# Genus, Species, and Subspecies Classification of _Salmonella_ Isolates by Proteomics
## 3 Results and Discussion  
The _Salmonella_ genus is comprised of two species, six subspecies, and more than 2600 serovars. The large number of highly homologous serovars makes subtyping a challenge. For example, genomic analysis of the reference _Salmonella_ strains listed in Figure 1B shows an average nucleotide identity (ANI; based on BLAST) [34] of >99% for strains within the same serovar, 99% for serovars within the same subspecies, 93% for subspecies within the same species, and 89% for species within the genus. A larger variation was observed when compared to a different genus; 80% ANI between _Salmonella_ and _E. coli_ and _Stigella_ and 66% when compared to other common foodborne bacteria listed in Table S1. Clearly, a sensitive method is required to distinguish between highly related _Salmonella_ isolates, especially for those below the species level. Here, we evaluate the feasibility and effectiveness of bottom-up proteomics for _Salmonella_ subtyping. Briefly, a tested isolate is lysed, proteolytically digested, analyzed by LC-MS/MS, and classified based on the comparison of experimentally identified peptide sequences to reference proteomes translated from genomes with known taxonomic ranks (Figure 1). The results for \(S\). Typhimurium str. SARA2 are shown in Figure 3. Of the 6767 peptides that were identified by LC-MS/MS, 6571 peptides matched to the correct reference proteome \(S\). Typhimurium SARA2 (D\({}_{\text{STI}}\)). The small difference between the two numbers, 196 peptides, is likely due to incomplete or incorrect genome annotation or, to a lesser extent, false positive peptide identifications. A slightly lower number of identified peptides match to the reference proteomes from different \(S\). Typhimurium strains (D\({}_{\text{STI}}\), str. SARA13, 6430 matches), due in part to the presence of a plasmid in the SARA2 strain. Fewer of the peptides identified in \(S\). Typhimurium str. SARA2 were present in reference proteomes from other \(S\). _enterica_ subsp. _enterica_ servovars (5900-6400 matches for D\({}_{\text{SS}}\), D\({}_{\text{SH}}\), D\({}_{\text{SP}}\), D\({}_{\text{SM}}\), and D\({}_{\text{SE}}\)). The presence of \(S\). Typhimurium peptides decreased markedly when the taxonomic difference of the reference proteome was more pronounced: approximately  
Figure 2: Schematic representation of bacterial classification by a proteomic approach. 5000 and 4800 peptides were common to different subspecies (D\({}_{\text{SA}}\)) and different species (D\({}_{\text{SB}}\)), respectively. Only approximately 3200 \(S\). Typhimurium peptide matches were found in the reference proteomes used for _E. coli._ and _Shigella_. The number dropped to <2400 when a strain from other common foodborne bacteria was compared. As shown in Figure 3, most \(S\). Typhimurium peptides are found in the reference proteomes of serovars from the same subspecies and the number of matches decreased as the taxonomic distance increased. Figure 4 displays a heatmap of the degree of similarity between each of the 19 isolates and the 24 reference proteomes, with red representing the highest degree of similarity and blue the lowest (detailed data are provided in Table S2). Each row in Figure 4 represents a tested isolate with each column representing its degree of similarity to a reference strain. Taking the _S. typhimurium_ str. SARA2 data in Figure 3 as an example in the top row: a similarity degree of 97% is shown to the database D\({}_{\text{STI}}\). Rows 2 to 15 are fourteen other _Salmonella_ isolates of various taxa. A similar trend was observed with the closest match to the correct serovar: SARA18: D\({}_{\text{STI}}\), SARA19: D\({}_{\text{STI}}\), SARA23: D\({}_{\text{SSI}}\), SARA24: D\({}_{\text{SSI}}\), SARA31: D\({}_{\text{SHI}}\), SARA36: D\({}_{\text{SHZ}}\), SARA39: D\({}_{\text{SHZ}}\), SARA50: D\({}_{\text{SPZ}}\), SARA63: D\({}_{\text{SMZ}}\), SE8a: D\({}_{\text{SEI}}\), SE22: D\({}_{\text{SE2}}\), SARC5: D\({}_{\text{SA1}}\), SARC6: D\({}_{\text{SA2}}\), and SARC12: D\({}_{\text{SB2}}\).