Bottom-up proteomics methods for strain-level typing and identification of bacteria

Abstract

Timely identification of bacteria is of vital importance in many areas of public health, biotechnology, forensics, and epidemiology. Mass spectrometry-based methods provide an attractive alternative to well-established microbiological procedures and are characterized by the relatively high speed of acquiring taxonomically relevant information. Bottom-up, shotgun-proteomics techniques allow for high-throughput sequencing of peptides from protease-digested cellular proteins using mass analysis of fragments from collision-induced dissociation of peptide ions. This peptide-centric technique uses database searching of product ion mass spectra against a database of a comprehensive list of protein sequences translated from protein-encoding open-reading frames found in bacterial genomes. The results of such searches allow assigning amino acid sequences to peptide fragmentation spectra and matching such peptides to reference microbial proteomes. Phylogenomic profiles of sequenced peptides may be then analyzed using numerical taxonomy tools to reveal strain identities up to subspecies level. The results thereof reveal not only the taxonomic position of an investigated strain but also allow for sequence-based subtyping of microbial strains based on expression of proteins associated with the virulence, antibiotic resistance, or with traditional serotyping methods.