Understanding the biological basis of MALDI-TOF mass spectrometry-based microbial identification

Microbial identification is critical to understanding the aetiological agent of disease, which provides the starting point for development of treatment strategies. Currently, nucleic acid-based identification methods remain dominant in identifying microbes to the species level. In complement, mass spectrometry-based approaches such as those based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have emerged to be robust, fast and accurate methods for identifying fungi, bacteria, and archaea to the species level. Specifically, algorithmic-based comparison of mass spectra of known and unknown microbes provides the basis for a positive identification in what is known as mass spectrum fingerprinting. However, the approach does not consider the identities of the profiled mass peaks in mass spectrum of bacterial species, which meant that the biological basis of the approach remains enigmatic. Using open access data of mass spectra of bacterial species from SpectraBank database, this study sought to examine the biological basis of MALDI-TOF MS microbial identification at different levels. Firstly, conservation of mass peaks between mass spectra of different strains of a species or between different species of a genus would point to the existence of common proteins that define the taxonomic classification of the species or genus. In this direction, results revealed that conserved mass peaks could be determined at the species and genus level, which suggested a biological basis for MALDI-TOF MS microbial identification. Specifically, Escherichia coli and Morganella morganii had the largest number of conserved mass peaks, while Staphylococcus aureus and Pseudomonas putida had the least. Presence of conserved mass peaks at the genus level revealed that a subset of proteins could demarcate the taxonomic boundary between the species and genus level. Secondly, given that ribosomal proteins are housekeeping proteins that have a high relative abundance in the cell, efforts were made in annotating ribosomal protein peaks in the mass spectra of different bacterial species. Results revealed that ribosomal protein peaks could be annotated in the mass spectra of many bacterial species, but not all profiled mass peaks belong to ribosomal proteins. Specifically, between 1 and 6 ribosomal protein peaks from the large and small ribosome subunits could be annotated in a given mass spectrum, with the rest of the profiled peaks of unknown provenance. Furthermore, the annotated ribosomal proteins could reproduce phylogenetic cluster groups similar to those found in the maximum likelihood phylogenetic tree of 16S rRNA, and thus, hold phylogenetic significance. Collectively, observation of conserved mass peaks at the species and genus levels as well as annotation of ribosomal protein mass peaks in MALDI-TOF mass spectra of bacterial species pointed to the biological basis underlying the microbial identification method. Future effort in annotating all the mass peaks profiled in the mass spectra of microbes would provide greater insights on the intersection between biology and the mass spectrometric view of a cell’s proteome.