Evaluation of the Limit of Detection of Bacteria by Tandem Mass Spectrometry Proteotyping and Phylopeptidomics.

Shotgun proteomics has proven to be an attractive alternative for identifying a pathogen and characterizing the antimicrobial resistance genes it produces. Because of its performance, proteotyping of microorganisms by tandem mass spectrometry is expected to become an essential tool in modern healthcare. Proteotyping microorganisms that have been isolated from the environment by culturomics is also a cornerstone for the development of new biotechnological applications. Phylopeptidomics is a new strategy that estimates the phylogenetic distances between the organisms present in the sample and calculates the ratio of their shared peptides, thus improving the quantification of their contributions to the biomass. Here, we established the limit of detection of tandem mass spectrometry proteotyping based on MS/MS data recorded for several bacteria. The limit of detection for Salmonella bongori with our experimental set-up is 4 × 104 colony-forming units from a sample volume of 1 mL. This limit of detection is directly related to the amount of protein per cell and therefore depends on the shape and size of the microorganism. We have demonstrated that identification of bacteria by phylopeptidomics is independent of their growth stage and that the limit of detection of the method is not degraded in presence of additional bacteria in the same proportion.

Mix24X, a Lab-Assembled Reference to Evaluate Interpretation Procedures for Tandem Mass Spectrometry Proteotyping of Complex Samples.

Correct identification of the microorganisms present in a complex sample is a crucial issue. Proteotyping based on tandem mass spectrometry can help establish an inventory of organisms present in a sample. Evaluation of bioinformatics strategies and tools for mining the recorded datasets is essential to establish confidence in the results obtained and to improve these pipelines in terms of sensitivity and accuracy. Here, we propose several tandem mass spectrometry datasets recorded on an artificial reference consortium comprising 24 bacterial species. This assemblage of environmental and pathogenic bacteria covers 20 different genera and 5 bacterial phyla. The dataset comprises difficult cases, such as the Shigella flexneri species, which is closely related to Escherichia coli, and several highly sequenced clades. Different acquisition strategies simulate real-life scenarios: from rapid survey sampling to exhaustive analysis. We provide access to individual proteomes of each bacterium separately to provide a rational basis for evaluating the assignment strategy of MS/MS spectra when recorded from complex mixtures. This resource should provide an interesting common reference for developers who wish to compare their proteotyping tools and for those interested in evaluating protein assignment when dealing with complex samples, such as microbiomes.

Assessing the ratio of Bacillus spores and vegetative cells by shotgun proteomics.

Mass spectrometry for rapid identification of microorganisms is expanding over the last years because this approach is quick. This methodology provides a decisive interest to fight against bioterrorism as it is applicable whatever the pathogen to be considered and often allows subtyping which may be crucial for confirming a massive and widespread attack with biological agents. Here, we present a methodology based on next-generation proteomics and tandem mass spectrometry for discovering numerous protein biomarkers allowing the discrimination of spores and vegetative cells of Bacillus atrophaeus, a biowarfare simulant. We propose a global quantitative evaluation of the two groups of discriminant biomarkers based on their aggregated normalized spectral abundance factors.