Abstract

Intact protein expression profiling has proven to be a powerful tool for bacterial subspecies differentiation. To facilitate typing, epidemiology, and trace-back of Salmonella contamination in the food supply, a minimum of serovar level differentiation is required. Subsequent identification and validation of marker proteins is integral to rapid screening development and to determining which proteins are subject to environmental pressure. Bacterial sequencing efforts have expanded the number of sequenced genomes available for single-nucleotide polymorphism (SNP) analyses, but annotation is often missing, start site errors are not uncommon, and the likelihood of expression is not known. In this work we show that the combination of intact protein expression profiles and top-down liquid chromatography−mass spectrometry (LC−MS/MS) facilitates the identification of proteins that result from expressed serovar specific nonsynonymous SNPs. Combinations of these marker proteins can be used in assays for rapid differentiation of bacteria. LC−MS generated intact protein expression profiles establish which bacterial protein masses differ across samples and can be determined without prior knowledge of the sample. Subsequent top-down LC−MS/MS is used to identify expressed proteins and their post-translational modifications (PTM), identify serovar specific markers, and validate genomic predicted orthologues as expressed biomarkers.