title: Thymol treatment of bacteria prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis aids in identifying certain bacteria at the subspecies level.
abstract: BACKGROUND
  The identification of bacteria based on mass spectra produced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has become routine since its introduction in 1996. The major drawback is that bacterial patterns produced by MALDI are dependent on sample preparation prior to analysis. This results in poor reproducibility in identifying bacterial types and between laboratories. The need for a more broadly applicable and useful sample handling procedure is warranted.
  
  
  METHODS
  Thymol was added to the suspension solvent of bacteria prior to MALDI analysis. The suspension solvent consisted of ethanol, water and TFA. The bacterium was added to the thymol suspension solvent and heated. An aliquot of the bacterial suspension was mixed directly with the matrix solution at a 9:1 ratio, matrix/bacteria solution, respectively. The mixture was then placed on the MALDI plate and allowed to air dry before MALDI analysis.
  
  
  RESULTS
  The thymol method improved the quality of spectra and number of peaks when compared to other sample preparation procedures studied. The bacterium-identifying biomarkers assigned to four strains of E. coli were statistically 95% reproducible analyzed on three separate days. The thymol method successfully differentiated between the four E. coli strains. In addition, the thymol procedure could identify nine out of ten S. enterica serovars over a 3-day period and nine S. Typhimurium strains from the other ten serovars 90% of the time over the same period.
  
  
  CONCLUSIONS
  The thymol method can identify certain bacteria at the sub-species level and yield reproducible results over time. It improves the quality of spectra by increasing the number of peaks when compared to the other sample preparation methods assessed in this study. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.
authors.affiliation: Division of Systems Biology/Innovative Safety and Technologies Branch, USFDA/National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR, 72079, USA.
authors.forename: Ricky D
authors.initials: RD
authors.lastname: Holland
authors.forename: Jon G
authors.initials: JG
authors.lastname: Wilkes
authors.forename: Willie M
authors.initials: WM
authors.lastname: Cooper
authors.forename: Pierre
authors.initials: P
authors.lastname: Alusta
authors.forename: Anna
authors.initials: A
authors.lastname: Williams
authors.forename: Bruce
authors.initials: B
authors.lastname: Pearce
authors.forename: Michael
authors.initials: M
authors.lastname: Beaudoin
authors.forename: Dan
authors.initials: D
authors.lastname: Buzatu
mesh_terms: D001419:Bacteria / Q000737:chemistry* / Q000145:classification*; D015373:Bacterial Typing Techniques / Q000379:methods*; D015415:Biomarkers / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013943:Thymol / Q000737:chemistry*
chemical_list: D015415:Biomarkers; D013943:Thymol