ABSTRACT
Clinical diagnostics and microbiology require high-throughput identification of microorganisms. Sample multiplexing prior to detection is an attractive means to reduce analysis costs and time-to-result. Recent studies have demonstrated the discriminative power of tandem mass spectrometry-based proteotyping. This technology can rapidly identify the most likely taxonomical position of any microorganism, even uncharacterized organisms. Here, we present a simplified label-free multiplexing method to proteotype isolates by tandem mass spectrometry that can identify six microorganisms in a single 20 min analytical run. The strategy involves the production of peptide fractions with distinct hydrophobicity profiles using spin column fractionation. Assemblages of different fractions can then be analyzed using mass spectrometry. Results are subsequently interpreted based on the hydrophobic characteristics of the peptides detected, which make it possible to link each taxon identified to the initial sample. The methodology was tested on 32 distinct sets of six organisms including several worst-scenario assemblages-with differences in sample quantities or the presence of the same organisms in multiple fractions-and proved to be robust. These results pave the way for the deployment of tandem mass spectrometry-based proteotyping in microbiology laboratories.
Subject(s)
Chemical Fractionation , Tandem Mass Spectrometry , Chromatography, LiquidABSTRACT
Rapid identification of microorganisms is essential for medical diagnostics, sanitary controls, and food safety. High-throughput analytical platforms currently rely on whole-cell MALDI-TOF mass spectrometry to process hundreds of samples per day. Although this technology has become a reference method, it is unable to process most environmental isolates and opportunistic pathogens due to an incomplete experimental spectrum database. In most cases, its discriminating power is limited to the species taxonomical rank. By recording much more sequence information at the peptide level, proteotyping by tandem mass spectrometry is able to identify the taxonomic position of any microorganism in the tree of life and can be highly discriminating at the subspecies level. We propose here a methodology for ultra-fast identification of microorganisms by tandem mass spectrometry based on direct sample infusion and a highly sensitive procedure for data processing and taxonomic identification. Results obtained on reference strains and hitherto uncharacterized bacterial isolates show identification to species level in 36 s of tandem mass spectrometry signal, 102 s when including the injection procedure. Flash proteotyping is highly discriminating, as it can provide information down to strain level. The methodology enables high throughput identification of isolates, opening up new prospects, particularly in culturomics, and diagnostics.
ABSTRACT
To meet clinical diagnostic needs and for general microbiological screening, it is essential to be able to accurately and rapidly identify any microorganisms from complex microbiota. To gain insight into the individual components of microbiota, culturomics has been proposed as a means to systematically test hundreds of possible cultivation conditions and generate numerous microbial isolates with very distinct characteristics. High-throughput identification methods must now be developed to quickly screen these isolates. Currently, most multiplexing methods involve labeling, which comes at a cost. In this paper, we present an innovative label-free multiplexing method for the identification of microorganisms using tandem mass spectrometry. The method is based on offline reversed-phase fractionation of individual peptidomes. Multiplexing is achieved by mixing fractions of staged hydrophobicity; thus, each sample is mapped to specific elution times. In this proof-of-concept study, multiplexed samples were analyzed by tandem mass spectrometry in a single run and microorganisms present in the mixture were resolved by phylopeptidomics proteotyping. Using this methodology, up to 21 microorganisms could be identified in a single 60 min run performed with a Q-Exactive HF high-resolution mass spectrometer, resulting in a rate of one microorganism identified per 3 min of mass spectrometry, without any need for the use of labeling reagents. This approach opens new perspectives for the application of high-throughput proteotyping of bacteria using tandem mass spectrometry in large culturomics projects.
