Direct Matrix-Assisted Laser Desorption– Ionisation (MALDI) Mass-Spectrometry Bacteria Profiling for Identifying and Characterizing Pathogens

Abstract

This study examines the features and limitations of direct Matrix-Assisted Laser Desorption–Ionisation (MALDI) mass-spectrometry profiling of bacterial cells for investigating a microbial population. The optimal laboratory protocol, including crude bacteria lyses by a solution of 50% acetonitrile, 2.5% trifluoroacetic acid, and using α-cyano-4-hydroxy cinnamic acid as the MALDI matrix, has been developed. Two different bacteria species were under investigation, and representative mass spectra from 278 strains of Neisseria gonorrhoeae and 22 strains of Helicobacter pylori have been analyzed. It’s known that both bacteria demonstrate a variable degree of polymorphism. For N. gonorrhoeae, the MALDI mass spectra that was collected possessed about 70 peaks, 20 of which were good reproducible ones. In spite of the fact that three peaks were found with differing spectra in some strains, little diversity in the N. gonorrhoeae population was revealed. This fact indicates the prospects in using direct MALDI mass-spectrometry profiling for gonococcus identification. In the case of H. pylori strains, the variety in the collected mass-spectra was shown to be essential. Only five peaks were present in more than 70% of strains, and a single mass value was common for all spectra. While these data call into question the possibility of the reliable species identification of H. pylori using this approach, the intraspecies differentiation of strains was offered. Good association between MALDI profile distributions and the region of strain isolation have been found. Thus, the suggested direct MALDI mass-spectrometry profiling strategy, coupled with special analysis software, seems promising for the species identification of N. gonorrhoeae but is assumed insufficient for H. pylori species determination. At the same time, this would create a very good chance for an epidemiological study of such variable bacteria as H. pylori.

Full Text

Modern microbiology and its applied branches require the development of new rapid and precise methods for identifying clinically significant pathogens and for describing their characteristic features such as virulence, antibiotic sensitivity, and strain group. The relative tolerance of MatrixAssisted Laser Desorption–Ionisation (MALDI) to contamination with salt and other impurities allows one to conduct a direct mass-spectrometry analysis of the microbial cell con116 | Acta naturae | № 1 2009 RE SEARC H ART ICLES The registrability of mass-spectra, unique and reproducible for families, genera, species, and subspecies of microorganisms, makes it possible to use mass-spectrometry for bacteria identification and typing, which was demonstrated for the first time in 1975 [1]. Despite the fact that a “whole cell” spectrum (without any separation of the cell components) reflects only an insignificant part of the cell proteome, it is demonstrative enough to characterize the cell taxonomic features, which was established for a number of bacteria [2, 3, 4, 5, 6]. It should be noted that this method does not involve the identification of separate microbial proteins and allows the application of a unique mass-profile for characterizing any microorganism on the “fingerprint pattern” principle [7]. Specific features and, at the same time, disadvantages of the considered approach when compared to the traditional methods are as follows: (1) a quite high sensitivity (105–106 of cells or 0.5 μg of cell culture), (2) simple sample preparation, (3) high measurement rate, and (4) the possibility of automating and robotizing all investigation stages. This study examines the advantages and disadvantages of using MALDI mass spectrometry to profile bacterial cells using such microbial populations as Helicobacter pylori and Neisseria gonorrhoeae, which are commonly referred to as microorganisms with a high genetic flexibility of genomes, as examples. Moreover, we set the task of estimating the variability of MALDI mass-profiles which were photographed according to the developed protocol within each bacterial population.