Proteogenomic Biomarkers for Identification of <i>Francisella</i> Species and Subspecies by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry

Abstract

<i>Francisella tularensis</i> is the causative agent of tularemia. Because some <i>Francisella</i> strains are very virulent, this species is considered by the Centers for Disease Control and Prevention to be a potential category A bioweapon. A mass spectrometry method to quickly and robustly distinguish between virulent and nonvirulent <i>Francisella</i> strains is desirable. A combination of shotgun proteomics and whole-cell matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry on the <i>Francisella tularensis</i> subsp. <i>holarctica</i> LVS defined three protein biomarkers that allow such discrimination: the histone-like protein HU form B, the 10 kDa chaperonin Cpn10, and the 50S ribosomal protein L24. We established that their combined detection by whole-cell MALDI-TOF spectrum could enable (i) the identification of <i>Francisella</i> species, and (ii) the prediction of their virulence level, i.e., gain of a taxonomical level with the identification of <i>Francisella tularensis</i> subspecies. The detection of these biomarkers by MALDI-TOF mass spectrometry is straightforward because of their abundance and the absence of other abundant protein species closely related in terms of <i>m</i>/<i>z</i>. The predicted molecular weights for the three biomarkers and their presence as intense peaks were confirmed with MALDI-TOF/MS spectra acquired on <i>Francisella philomiragia</i> ATCC 25015 and on <i>Francisella tularensis</i> subsp. <i>tularensis</i> CCUG 2112, the most virulent <i>Francisella</i> subspecies