Shotgun mass mapping of Lactobacillus species and subspecies from caries related isolates by MALDI‐MS

Abstract

A taxonomical study of 90 isolates of lactobacilli isolated from soft and hard carious dentine of 70 deciduous molars is presented. The Lactobacillus strains were determined by shotgun mass mapping (SMM). This method based on MALDI-MS analysis of Lactobacillus isolates treated with trypsin followed by database comparison against a library of mass spectra derived from 20 reference strains. The SMM method allowed to discriminate different Lactobacillus subspecies. The method was used to analyse Lactobacillus isolates of unknown identity derived from carious dentine. Application of the SMM method to isolates from hard carious dentine revealed a nearly similar distribution of L. paracasei ss paracasei (29%), L. paracasei ss tolerans (32%) and L. casei ss rhamnosus (23%) as dominant subspecies. On the other hand, samples derived from soft carious dentine showed a clear bias only to L. paracasei ss paracasei (60%), whereas L. paracasei ss tolerans (14%) and L. casei ss rhamnosus (12%) were clear minorities. Compared to existent methods, SMM has unique potential for the analysis of Lactobacillus strains on subspecies level.

The essence on mass spectrometry based microbial diagnostics

“…Furthermore, mass spectrometry based bacterial identification has been largely successfully validated for comparison with conventional automated bacterial identification techniques and could produce very convincing intralaboratory and interlaboratory reproducibility [25 ,26]. The ability to differentiate closely related bacteria by mass patterns might also be improved by increasing the number of peptides using in addition to protein extraction proteases such as trypsin to digest bacterial proteomes [27]. The resulting protein fragment patterns can be compared against reference mass spectra produced by protease digestion of bacterial reference protein extracts.…”

Comparison of methods for simultaneous identification of bacterial species and determination of metabolic activity by protein‐based stable isotope probing (Protein‐SIP) experiments

“…39 or to characterise clinical isolates of lactobacilli. 16 Using SMM, P. putida specific peptides were obtained. The incorporation of heavy isotopes into peptides was again detected and calculated by comparison of mass spectra from unlabelled, 15 N-labelled and 13 C-labelled samples acquired by MALDI-MS (Fig.…”

“…However, if bacterial references are available, SMM can be used to identify bacteria down to the subspecies level, as demonstrated. 16 The advantage of analysing peptides instead of whole proteins is that fragmentation techniques enable specific sequence information and phylogenetic information to be derived simultaneously. Very promising methods for detecting and identifying active species in microbial communities are based on the use of substrates labelled with stable isotopes, e.g.…”

Species determination of Culicoides biting midges via peptide profiling using matrix-assisted laser desorption ionization mass spectrometry

“…In this procedure, crude extracts from whole cells or biopsies are subjected to proteolytic hydrolysis by trypsin without any pre-fractionation, and the resulting peptide-containing mixtures may be subjected to MALDI-MS analysis without additional clean-up steps [24]. Spectra recorded by this strategy have been used to detect the presence of cancer in cells [43, 44] or for bacteria species identification [45, 46]. Although analyzing peptides instead of whole proteins requires a somewhat more elaborate sample preparation, this approach offers several advantages over conventional analyses.…”