Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Can Precisely Discriminate the Lineages of Listeria monocytogenes and Species of Listeria

Abstract

The genetic lineages of Listeria monocytogenes and other species of the genus Listeria are correlated with pathogenesis in humans. Although matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a prevailing tool for rapid and reliable microbial identification, the precise discrimination of Listeria species and lineages remains a crucial issue in clinical settings and for food safety. In this study, we constructed an accurate and reliable MS database to discriminate the lineages of L. monocytogenes and the species of Listeria (L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, L. grayi, and L. rocourtiae) based on the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum (S10-GERMS) proteotyping method, which relies on both genetic information (genomics) and observed MS peaks in MALDI-TOF MS (proteomics). The specific set of eight biomarkers (ribosomal proteins L24, L6, L18, L15, S11, S9, L31 type B, and S16) yielded characteristic MS patterns for the lineages of L. monocytogenes and the different species of Listeria, and led to the construction of a MS database that was successful in discriminating between these organisms in MALDI-TOF MS fingerprinting analysis followed by advanced proteotyping software Strain Solution analysis. We also confirmed the constructed database on the proteotyping software Strain Solution by using 23 Listeria strains collected from natural sources.

Fig 1. Theoretical MS database constructed with publically available L. monocytogenes strains and analysis with Strain Solution software.

A. The theoretical m/z of the ribosomal proteins in S10-spc-alpha operon and an additional three biomarker candidates are shown. Circles in ‘Detection’ means the corresponding peaks are detectable in all strains with default analytical condition (threshold offset: 0.015 mV; threshold response: 1.200) in the AXIMA system. The undetected or weak peaks that we could not always find are marked with an “x”. Triangles indicate that MS differences in each strain or the other protein peaks could not be distinguished from one another with a 500 ppm tolerance, though putative peaks were detected. Characteristic MS values were colored. B. The selected three biomarkers registered as the standard database in Strain Solution. C. The results obtained from the Strain Solution analysis.

Fig 2. Theoretical MS database for Listeria species.

^aThe MS [M + H]⁺ plus m/z 17 of the theoretically calculated value is shown based on the observed MS peak. ^bThe MS [M + H]⁺ plus m/z 14 of the theoretically calculated value is shown because it appeared to be methylated.