AIMS: To propose a universal workflow of sample preparation method for the identification of highly pathogenic bacteria by MALDI-TOF MS. METHODS AND RESULTS: Fifteen bacterial species, including highly virulent Gram-positive (Bacillus anthracis and Clostridium botulinum) and Gram-negative bacteria (Brucella melitensis, Burkholderia mallei, Francisella tularensis, Shigella dysenteriae, Vibrio cholerae, Yersinia pestis and Legionella pneumophila), were employed in the comparative study of four sample preparation methods compatible with MALDI-TOF MS. The yield of bacterial proteins was determined by spectrophotometry, and the quality of the mass spectra, recorded in linear mode in the range of 2000-20,000 Da, was evaluated with respect to the information content (number of signals) and quality (S/N ratio). CONCLUSIONS: Based on the values of protein concentration and spectral quality, the method using combination of ethanol treatment followed by extraction with formic acid and acetonitrile was the most efficient sample preparation method for the identification of highly pathogenic bacteria using MALDI-TOF MS. SIGNIFICANCE AND IMPACT OF THE STUDY: The method using ethanol/formic acid generally shows the highest extraction efficacy and the spectral quality with no detrimental effect caused by storage. Thus, this can be considered as a universal sample preparation method for the identification of highly virulent micro-organisms by MALDI-TOF mass spectrometry.
