Abstract
Treponema pallidum subsp. pallidum (T. pallidum), the fastidious causative agent of syphilis, has become more accessible for research with its recently developed in vitro cultivation method. In this work, the proteomes of seven T. pallidum strains (Nichols-like: DAL-1, Haiti B, and Madras; SS14-like: SS14, Mexico A, Philadelphia 1, and Grady), cultivated in vitro, were analyzed in biological triplicates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS data were processed against their corresponding genomes using various annotation algorithms (DFAST, PGAP, Prodigal, Prokka, RAST, GeneMarkS, and manual GenBank annotation). Additionally, ORFfinder was used to predict all ORFs encoding polypeptides exceeding 50 amino acids. While the RAST algorithm predicted the highest number of genes per genome, GeneMarkS offered the best coverage of annotated genes (up to 88.9%). By combining annotations from seven T. pallidum strains, we identified 911 unique treponemal proteins (74.9% of 1216 predicted sequences). The confidence of protein identifications was high, with 85.5% identified by two or more peptides and 72.4% by three or more peptides. Overall, 51 proteins showed statistically significant quantitative differences in intensity across T. pallidum strains. Furthermore, our proteome analysis revealed detectable quantitative proteomic differences between strains in the Nichols-like and SS14-like groups.