, Volume 8, Issue 4, pp 259–268 | Cite as

Identification of archaea and some extremophilic bacteria using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

Original Paper


Archaea and a number of groups of environmentally important bacteria, e.g., sulfate-reducing bacteria, anoxygenic phototrophs, and some thermophiles, are difficult to characterize using current methods developed for phenotypically differentiating heterotrophic bacteria. We have evaluated matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) as a rapid method for identifying different groups of extremophilic prokaryotes using a linear mass spectrometer (Micromass, UK). The instrument is designed to acquire mass-spectral patterns from prokaryotic cell-wall components between masses of 500 and 10,000 Da in a statistically robust manner and create a database that can be used for identification. We have tested 28 archaea (10 genera, 20 spp.) and 42 bacteria (25 genera, 37 spp.) and found that all species yield reproducible, unique mass-spectral profiles. As a whole, the profiles for the archaea had fewer peaks and showed less differentiation compared to the bacteria, perhaps reflecting fundamental differences in cell-wall structure. The halophilic archaea all had consistent patterns that showed little differentiation; however, the software was able to consistently distinguish Halobacterium salinarium, Halococcus dombrowski, and Haloarcula marismortui from one another, although it could not always correctly distinguish four strains of Hb. salinarium from one another. The method was able to reliably identify 105 cells of either Albidovulum inexpectatum or Thermococcus litoralis and could detect as low as 103 cells. We found that the matrix, alpha-cyano-4-hydroxy-cinnamic acid yielded better spectra for archaea than 5-chloro-2-mercapto-benzothiazole. Overall, the method was rapid, required a minimum of sample processing, and was capable of distinguishing and identifying a very diverse group of prokaryotes.


Archaea Bacteria Extremophiles Identification Intact cell MALDI-TOF 



We thank Dave Cleland for help with culture work and Dr. Jason Bannan, Thuy Penella, and Michelle Pignone for discussions and help in using the MALDI-TOF. We thank Drs. Tim Lilburn and Yufeng Wang for comments on an earlier draft of the manuscript. We are indebted to Dr. Therese McKenna of Micromass-Waters for training and assistance in using the MALDI, and Jason Watson of Micromass-Waters for expert technical assistance. Micromass-Waters provided the ATCC with use of the instrument, but there are no financial obligations between the ATCC and Micromass-Waters. This work was supported in part by a grant (DBI-0090224) from the NSF to the ATCC.


  1. Albuquerque L, Santos J, Travassos P, Fernanda Nobre M, Rainey FA, Wait R, Empadinhas N, Silva MT, da Costa MS (2002) Albidovulum inexpectatum gen. nov., sp. nov., a nonphotosynthetic and slightly thermophilic bacterium from a marine hot spring that is very closely related to members of the photosynthetic genus Rhodovulum. Appl Environ Microbiol 68:4266–4273CrossRefPubMedGoogle Scholar
  2. Arnold RJ, Karty JA, Ellington AD, Reilly JP (1999) Monitoring the growth of a bacteria culture by MALDI-MS of whole cells. Anal Chem 71:1990–1996CrossRefPubMedGoogle Scholar
  3. Boone DR, Castenholtz RW (ed) (2001) Bergey’s manual of systematic bacteriology. Springer, Berlin Heidelberg New YorkGoogle Scholar
  4. Bright JJ, Claydon MA, Soufian M, Gordon DB (2002) Rapid typing of bacteria using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and pattern recognition software. J Microbiol Methods 48:127–138CrossRefPubMedGoogle Scholar
  5. Claydon MA, Davey SN, Edwards-Jones V, Gordon, DB (1996) The rapid identification of intact microorganisms using mass spectrometry. Nat Biotechnol 14:1584–1586PubMedGoogle Scholar
  6. Demirev PA, Ho Y, Ryzhov V, Fenselau C (1999) Microorganism identification by mass spectrometry and protein database searches. Anal Chem 71:2732–2738CrossRefPubMedGoogle Scholar
  7. Evason DJ, Claydon MA, Gordon DB (2001) Exploring the limits of bacterial identification by intact-cell mass spectrometry. J Am Soc Mass Spectrom 12:49–54CrossRefPubMedGoogle Scholar
  8. Fastner J, Erhard M, von Dohren H (2001) Determination of oligopeptide diversity within a natural population of Microcystis ssp. (cyanobacteria) by typing single colonies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Appl Environ Microbiol 67:5069–5076CrossRefPubMedGoogle Scholar
  9. Fenselau C, Demirov PA (2001) Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom Rev 20:157–171CrossRefPubMedGoogle Scholar
  10. Hathout Y, Demirev PA, Ho Y-P, Bundy JL, Ryzhov V, Sapp L, Stutler J, Jackman J, Fensleau C (1999) Identification of Bacillus spores by MALDI mass spectrometry. Appl Environ Microbiol 65:4313–4319PubMedGoogle Scholar
  11. Holland R, Wilkes JG, Rafii F, Sutherland JB, Persons CC, Voorhees KJ, Lay JO Jr (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 10:1227–1232CrossRefPubMedGoogle Scholar
  12. Jarman KH CS, Saenz AJ, Petersen CE, Valentine NB, Kingsley MT, Wahl KL (2000) An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 72:1217–1223CrossRefPubMedGoogle Scholar
  13. Kandler O, Konig H (1998) Cell wall polymers in archaea (archaebacteria). Cell Mol Life Sci 54:305–308CrossRefPubMedGoogle Scholar
  14. Korn-Wendisch F, Kutzner HJ (2003) The family Streptomycetacae. In: Dworkin M (ed) The prokaryotes. Springer, Berlin Heidelberg New YorkGoogle Scholar
  15. Krishnamurthy T, Ross PL (1996) Rapid identification of bacteria by direct matrix-assisted laser desorption/ionization mass spectrometric analysis of whole cells. Rapid Commun Mass Spectrom 10:1992–1996CrossRefPubMedGoogle Scholar
  16. Krishnamurthy T, Rajamani U, Ross PL, Jabbour R, Nair H, Eng J, Yates J, David MT, Stahl DC, Lee TD (2000) Mass spectral investigations on microorganisms. J Toxicol Toxin Rev 19:95–117CrossRefGoogle Scholar
  17. Lay JO Jr (2000) MALDI-TOF mass spectrometry and bacterial taxonomy. Trends Anal Chem 19:507–516CrossRefGoogle Scholar
  18. Lay JO Jr (2001) MALDI-TOF Mass spectrometry of bacteria. Mass Spectrom Rev 20:172–194Google Scholar
  19. Neuner A (1990) Thermococcus litoralis sp. nov.: a new species of extremely thermophilic marine archaebacteria. Arch Microbiol 153:205–207Google Scholar
  20. Pikuta E, Hoover RB, Marsic D, Whitman W, Cleland D, Krader P (2003) Desulfonatronum paiuteum sp. novel, a new alkaliphilic, sulfate-reducing bacterium, isolated from Mono Lake, California. Int J System Evol Microbiol 53:815–821CrossRefGoogle Scholar
  21. Saenz AJ, Peterson CE, Valentine NB, Gantt SL, Jarman KH, Kingsley MT, Wahl KL (1999) Reproducibility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for replicate bacterial culture analysis. Rapid Commun Mass Spectrom 13:1580–1585CrossRefPubMedGoogle Scholar
  22. Wahl KL, Wunschel SC, Jarman KH, Valentine, NB, Petersen CE, Kingsley, MT, Zartolas KA, Saenz AJ (2002) Analysis of microbial mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Chem 74:6191–6199PubMedGoogle Scholar
  23. Walker J, Fox AJ, Edwards-Jones V, Gordon DB (2002) Intact cell mass spectrometry (ICMS) used to type methicillin-resistant Staphylococcus aureus: media effects and inter-laboratory reproducibility. J Microbiol Methods 48:117–126CrossRefPubMedGoogle Scholar
  24. Watson JT (1997) Introduction to mass spectrometry, 3rd edn. Lippincott-Raven, PhiladelphiaGoogle Scholar
  25. Whitman WB, Boone DR, Koga Y (2001) Family II. Methanocoaldococcaceae fam. nov. In: Boone DR (ed) Bergey’s manual of systematic bacteriology: the archaea and the deeply branching and phototrophic bacteria. Springer, Berlin Heidelberg New York, pp 242–246Google Scholar
  26. Zillig W, Holz I, Janekovic D, Schafer W, Reiter WD (1983) The archaebacterium Thermococcus celer represents a novel genus within the thermophilic branch of the archaebacteria. Syst Appl Microbiol 4:88–94Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.American Type Culture CollectionManassasUSA

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