Beyond the Matrix-Assisted Laser Desorption Ionization (MALDI) Biotyping Workflow: in Search of Microorganism-Specific Tryptic Peptides Enabling Discrimination of Subspecies

Abstract

A well-accepted method for identification of microorganisms uses matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled to analysis software which identifies and classifies the organism according to its ribosomal protein spectral profile. The method, called MALDI biotyping, is widely used in clinical diagnostics and has partly replaced conventional microbiological techniques such as biochemical identification due to its shorter time to result (minutes for MALDI biotyping versus hours or days for classical phenotypic or genotypic identification). Besides its utility for identifying bacteria, MS-based identification has been shown to be applicable also to yeasts and molds. A limitation to this method, however, is that accurate identification is most reliably achieved on the species level on the basis of reference mass spectra, making further phylogenetic classification unreliable. Here, it is shown that combining tryptic digestion of the acid/organic solvent extracted (classical biotyping preparation) and resolubilized proteins, nano-liquid chromatography (nano-LC), and subsequent identification of the peptides by MALDI-tandem TOF (MALDI-TOF/TOF) mass spectrometry increases the discrimination power to the level of subspecies. As a proof of concept, using this targeted proteomics workflow, we have identified subspeciesspecific biomarker peptides for three Salmonella subspecies, resulting in an extension of the mass range and type of proteins investigated compared to classical MALDI biotyping. This method therefore offers rapid and cost-effective identification and classification of microorganisms at a deeper taxonomic level.

Use of MALDI-TOF mass spectrometry fingerprinting to characterize Enterococcus spp. and Escherichia coli isolates

“…As previously stated, several reasons could explain the difficulty in distinguishing between the different enterococcal species present in this study. The workflow of the classical MALDI biotyping to discriminate at the species level revealed that about 10 times more proteins were detected compared to a classical MALDI-TOF MS protocol [40], like the one used in this work. Furthermore, although some improvements have been made in bacterial identification, a broadly applicable and useful sample preparation method prior to MALDI analysis is needed [31].…”

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Burkholderia pseudomallei from Asia and Australia and differentiation between Burkholderia species

“…Extracts were analyzed in triplicate in both assays. Spectra with maximal absolute peak intensities ranging from 10 3 to 10 4 arbitrary units were considered for evaluation [42, 43]. …”

MALDI-TOF mass spectroscopy of yeasts and filamentous fungi for research and diagnostics in the agricultural value chain

“…As for the highly sensitive species identification, discussed above, sample processing (e.g., tryptic digestion, acidic/organic extraction, nano-liquid chromatography) can greatly increase the sensitivity and specificity of a MALDI-TOF MS method and thus enable subspecies identification [66,75]. A further possibility is the functional modification of the MALDI target plates themselves, which was for example performed with antibodies for direct immunoaffinity MALDI-TOF MS of haptoglobin or by dioxide coating in order to enrich phosphopeptides [76,77].…”