A Proteogenomic Resource Enabling Integrated Analysis of Genotype-Proteotype-Phenotype Relationships.

is an opportunistic foodborne pathogen responsible for listeriosis, a potentially fatal foodborne disease. Many different strains and serotypes exist, but a proteogenomic resource that bridges the gap in our molecular understanding of the relationships between the genotypes and phenotypes via proteotypes is still missing. Here, we devised a next-generation proteogenomics strategy that enables the community to rapidly proteotype strains and relate this information back to the genotype. Based on sequencing and assembly of the two most commonly used model strains, EGD-e and ScottA, we established two comprehensive proteogenomic databases. A genome comparison established core- and strain-specific genes potentially responsible for virulence differences. Next, we established a DIA/SWATH-based proteotyping strategy, including a new and robust sample preparation workflow, that enables the reproducible, sensitive, and relative quantitative measurement of proteotypes. This reusable and publicly available DIA/SWATH library covers 70% of open reading frames of and represents the most extensive spectral library for proteotype analysis to date. We used these two new resources to investigate the proteotype in states mimicking the upper gastrointestinal passage. Exposure of to bile salts at 37 °C, which simulates conditions encountered in the duodenum, showed significant proteotype perturbations including an increase of FlaA, the structural protein of flagella. Given that is known to lose its flagella above 30 °C, this was an unexpected finding. The formation of flagella, which might have implications on infectivity, was validated by parallel reaction monitoring and light and scanning electron microscopy. transcript levels did not change significantly upon exposure to bile salts at 37 °C, suggesting regulation at the post-transcriptional level. Together, these analyses provide a comprehensive proteogenomic resource and toolbox for the community enabling the analysis of genotype-proteotype-phenotype relationships.