in hearing 23 plenary talks and 36 submitted papers and posters. The book ‘‘Analytical Microbiology Methods: Chromatography and Mass Spectrometry’’ was loosely based on some of the presentations and discussions at the meeting. Each chapter described specific methodology and applications in the context of the relevant scientific background (Fox et al., 1990). It was noted, in the preface to the book, that ‘‘The advances in analytical microbiology have the potential to stimulate a revolution of improved methods for automated and rapid identification of microorganisms, characterization of microbial products and constituents and trace detection of microbial chemicals’’. This has been well documented at the three succeeding ‘‘interface’’ meetings. A tradition has been established in that
special issues of the Journal of Microbiological Methods (JMM) serve as platforms for original articles summarizing some of the major presentations at each meeting: Chromatography and mass spectrometry in microbiology (Tunlid and Larsson, 1992) and Analytical chemistry in environmental microbiology (White, 1996). The current special issue (Mass spectrometry and chromatography in biotechnology and clinical/ environmental microbiology (Guezennec, 2000)) serves as the third in the JMM series. Although, not associated directly with the ‘‘interface’’ series, an important book edited by Catherine Fenselau deserves attention in helping the analytical microbiology area move forward (Fenselau, 1994). It is interesting to see the dramatic changes that have occurred since the first ‘‘interface’’ meeting. Profiling of fatty acid monomers, released from membrane phospholipids of whole cells, using gas chromatography (GC) with flame ionization detection (FID) is still unequivocally the most widely used analytical method for bacterial speciation. As we are all aware, prior to GC analysis, fatty acids are released by methanolysis and subsequently converted to methyl esters and are thus referred to as fatty acid method esters (FAMES). The procedure, although not complicated, still requires several hours of manual derivatization prior to the automated GC analysis. At the time of the first ‘‘interface’’ meeting in 1987, this technique had certainly reached maturity as nicely described by Moss (1990). Although less widely used, carbohydrate profiling of whole cell hydrolysates using gas chromatographymass spectrometry (GC-MS) provides complementary information to FAMES and was also well established in 1987 (for review and update see Fox, 1999, 2000).
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Preface
Procedures developed for GC-FID (1960s – 1970s) were readily adapted for GC-MS analysis (from 1978 into the 1980s). Total ion spectra can also be used to identify each fatty acid component in a FAME profile. In the selected ion monitoring (SIM) MS mode, simple chromatograms free of background interferences from other components of the bacterial cell are generated. This helps greatly in trace analysis of fatty acids in complex environmental matrices (Tunlid and White, 1990). Indeed, developments in GC-MS methodology dominated the first and second interface meetings in 1987 and 1991. The 1995 meeting was perhaps a turning point. Clearly GC-MS as a technique was being widely used, for trace detection of fatty acids rather than developed, and this was confirmed by the large number of presentations at ISIAM 2000. Additionally, hydroxy fatty acids and carbohydrates (most notably muramic acid) have also been extensively used to quantitate the levels of bacteria and their cell envelope constituents (lipopolysaccharide [LPS] and peptidoglycan [PG]) in complex clinical and environmental matrices (Larsson and Saraf, 1997; Saraf and Larsson, 1998; Fox, 1999, 2000). High resolution chromatographic separations coupled with selective clean-up steps are important in improving the specificity of the detection of chemical markers in complex matrices. However, chromatographic separation is not sufficient to eliminate extraneous peaks when non-selective detectors are employed. The use of the mass spectrometer as a selective GC detector (i.e. GC-MS analysis in selected ion monitoring, SIM, mode) greatly in diminishing background noise by focusing only on ion(s) that are present in the compound of interest. However, even when using SIM, it is not uncommon to find extraneous background peaks. The tandem mass spectrometer, as a GC detector, provides even greater specificity in detecting trace amounts of chemical markers in complex matrices when used in multiple reaction monitoring (MRM) mode. Tandem mass spectrometry has the added advantage of generating a total ion mass spectrum from a selected precursor ion (product ion spectrum). The resulting product ion spectrum can be used for a definitive identification of the compound of interest at trace levels (Fox et al., 1995, 1996a,b; Saraf and Larsson, 1996, 1998; Larsson and Saraf, 1997; Krahmer et al., 1999a; Bal and Larsson, 2000). Both Swedish and US groups
interested in developments in GC-MS/MS participated in ISIAM 2000. In bacterial cells, marker compounds are present at the part per hundred to part per thousand level. In environmental samples, which represent a complex mixture of components, such markers are often present at the part per ten thousand to part per hundred thousand level. In certain clinical samples, in some instances, these markers may be present as low as parts per 100 million. Absolute identification of these markers in certain clinical and environmental samples is an exacting analytical task requiring sophisticated instrumentation. GC-MS/MS has currently been used with the greatest degree of sensitivity and specificity (Kozar et al., 2000). MRM and generation of product ion spectra both involve three discrete mass analysis steps. The first stage involves selection of a precursor ion. This instrumental clean-up removes other ions. The precursor ion is then fragmented by collision induced dissociation (CID) using an inert gas. In the third stage, all precursor ions can be collected (product ion spectrum) or a single product ion is selected (MRM). Both SIM GC-MS and MRM GC-MS/MS analyses allow excellent quantitation of such chemical markers, but the latter provides much greater confidence in trace analysis (Krahmer et al., 1999a). Two types of instruments are commonly used in GC-MS/MS analysis: ion traps and triple quadrupoles. In triple quadrupole instruments, the three stages of analysis are performed using three distinct quadrupole mass analyzers. There is some decrease in sensitivity due to loss of ions in transmission through the three quadrupoles (‘‘separation in space’’). In ion trap tandem mass spectrometers, the three stages occur in the same mass analyzer (‘‘separation in time’’). This dramatically simplifies the instrument and its cost. Furthermore, sensitivity of MS/MS analysis is improved, particularly in product ion spectrum mode (Krahmer et al., 1999a). However, in trace quantitative analysis of carbohydrates, in MRM mode, it was observed the ion trap is less precise than the triple quadrupole. However, the low cost, ease of use of the ion trap and its power for absolute identification (product ion spectrum) makes it use extremely attractive for diagnostic applications. MRM GC-MS/MS has great utility for determining the levels of bacterial contamination for both clinical
Preface
and environmental analyses. For example, muramic acid, ergosterol and hydroxy fatty acid levels (markers respectively for bacterial PG, Gram negative bacterial LPS and fungi) serve as a useful measure of biopollution of indoor air (Saraf and Larsson, 1998). GCMS/MS is also a powerful tool for detection of bacteria or their constituents in mammalian body fluids and tissues which are sterile in the absence of infection. Muramic acid is not synthesized by mammalian enzyme systems. As we learned at ISIAM 2000 (Szponar and Larsson), hydroxy fatty acids are present in normal blood. The presence of a hydroxy fatty acid ‘‘background’’ may limit the usefulness of hydroxy fatty acids in clinical applications. An automated derivatization instrument has been developed for sugar profiling by GC-MS. The instrument could be readily adapted for other applications. The core of the automated derivatization machine, where chemical reactions and evaporations are performed, consists of a custom built manifold with 21 glass chambers, to each of which a test tube is attached. The manifold is seated in a movable heating block. A series of electrically driven solenoid valves are attached in-line with the manifold. A set of solvent valves control the input of solvent and/or nitrogen gas to each sample chamber. A set of gas valves control output to atmosphere or vacuum. Additionally, closure of all valves allow the samples to be sealed in a closed chamber (Steinberg and Fox, 1999). High performance liquid chromatography (LC) analysis coupled with electrospray ionization (ESI) MS and MS/MS is usually performed without prior derivatization simplifying sample preparation. However, GC-MS and GC-MS/MS are often less demanding for routine carbohydrate analysis. Furthermore, the sensitivity of GC-MS/MS for trace analysis of sugars present in complex matrices is currently unmatched by LC-MS/MS (Simpson et al., 1990; Conboy and Henion, 1992; Shahgholi et al., 1997; Wunschel et al., 1997). Simplification of manual steps (with custom built devices) and, more importantly, development of an automated derivatization instrument for GC-based analysis makes sample preparation potentially only a little more involved, than for LC analysis, but the other advantages of GC are retained. At the 1995 meeting, among soft ionization MS techniques, for analysis of small phospholipids and glycolipids, fast atom bombardment and laser desorp-
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tion ionization dominated the meeting. There were only a few talks, introducing analysis of larger molecules, including peptidoglycan (PG) oligomers and DNA, using more sensitive techniques—ESI MS and matrix-assisted laser desorption-time of flight (MALDITOF) MS. A notable presentation by Gunter Allmaier was built on elegantly at ISAIM 2000 (for earlier work, see Zenker et al., 1998). It has only been in the past 5 years with the widespread use of ESI and MALDI-TOF MS that ‘‘sensitive’’ analysis of native polar molecules (including proteins and DNA) can be achieved by mass spectrometry. Both ESI and MALDI-TOF MS show great promise in that rapid introduction of macromolecular chemical markers from microbes into the MS can be achieved without their destruction. Phospholipids can be ionized as intact entities for MS or MS/MS analysis. On MS/MS analysis, in the negative ion mode, individual fatty acids are observed in product ion spectra. The class of the phospholipid can be determined by summing the masses of individual fatty acids and subtracting the value from the mass of the parent (molecular) ion. MS analysis of intact microbial phospholipids had been demonstrated earlier using laser desorption (Platt et al., 1988) and fast atom bombardment MS analysis (Cole and Enke, 1991). Dramatic improvements in the sensitivity of phospholipid analysis have been achieved using ESI MS and MS/MS (Smith et al., 1995; Black et al., 1997). Surprisingly, there were only limited application of these developments presented at ISIAM 2000. In contrast, MALDI-TOF MS analysis of higher molecular mass bacterial whole cell proteins was a major new addition at ISIAM 2000. MALDI-TOF MS analysis of proteins was introduced several years earlier (Cain et al., 1994; Claydon et al., 1996; Holland et al., 1996; Krishnamurphy et al., 1996). Oral presentations focused on computer software for comparison of mass spectra. The power of the technique resides in the minimal sample preparation and the possibility of rapid computer assisted data handling. However, there were several questions and comments about sample handling and effect on mass spectra which appeared to be not totally resolved. Additionally, issues of long-term reproducibility both within and between laboratories were of concern. This technique appears to have a promising future. However, the discussion smacked very much of de´ja` vu of an earlier technique—pyrolysis mass spectrometry.
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Preface
Pyrolysis MS also involves minimal samples preparation. A bacterial suspension is inserted using a pyrolysis probe into the mass spectrometer. High temperature in the absence of oxygen (pyrolysis) converts macromolecules into volatile breakdown constituents involving depolymerization, dehydration, rearrangements, and bond scission reactions. Complex mass spectra are produced. Since all components of the cell (including nucleic acid, proteins, lipids and sugars) contribute to the mass spectra, it is generally difficult to assign the origin of peaks to specific molecular sources within the cell (Helyer et al., 1997). However, sometimes, specific components can be identified (e.g. fatty acids) in the spectra. The use of MS/MS aids greatly in this process (Deluca et al., 1990). Pyrolysis MS has proven useful in batch analysis both at the species and strain level. However, direct comparisons between laboratories have often proven difficult. This has made pyrolysis MS a fringe technique, often outside the microbiological mainstream. It is genuinely hoped that MALDI-TOF MS profiling does not share the same fate. LC is readily interfaced prior to ESI MS analysis and allows identification of proteins not readily achieved by MALDI-TOF MS (Krishnamurphy et al., 1999; Kawano et al., 2000). With the availability of bench top ion trap instruments, there are even more exciting possibilities for LC-MS/MS analysis. Comparison of sequence variability in the intergenic spacer region (ISR) between the 16S and 23S ribosomal RNA (rRNA) genes is useful for differentiating bacterial species. This region has been shown to vary in size and sequence even within closely related taxonomic groups. A complementary technique, 16S rRNA sequencing (the current ‘‘gold standard’’), tends to group closely related species, demonstrating their relatedness but not distinguishing them. For comparison of sequence variability within the entire ISR, PCR using a primer pair corresponding to conserved sequences of the 16S and 23 rRNA genes is performed. Agarose gel electrophoresis of the PCR products is commonly employed for determination of size of these PCR products by relative electrophoretic mobility. However, for closely related species, PCR products of ISRs are sufficiently similar in size that sequencing has previously been required for differentiation (Johnson et al., 2000). Precise determination of the MW of the PCR products, of portions of the ISR, allows the closely related species B. subtilis and B. atrophaeus to
be readily distinguished, 114 and 119 bp, respectively (Johnson et al., 2000). These developments were summarized at ISIAM 2000. Mass spectrometric analysis of PCR products is a new approach that allows more precise MW determination than gel electrophoresis (Tang et al., 1994; Liu et al., 1995; Doltycz et al., 1995; Muddiman et al., 1996; Naito et al., 1995; Hurst et al., 1996; Krahmer et al., 1999b; Johnson et al., 2000). Although impressive results have been achieved with high resolution mass spectrometers (Muddiman et al., 1996; Naito et al., 1995), routine analysis of PCR products is likely to occur with less expensive instruments (e.g. quadrupole or time-of-flight). Mass spectrometers differentiate PCR products by differences in the mass-divided-bycharge (m/z) of their ions. ESI produces multiple charged ions. Thus, m/z is relatively low even for large molecules such as PCR products. Quadrupole mass spectrometers, which usually have an upper m/z of 2000– 4000, are used most commonly in conjunction with ESI. In conclusion, MALDI-TOF MS and ESI MS analysis have taken over the imagination of many analytical chemists and microbiologists working at the ‘‘interface’’. As noted above, LC-ESI MS, LS-MS/MS and MS/MS provide exciting opportunities in analysis of other oligomers and polymers (including phospholipids, peptidoglycan fragments, proteins and PCR products). However, it is worthy of note that GCbased techniques (for analysis of fatty acids and sugars) are still widely used and have become even more powerful with the advent of GC-MS/MS. There are, thus, a wide variety of successful mass spectrometry- and chromatography-based techniques currently available. No one method is suitable for every application. With careful consideration of the various possibilities, technology is available that fits well the problem of interest. There is now a strong base on which to build further developments. Indeed, the future for analytical microbiology appears bright. References Bal, K., Larsson, L., 2000. New and simple procedure for the determination of muramic acid in chemically complex environments by gas chromatography-ion trap tandem mass spectrometry. J. Chromatogr., B 738, 57 – 65. Black, G.E., Snyder, A., Heroux, K., 1997. Chemotaxonomic diffe-
Preface rentiation between the Bacillus cereus group and Bacillus subtilis by phospholipid extracts analyzed with electospray tandem mass spectrometry. J. Microbiol. Methods 28, 187 – 200. Cain, T., Lubman, D., Weber, J., 1994. Differentiation of bacteria using protein profiles from matrix assisted laser desorption/ionization time of flight mass spectrometry. Rapid Commun. Mass Spectrom. 8, 1026 – 1030. Claydon, M., Davey, S., Edwards-Jones, V., Gordon, D., 1996. The rapid identification of intact microorganisms using mass spectrometry. Nat. Biotechnol. 14, 1584 – 1586. Cole, M., Enke, C., 1991. Direct determination of phospholipid structures in microorganisms by fast atom bombardment triple quadropole mass spectrometry. Anal. Chem. 63, 1032 – 1038. Conboy, J.J., Henion, J., 1992. High performance anion exchange chromatography coupled with mass spectrometry for the determination of carbohydrates. Biol. Mass Spectrom. 21, 397 – 407. DeLuca, S., Sarver, E., Harrington, P., Vorhees, K., 1990. Direct analysis of bacterial fatty acids by curie-point pyrolysis tandem mass spectrometry. Anal. Chem. 62, 1465 – 1472. Doltycz, M., Hurst, G., Habibi Goudarzi, S., McLuckey, S., Tang, K., Chen, C., Uziel, M., Jacobson, K.B., Woychik, R., Buchanan, M., 1995. Analysis of polymerase chain reaction-amplified DNA products by mass spectrometry using laser desorption and electrospray: current status. Anal. Biochem. 230, 205 – 214. Fenselau, C. (Ed.), 1994. Mass Spectrometry for the Characterization of Microorganisms. American Chemical Society, Washington, DC. Fox, A., 1999. Review. Carbohydrate profiling of bacteria by gas chromatography-mass spectrometry and their trace detection in complex matrices by gas chromatography-tandem mass spectrometry. J. Chromatogr., A 843, 287 – 300. Fox, A., 2000. Profiling and trace detection of bacterial cellular carbohydrates. In: Doyle, R. (Ed.), Glyocomicrobiology. Plenum, New York, NY, pp. 341 – 357. Fox, A., Morgan, S., Larsson, L., Odham, G. (Eds.), 1990. Analytical Microbiology Methods: Chromatography and Mass Spectrometry. Plenum, New York, NY. Fox, A., Wright, L., Fox, K., 1995. Gas chromatography tandem mass spectrometry for detection of muramic acid, a peptidoglycan marker in organic dust. J. Microbiol. Methods 22, 11 – 26. Fox, A., Krahmer, M., Harrelson, D., 1996a. Monitoring muramic acid in air (after alditol acetate derivatization) using a gas chromatograph-ion trap tandem mass spectrometer. J. Microbiol. Methods 27, 129 – 138. Fox, A., Fox, K., Christensson, B., Krahmer, M., Harrelson, D., 1996b. Absolute identification of muramic acid at trace levels in human septic fluids in vivo and absence in aseptic fluids. Infect. Immun. 64, 3911 – 3955. Helyer, R., Kelley, T., Berkeley, R., 1997. Pyrolysis mass spectrometry studies on Bacillus anthracis, Bacillus cereus and their close relatives. Zbl. Bact. 285, 319 – 328. Holland, R.D., Wilkes, J.G., Rafii, F., Sutherland, J.B., Persons, C.C., Vorhees, K.J., Lay, J.O., 1996. Rapid identification of intact whole bacteria based on spectral patterns using matrix assisted laser desorption/ionization with time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 10, 1227 – 1232. Hurst, G., Doktycz, M., Vass, A., Buchanan, M., 1996. Detection of
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bacterial DNA polymerase chain reaction products by matrix assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 10, 377 – 382. Johnson, Y.A., Nagpal, M., Krahmer, M.T., Fox, K.F., Fox, A., 2000. Precise molecular weight determination of PCR products of the rRNA intergenic region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli. J. Microbiol. Methods 40, 241 – 254. Kawano, Y., Ito, Y., Yamakawa, Y., Yamashino, T., Horii, T., Hasegawa, T., Ohta, M., 2000. Rapid isolation and identification of staphylocooccal exoproteins by reverse phase high performance liquid chromatography-electrospray ionization mass spectrometry. FEMS Micro. Lett. 189, 103 – 118. Kozar, M., Krahmer, M., Fox, A., Gray, B., 2000. Failure to detect muramic acid in normal rat tissues but detection in cerebrospinal fluid from patients with pneumococcal meningitis. Infect. Immun. 68, 4688 – 4698. Krahmer, M., Fox, K., Fox, A., 1999a. Comparison of ion trap and triple quadrupole GC-MS/MS in the quantitative and qualitative trace analysis of muramic acid in complex matrices. Int. J. Mass Spectrom. 190/191, 321 – 329. Krahmer, M.T., Johnson, Y.A., Walters, J.J., Fox, K.F., Fox, A., Nagpal, M., 1999b. Electrospray quadrupole mass spectrometry analysis of model oligonucleotides and polymerase chain reaction products: determination of base substitutions, nucleotide additions/deletions, and chemical modifications. Anal. Chem. 71, 2893 – 2900. Krishnamurphy, T., Ross, P., Rajamani, U., 1996. Detection of pathogenic and non-pathogenic bacteria by matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Methods Commun. Mass Spectrom. 10, 883 – 888. Krishnamurphy, T., Davis, M.T., Stahl, D.C., Lee, T.D., 1999. Liquid chromatography/microspray massspectrometry for bacterial investigation. Rapid Commun. Mass Spectrom. 13, 39 – 49. Larsson, L., Saraf, A., 1997. Review. Use of gas chromatographyion trap mass spectrometry for the detection and characterization of microorganisms in complex samples. Mol. Biotechnol. 7, 279 – 287. Liu, Y.-H., Bai, J., Zhu, Y., Liang, X., Siemieniak, D., Venta, P.J., Lubman, D.M., 1995. Rapid screening of genetic polymorphisms using buccal DNA with detection by matrix assisted laser desorption/ionization mass spectrometry. Rapid Comm. Mass Spectrom. 9, 735 – 743. Moss, W., 1990. The use of cellular fatty acids for identification of microorganisms. In: Fox, A., Morgan, S., Larsson, L., Odham, G. (Eds.), Analytical Microbiology Methods: Chromatography and Mass Spectrometry. Plenum, New York, NY, pp. 59 – 70. Muddiman, D.C., Wunschel, D.S., Liu, C., Pasa-Tolic, L., Fox, K.F., Fox, A., Anderson, G.A., Smith, R.D., 1996. Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry. Anal. Chem. 68, 3705 – 3712. Naito, Y., Ishikawa, K., Koga, Y., Tsuneyoshi, T., Terunuma, H., Arakawa, R., 1995. Molecular mass measurement of polymerase chain reaction products amplified from human blood DNA by electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 9, 1484 – 1486.
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Platt, J., Uy, O., Heller, D., Cotter, R., Fenselau, C., 1988. Computer-based linear regression analysis of desorption mass spectra of microorganisms. Anal. Chem. 60, 1415 – 1419. Saraf, A., Larsson, L., 1996. Use of a gas chromatography ion trap tandem mass spectrometer for the determination of chemical markers of microorganisms in organic dust. J. Mass Spectrom. 31, 389 – 396. Saraf, A., Larsson, L., 1998. Identification of microorganisms by mass spectrometry. In: Karjalainen, E.J., Hesso, A.E., Jalonen, J.E., Karjalainen, U.P. (Eds.), Advances in Mass Spectrometry, vol. 14. Elsevier, Amsterdam, Holland, pp. 445 – 455. Shahgholi, M., Ohorodnik, S., Callahan, J., Fox, A., 1997. Trace detection of underivatized muramic acid in environmental dust samples by microcolumn liquid chromatography-electrospray tandem mass spectrometry. Anal. Chem. 69, 1956 – 1960. Simpson, R.C., Fenselau, C.C., Hardy, M.R., Townsend, R.R., Lee, Y.C., Cotter, R.J., 1990. Adaptation of a thermospray liquid chromatography/mass spectrometry interface for use with alkaline exchange liquid chromatography of carbohydrates. Anal. Chem. 62, 248 – 252. Smith, P., Snyder, A.P., Harden, C.S., 1995. Characterization of bacterial phospholipids by electrospray ionization tandem mass spectrometry. Anal. Chem. 67, 1824 – 1830. Steinberg, P., Fox, A., 1999. Automated derivatization instrument: preparation of alditol acetates for analysis of bacterial carbohydrates using gas chromatography-mass spectrometry. Anal. Chem. 71, 1914 – 1917. Tang, K., Tarenko, N., Allman, S., Chang, L., Chen, C., 1994. Detection of 500-nucleotide DNA by laser desorption mass spectrometry. Rapid Commun. Mass Spectrom. 8, 727 – 730.
Tunlid, A., White, D.C., 1990. Use of lipid biomarkers in environmental samples. In: Fox, A., Morgan, S., Larsson, L., Odham, G. (Eds.), Plenum, New York, NY, pp. 59 – 70. Tunlid, A., Larsson, L. (Eds.), 1992. Chromatography and mass spectrometry in microbiology. J. Microbiol. Methods 15, 145 – 248. White, D.C. (Ed.), 1996. Analytical chemistry in environmental microbiology. J. Microbiol. Methods 25, 101 – 195. Wunschel, D., Fox, K., Fox, A., Nagpal, M., Kim, K., Stewart, G., Shahgholi, M., 1997. Quantitative analysis of neutral and acidic sugars in whole bacterial cell hydrolysates using high performance anion exchange liquid chromatography electrospray ionization tandem mass spectrometry. J. Chromatogra A. 776, 205 – 219. Zenker, A., Planzagl, B., Lo¨ffelhardt, W., Allmaier, G., 1998. Negative and positive ion matrix assisted laser desorption ionization mass spectrometry of peptidoglycan fragments after size fractionation and reversed phase high performance liquid chromatography. J. Microbiol. Methods 32, 237 – 246.
Alvin Fox * Department of Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, SC 29208, USA E-mail address: [email protected]
*
Tel.: +1-803-733-3288; fax: +1-803-733-3192.
Preface
A perspective on the fourth International Symposium on the Interface between Analytical Chemistry and Microbiology (ISIAM 2000) ISIAM 2000 is the 4th in the series and we opportunistically look to the future towards ISIAM 2004. Having been one of the few present at the entire series to date, I was given the honor by the French organizers of helping to close the meeting and give an impromptu summary of where we are. Having now had a little more time for thought, I attempt here to provide a little deeper perspective. The First International Symposium on the Interface between Analytical Chemistry and Microbiology: Applications of Chromatography and Mass spectrometry was held in June 1987 at the University of South Carolina, Columbia, SC. The purpose of the ‘‘interface’’ meeting was to forge connections between analytical chemists and microbiologists that are using chromatography and mass spectrometry to solve common problems. The goals were admirably fulfilled. Nearly 100 participants from seven European countries, Japan and the United States participated in hearing 23 plenary talks and 36 submitted papers and posters. The book ‘‘Analytical Microbiology Methods: Chromatography and Mass Spectrometry’’ was loosely based on some of the presentations and discussions at the meeting. Each chapter described specific methodology and applications in the context of the relevant scientific background (Fox et al., 1990). It was noted, in the preface to the book, that ‘‘The advances in analytical microbiology have the potential to stimulate a revolution of improved methods for automated and rapid identification of microorganisms, characterization of microbial products and constituents and trace detection of microbial chemicals’’. This has been well documented at the three succeeding ‘‘interface’’ meetings. A tradition has been established in that
special issues of the Journal of Microbiological Methods (JMM) serve as platforms for original articles summarizing some of the major presentations at each meeting: Chromatography and mass spectrometry in microbiology (Tunlid and Larsson, 1992) and Analytical chemistry in environmental microbiology (White, 1996). The current special issue (Mass spectrometry and chromatography in biotechnology and clinical/ environmental microbiology (Guezennec, 2000)) serves as the third in the JMM series. Although, not associated directly with the ‘‘interface’’ series, an important book edited by Catherine Fenselau deserves attention in helping the analytical microbiology area move forward (Fenselau, 1994). It is interesting to see the dramatic changes that have occurred since the first ‘‘interface’’ meeting. Profiling of fatty acid monomers, released from membrane phospholipids of whole cells, using gas chromatography (GC) with flame ionization detection (FID) is still unequivocally the most widely used analytical method for bacterial speciation. As we are all aware, prior to GC analysis, fatty acids are released by methanolysis and subsequently converted to methyl esters and are thus referred to as fatty acid method esters (FAMES). The procedure, although not complicated, still requires several hours of manual derivatization prior to the automated GC analysis. At the time of the first ‘‘interface’’ meeting in 1987, this technique had certainly reached maturity as nicely described by Moss (1990). Although less widely used, carbohydrate profiling of whole cell hydrolysates using gas chromatographymass spectrometry (GC-MS) provides complementary information to FAMES and was also well established in 1987 (for review and update see Fox, 1999, 2000).
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 ( 0 1 ) 0 0 3 11 - 6
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Preface
Procedures developed for GC-FID (1960s – 1970s) were readily adapted for GC-MS analysis (from 1978 into the 1980s). Total ion spectra can also be used to identify each fatty acid component in a FAME profile. In the selected ion monitoring (SIM) MS mode, simple chromatograms free of background interferences from other components of the bacterial cell are generated. This helps greatly in trace analysis of fatty acids in complex environmental matrices (Tunlid and White, 1990). Indeed, developments in GC-MS methodology dominated the first and second interface meetings in 1987 and 1991. The 1995 meeting was perhaps a turning point. Clearly GC-MS as a technique was being widely used, for trace detection of fatty acids rather than developed, and this was confirmed by the large number of presentations at ISIAM 2000. Additionally, hydroxy fatty acids and carbohydrates (most notably muramic acid) have also been extensively used to quantitate the levels of bacteria and their cell envelope constituents (lipopolysaccharide [LPS] and peptidoglycan [PG]) in complex clinical and environmental matrices (Larsson and Saraf, 1997; Saraf and Larsson, 1998; Fox, 1999, 2000). High resolution chromatographic separations coupled with selective clean-up steps are important in improving the specificity of the detection of chemical markers in complex matrices. However, chromatographic separation is not sufficient to eliminate extraneous peaks when non-selective detectors are employed. The use of the mass spectrometer as a selective GC detector (i.e. GC-MS analysis in selected ion monitoring, SIM, mode) greatly in diminishing background noise by focusing only on ion(s) that are present in the compound of interest. However, even when using SIM, it is not uncommon to find extraneous background peaks. The tandem mass spectrometer, as a GC detector, provides even greater specificity in detecting trace amounts of chemical markers in complex matrices when used in multiple reaction monitoring (MRM) mode. Tandem mass spectrometry has the added advantage of generating a total ion mass spectrum from a selected precursor ion (product ion spectrum). The resulting product ion spectrum can be used for a definitive identification of the compound of interest at trace levels (Fox et al., 1995, 1996a,b; Saraf and Larsson, 1996, 1998; Larsson and Saraf, 1997; Krahmer et al., 1999a; Bal and Larsson, 2000). Both Swedish and US groups
interested in developments in GC-MS/MS participated in ISIAM 2000. In bacterial cells, marker compounds are present at the part per hundred to part per thousand level. In environmental samples, which represent a complex mixture of components, such markers are often present at the part per ten thousand to part per hundred thousand level. In certain clinical samples, in some instances, these markers may be present as low as parts per 100 million. Absolute identification of these markers in certain clinical and environmental samples is an exacting analytical task requiring sophisticated instrumentation. GC-MS/MS has currently been used with the greatest degree of sensitivity and specificity (Kozar et al., 2000). MRM and generation of product ion spectra both involve three discrete mass analysis steps. The first stage involves selection of a precursor ion. This instrumental clean-up removes other ions. The precursor ion is then fragmented by collision induced dissociation (CID) using an inert gas. In the third stage, all precursor ions can be collected (product ion spectrum) or a single product ion is selected (MRM). Both SIM GC-MS and MRM GC-MS/MS analyses allow excellent quantitation of such chemical markers, but the latter provides much greater confidence in trace analysis (Krahmer et al., 1999a). Two types of instruments are commonly used in GC-MS/MS analysis: ion traps and triple quadrupoles. In triple quadrupole instruments, the three stages of analysis are performed using three distinct quadrupole mass analyzers. There is some decrease in sensitivity due to loss of ions in transmission through the three quadrupoles (‘‘separation in space’’). In ion trap tandem mass spectrometers, the three stages occur in the same mass analyzer (‘‘separation in time’’). This dramatically simplifies the instrument and its cost. Furthermore, sensitivity of MS/MS analysis is improved, particularly in product ion spectrum mode (Krahmer et al., 1999a). However, in trace quantitative analysis of carbohydrates, in MRM mode, it was observed the ion trap is less precise than the triple quadrupole. However, the low cost, ease of use of the ion trap and its power for absolute identification (product ion spectrum) makes it use extremely attractive for diagnostic applications. MRM GC-MS/MS has great utility for determining the levels of bacterial contamination for both clinical
Preface
and environmental analyses. For example, muramic acid, ergosterol and hydroxy fatty acid levels (markers respectively for bacterial PG, Gram negative bacterial LPS and fungi) serve as a useful measure of biopollution of indoor air (Saraf and Larsson, 1998). GCMS/MS is also a powerful tool for detection of bacteria or their constituents in mammalian body fluids and tissues which are sterile in the absence of infection. Muramic acid is not synthesized by mammalian enzyme systems. As we learned at ISIAM 2000 (Szponar and Larsson), hydroxy fatty acids are present in normal blood. The presence of a hydroxy fatty acid ‘‘background’’ may limit the usefulness of hydroxy fatty acids in clinical applications. An automated derivatization instrument has been developed for sugar profiling by GC-MS. The instrument could be readily adapted for other applications. The core of the automated derivatization machine, where chemical reactions and evaporations are performed, consists of a custom built manifold with 21 glass chambers, to each of which a test tube is attached. The manifold is seated in a movable heating block. A series of electrically driven solenoid valves are attached in-line with the manifold. A set of solvent valves control the input of solvent and/or nitrogen gas to each sample chamber. A set of gas valves control output to atmosphere or vacuum. Additionally, closure of all valves allow the samples to be sealed in a closed chamber (Steinberg and Fox, 1999). High performance liquid chromatography (LC) analysis coupled with electrospray ionization (ESI) MS and MS/MS is usually performed without prior derivatization simplifying sample preparation. However, GC-MS and GC-MS/MS are often less demanding for routine carbohydrate analysis. Furthermore, the sensitivity of GC-MS/MS for trace analysis of sugars present in complex matrices is currently unmatched by LC-MS/MS (Simpson et al., 1990; Conboy and Henion, 1992; Shahgholi et al., 1997; Wunschel et al., 1997). Simplification of manual steps (with custom built devices) and, more importantly, development of an automated derivatization instrument for GC-based analysis makes sample preparation potentially only a little more involved, than for LC analysis, but the other advantages of GC are retained. At the 1995 meeting, among soft ionization MS techniques, for analysis of small phospholipids and glycolipids, fast atom bombardment and laser desorp-
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tion ionization dominated the meeting. There were only a few talks, introducing analysis of larger molecules, including peptidoglycan (PG) oligomers and DNA, using more sensitive techniques—ESI MS and matrix-assisted laser desorption-time of flight (MALDITOF) MS. A notable presentation by Gunter Allmaier was built on elegantly at ISAIM 2000 (for earlier work, see Zenker et al., 1998). It has only been in the past 5 years with the widespread use of ESI and MALDI-TOF MS that ‘‘sensitive’’ analysis of native polar molecules (including proteins and DNA) can be achieved by mass spectrometry. Both ESI and MALDI-TOF MS show great promise in that rapid introduction of macromolecular chemical markers from microbes into the MS can be achieved without their destruction. Phospholipids can be ionized as intact entities for MS or MS/MS analysis. On MS/MS analysis, in the negative ion mode, individual fatty acids are observed in product ion spectra. The class of the phospholipid can be determined by summing the masses of individual fatty acids and subtracting the value from the mass of the parent (molecular) ion. MS analysis of intact microbial phospholipids had been demonstrated earlier using laser desorption (Platt et al., 1988) and fast atom bombardment MS analysis (Cole and Enke, 1991). Dramatic improvements in the sensitivity of phospholipid analysis have been achieved using ESI MS and MS/MS (Smith et al., 1995; Black et al., 1997). Surprisingly, there were only limited application of these developments presented at ISIAM 2000. In contrast, MALDI-TOF MS analysis of higher molecular mass bacterial whole cell proteins was a major new addition at ISIAM 2000. MALDI-TOF MS analysis of proteins was introduced several years earlier (Cain et al., 1994; Claydon et al., 1996; Holland et al., 1996; Krishnamurphy et al., 1996). Oral presentations focused on computer software for comparison of mass spectra. The power of the technique resides in the minimal sample preparation and the possibility of rapid computer assisted data handling. However, there were several questions and comments about sample handling and effect on mass spectra which appeared to be not totally resolved. Additionally, issues of long-term reproducibility both within and between laboratories were of concern. This technique appears to have a promising future. However, the discussion smacked very much of de´ja` vu of an earlier technique—pyrolysis mass spectrometry.
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Pyrolysis MS also involves minimal samples preparation. A bacterial suspension is inserted using a pyrolysis probe into the mass spectrometer. High temperature in the absence of oxygen (pyrolysis) converts macromolecules into volatile breakdown constituents involving depolymerization, dehydration, rearrangements, and bond scission reactions. Complex mass spectra are produced. Since all components of the cell (including nucleic acid, proteins, lipids and sugars) contribute to the mass spectra, it is generally difficult to assign the origin of peaks to specific molecular sources within the cell (Helyer et al., 1997). However, sometimes, specific components can be identified (e.g. fatty acids) in the spectra. The use of MS/MS aids greatly in this process (Deluca et al., 1990). Pyrolysis MS has proven useful in batch analysis both at the species and strain level. However, direct comparisons between laboratories have often proven difficult. This has made pyrolysis MS a fringe technique, often outside the microbiological mainstream. It is genuinely hoped that MALDI-TOF MS profiling does not share the same fate. LC is readily interfaced prior to ESI MS analysis and allows identification of proteins not readily achieved by MALDI-TOF MS (Krishnamurphy et al., 1999; Kawano et al., 2000). With the availability of bench top ion trap instruments, there are even more exciting possibilities for LC-MS/MS analysis. Comparison of sequence variability in the intergenic spacer region (ISR) between the 16S and 23S ribosomal RNA (rRNA) genes is useful for differentiating bacterial species. This region has been shown to vary in size and sequence even within closely related taxonomic groups. A complementary technique, 16S rRNA sequencing (the current ‘‘gold standard’’), tends to group closely related species, demonstrating their relatedness but not distinguishing them. For comparison of sequence variability within the entire ISR, PCR using a primer pair corresponding to conserved sequences of the 16S and 23 rRNA genes is performed. Agarose gel electrophoresis of the PCR products is commonly employed for determination of size of these PCR products by relative electrophoretic mobility. However, for closely related species, PCR products of ISRs are sufficiently similar in size that sequencing has previously been required for differentiation (Johnson et al., 2000). Precise determination of the MW of the PCR products, of portions of the ISR, allows the closely related species B. subtilis and B. atrophaeus to
be readily distinguished, 114 and 119 bp, respectively (Johnson et al., 2000). These developments were summarized at ISIAM 2000. Mass spectrometric analysis of PCR products is a new approach that allows more precise MW determination than gel electrophoresis (Tang et al., 1994; Liu et al., 1995; Doltycz et al., 1995; Muddiman et al., 1996; Naito et al., 1995; Hurst et al., 1996; Krahmer et al., 1999b; Johnson et al., 2000). Although impressive results have been achieved with high resolution mass spectrometers (Muddiman et al., 1996; Naito et al., 1995), routine analysis of PCR products is likely to occur with less expensive instruments (e.g. quadrupole or time-of-flight). Mass spectrometers differentiate PCR products by differences in the mass-divided-bycharge (m/z) of their ions. ESI produces multiple charged ions. Thus, m/z is relatively low even for large molecules such as PCR products. Quadrupole mass spectrometers, which usually have an upper m/z of 2000– 4000, are used most commonly in conjunction with ESI. In conclusion, MALDI-TOF MS and ESI MS analysis have taken over the imagination of many analytical chemists and microbiologists working at the ‘‘interface’’. As noted above, LC-ESI MS, LS-MS/MS and MS/MS provide exciting opportunities in analysis of other oligomers and polymers (including phospholipids, peptidoglycan fragments, proteins and PCR products). However, it is worthy of note that GCbased techniques (for analysis of fatty acids and sugars) are still widely used and have become even more powerful with the advent of GC-MS/MS. There are, thus, a wide variety of successful mass spectrometry- and chromatography-based techniques currently available. No one method is suitable for every application. With careful consideration of the various possibilities, technology is available that fits well the problem of interest. There is now a strong base on which to build further developments. Indeed, the future for analytical microbiology appears bright. References Bal, K., Larsson, L., 2000. New and simple procedure for the determination of muramic acid in chemically complex environments by gas chromatography-ion trap tandem mass spectrometry. J. Chromatogr., B 738, 57 – 65. Black, G.E., Snyder, A., Heroux, K., 1997. Chemotaxonomic diffe-
Preface rentiation between the Bacillus cereus group and Bacillus subtilis by phospholipid extracts analyzed with electospray tandem mass spectrometry. J. Microbiol. Methods 28, 187 – 200. Cain, T., Lubman, D., Weber, J., 1994. Differentiation of bacteria using protein profiles from matrix assisted laser desorption/ionization time of flight mass spectrometry. Rapid Commun. Mass Spectrom. 8, 1026 – 1030. Claydon, M., Davey, S., Edwards-Jones, V., Gordon, D., 1996. The rapid identification of intact microorganisms using mass spectrometry. Nat. Biotechnol. 14, 1584 – 1586. Cole, M., Enke, C., 1991. Direct determination of phospholipid structures in microorganisms by fast atom bombardment triple quadropole mass spectrometry. Anal. Chem. 63, 1032 – 1038. Conboy, J.J., Henion, J., 1992. High performance anion exchange chromatography coupled with mass spectrometry for the determination of carbohydrates. Biol. Mass Spectrom. 21, 397 – 407. DeLuca, S., Sarver, E., Harrington, P., Vorhees, K., 1990. Direct analysis of bacterial fatty acids by curie-point pyrolysis tandem mass spectrometry. Anal. Chem. 62, 1465 – 1472. Doltycz, M., Hurst, G., Habibi Goudarzi, S., McLuckey, S., Tang, K., Chen, C., Uziel, M., Jacobson, K.B., Woychik, R., Buchanan, M., 1995. Analysis of polymerase chain reaction-amplified DNA products by mass spectrometry using laser desorption and electrospray: current status. Anal. Biochem. 230, 205 – 214. Fenselau, C. (Ed.), 1994. Mass Spectrometry for the Characterization of Microorganisms. American Chemical Society, Washington, DC. Fox, A., 1999. Review. Carbohydrate profiling of bacteria by gas chromatography-mass spectrometry and their trace detection in complex matrices by gas chromatography-tandem mass spectrometry. J. Chromatogr., A 843, 287 – 300. Fox, A., 2000. Profiling and trace detection of bacterial cellular carbohydrates. In: Doyle, R. (Ed.), Glyocomicrobiology. Plenum, New York, NY, pp. 341 – 357. Fox, A., Morgan, S., Larsson, L., Odham, G. (Eds.), 1990. Analytical Microbiology Methods: Chromatography and Mass Spectrometry. Plenum, New York, NY. Fox, A., Wright, L., Fox, K., 1995. Gas chromatography tandem mass spectrometry for detection of muramic acid, a peptidoglycan marker in organic dust. J. Microbiol. Methods 22, 11 – 26. Fox, A., Krahmer, M., Harrelson, D., 1996a. Monitoring muramic acid in air (after alditol acetate derivatization) using a gas chromatograph-ion trap tandem mass spectrometer. J. Microbiol. Methods 27, 129 – 138. Fox, A., Fox, K., Christensson, B., Krahmer, M., Harrelson, D., 1996b. Absolute identification of muramic acid at trace levels in human septic fluids in vivo and absence in aseptic fluids. Infect. Immun. 64, 3911 – 3955. Helyer, R., Kelley, T., Berkeley, R., 1997. Pyrolysis mass spectrometry studies on Bacillus anthracis, Bacillus cereus and their close relatives. Zbl. Bact. 285, 319 – 328. Holland, R.D., Wilkes, J.G., Rafii, F., Sutherland, J.B., Persons, C.C., Vorhees, K.J., Lay, J.O., 1996. Rapid identification of intact whole bacteria based on spectral patterns using matrix assisted laser desorption/ionization with time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 10, 1227 – 1232. Hurst, G., Doktycz, M., Vass, A., Buchanan, M., 1996. Detection of
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bacterial DNA polymerase chain reaction products by matrix assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 10, 377 – 382. Johnson, Y.A., Nagpal, M., Krahmer, M.T., Fox, K.F., Fox, A., 2000. Precise molecular weight determination of PCR products of the rRNA intergenic region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli. J. Microbiol. Methods 40, 241 – 254. Kawano, Y., Ito, Y., Yamakawa, Y., Yamashino, T., Horii, T., Hasegawa, T., Ohta, M., 2000. Rapid isolation and identification of staphylocooccal exoproteins by reverse phase high performance liquid chromatography-electrospray ionization mass spectrometry. FEMS Micro. Lett. 189, 103 – 118. Kozar, M., Krahmer, M., Fox, A., Gray, B., 2000. Failure to detect muramic acid in normal rat tissues but detection in cerebrospinal fluid from patients with pneumococcal meningitis. Infect. Immun. 68, 4688 – 4698. Krahmer, M., Fox, K., Fox, A., 1999a. Comparison of ion trap and triple quadrupole GC-MS/MS in the quantitative and qualitative trace analysis of muramic acid in complex matrices. Int. J. Mass Spectrom. 190/191, 321 – 329. Krahmer, M.T., Johnson, Y.A., Walters, J.J., Fox, K.F., Fox, A., Nagpal, M., 1999b. Electrospray quadrupole mass spectrometry analysis of model oligonucleotides and polymerase chain reaction products: determination of base substitutions, nucleotide additions/deletions, and chemical modifications. Anal. Chem. 71, 2893 – 2900. Krishnamurphy, T., Ross, P., Rajamani, U., 1996. Detection of pathogenic and non-pathogenic bacteria by matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Methods Commun. Mass Spectrom. 10, 883 – 888. Krishnamurphy, T., Davis, M.T., Stahl, D.C., Lee, T.D., 1999. Liquid chromatography/microspray massspectrometry for bacterial investigation. Rapid Commun. Mass Spectrom. 13, 39 – 49. Larsson, L., Saraf, A., 1997. Review. Use of gas chromatographyion trap mass spectrometry for the detection and characterization of microorganisms in complex samples. Mol. Biotechnol. 7, 279 – 287. Liu, Y.-H., Bai, J., Zhu, Y., Liang, X., Siemieniak, D., Venta, P.J., Lubman, D.M., 1995. Rapid screening of genetic polymorphisms using buccal DNA with detection by matrix assisted laser desorption/ionization mass spectrometry. Rapid Comm. Mass Spectrom. 9, 735 – 743. Moss, W., 1990. The use of cellular fatty acids for identification of microorganisms. In: Fox, A., Morgan, S., Larsson, L., Odham, G. (Eds.), Analytical Microbiology Methods: Chromatography and Mass Spectrometry. Plenum, New York, NY, pp. 59 – 70. Muddiman, D.C., Wunschel, D.S., Liu, C., Pasa-Tolic, L., Fox, K.F., Fox, A., Anderson, G.A., Smith, R.D., 1996. Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry. Anal. Chem. 68, 3705 – 3712. Naito, Y., Ishikawa, K., Koga, Y., Tsuneyoshi, T., Terunuma, H., Arakawa, R., 1995. Molecular mass measurement of polymerase chain reaction products amplified from human blood DNA by electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 9, 1484 – 1486.
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Platt, J., Uy, O., Heller, D., Cotter, R., Fenselau, C., 1988. Computer-based linear regression analysis of desorption mass spectra of microorganisms. Anal. Chem. 60, 1415 – 1419. Saraf, A., Larsson, L., 1996. Use of a gas chromatography ion trap tandem mass spectrometer for the determination of chemical markers of microorganisms in organic dust. J. Mass Spectrom. 31, 389 – 396. Saraf, A., Larsson, L., 1998. Identification of microorganisms by mass spectrometry. In: Karjalainen, E.J., Hesso, A.E., Jalonen, J.E., Karjalainen, U.P. (Eds.), Advances in Mass Spectrometry, vol. 14. Elsevier, Amsterdam, Holland, pp. 445 – 455. Shahgholi, M., Ohorodnik, S., Callahan, J., Fox, A., 1997. Trace detection of underivatized muramic acid in environmental dust samples by microcolumn liquid chromatography-electrospray tandem mass spectrometry. Anal. Chem. 69, 1956 – 1960. Simpson, R.C., Fenselau, C.C., Hardy, M.R., Townsend, R.R., Lee, Y.C., Cotter, R.J., 1990. Adaptation of a thermospray liquid chromatography/mass spectrometry interface for use with alkaline exchange liquid chromatography of carbohydrates. Anal. Chem. 62, 248 – 252. Smith, P., Snyder, A.P., Harden, C.S., 1995. Characterization of bacterial phospholipids by electrospray ionization tandem mass spectrometry. Anal. Chem. 67, 1824 – 1830. Steinberg, P., Fox, A., 1999. Automated derivatization instrument: preparation of alditol acetates for analysis of bacterial carbohydrates using gas chromatography-mass spectrometry. Anal. Chem. 71, 1914 – 1917. Tang, K., Tarenko, N., Allman, S., Chang, L., Chen, C., 1994. Detection of 500-nucleotide DNA by laser desorption mass spectrometry. Rapid Commun. Mass Spectrom. 8, 727 – 730.
Tunlid, A., White, D.C., 1990. Use of lipid biomarkers in environmental samples. In: Fox, A., Morgan, S., Larsson, L., Odham, G. (Eds.), Plenum, New York, NY, pp. 59 – 70. Tunlid, A., Larsson, L. (Eds.), 1992. Chromatography and mass spectrometry in microbiology. J. Microbiol. Methods 15, 145 – 248. White, D.C. (Ed.), 1996. Analytical chemistry in environmental microbiology. J. Microbiol. Methods 25, 101 – 195. Wunschel, D., Fox, K., Fox, A., Nagpal, M., Kim, K., Stewart, G., Shahgholi, M., 1997. Quantitative analysis of neutral and acidic sugars in whole bacterial cell hydrolysates using high performance anion exchange liquid chromatography electrospray ionization tandem mass spectrometry. J. Chromatogra A. 776, 205 – 219. Zenker, A., Planzagl, B., Lo¨ffelhardt, W., Allmaier, G., 1998. Negative and positive ion matrix assisted laser desorption ionization mass spectrometry of peptidoglycan fragments after size fractionation and reversed phase high performance liquid chromatography. J. Microbiol. Methods 32, 237 – 246.
Alvin Fox * Department of Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, SC 29208, USA E-mail address: [email protected]
*
Tel.: +1-803-733-3288; fax: +1-803-733-3192.