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