PECULIARITIES OF MASS SPECTROMETRIC ANALYSIS OF BRUCELLA S- AND L-FORMS
Infekciâ i Immunitet
Zoonotic and parasitic infections and their analysis were carried out using Microflex LT (Brucker) and its programs v. 3.3. 64 and v. 3.3.65. At the first stage we created 2 databases of mass spectra of reference strains: 1) saprophytes of the genus Bacillus and 2) strains of B. anthracis. When carrying out "blind" tests we revealed that fragments of peptide complexes over the range 2-12 000 Da in all representatives of both groups practically did not differ because of high degree of affinity.
... egree of affinity. Thus, strains of closely related saprophytes were identified as B. anthracis and strains with high indicator SV on the contrary as saprophytes. When all spectra of cultures of both groups were pooled, identification became more correct, allowing to obtain the highest values of SV for strains of one species. The most optimum results of specific identification were obtained when identification of cultures was carried out using the program MALDI Biotyper RIC and construction of MsP-dendrogram was carried out using the program FlexAnalysis. In obtained dendrograms samples under study were clearly clustered with one of bacilli species represented in the base. Thus, perfection of the scheme of reliable identification of B. anthracis, including accurate differentiation from closely related bacilli on the basis of MALDI TOF MS continues to remain urgent. The use of the sensitive and rapid method of MALDI-TOF MS for identification of cultures of the causative agent of anthrax requires not only strict specificity, but also universality for all strains irrespective of their intraspecific variability of phenotypic properties. The aim of the work was to reveal species-specific signals, common to all B. anthracis strains with various complexes of phenotypic properties. We used 37 strains which included strains atypical in capsule formation, toxin production, nutritional requirements, activities of protease, lecithinase and hemolysins, ability to hydrolyze carbohydrates, as well as strains with different MLVA-and SNP-genotypes. Samples were prepared by lysis of 18-hour cultures in 80% TFA followed by ultra-micro-centrifuge filtration. The studies were carried out using Microflex LT instrument (Bruker). Collection of mass spectra and analysis of data were carried out using the programs v. 3.3.64 and v. 3.3.65. Analysis of spectra for frequency of signals was carried out using the program Microbe MS. The occurrence of various combinations of phenotypic properties made it possible to discriminate 11 phenotypes. Individual spectra of each of these phenotypes (20 spectra of each strain) were analyzed and peak frequency was determined. For the further analysis we used peaks occurring at the frequency ≥ 95%, with their numbers in various groups varying from 2 to 32. When comparing the peak frequency of all the 11 phenotypic groups we revealed the absence of common peaks with the frequency ≥ 95%. The distribution of signals which were identified in all the groups most often were as follows: 2601 . Earlier these peaks were not considered as specific markers of B. anthracis. The approach to choose markers we used when analyzing strains with a great number of phenotypic groups, including rare strains, may account for this. Markers of the system of ribosomal proteins, SASP and histone proteins, earlier described as species-specific markers, also occur in the spectra of strains from various groups, but at much lower frequency, and that may be connected with the production of various proteins or with various levels of their expression. Thus, selection of species-specific peaks for identification of B. anthracis strains should be carried out taking into account the variability of their biological properties. 4.15