Mass Spectrometry-Based Analysis of Surface Proteins in Clinical Strains: Identification of Promising k-mer Targets for Diagnostics.

Surface proteins of Gram-positive bacteria are critical for adherence to host tissues, evasion of the immune system, and interaction with the environment. They can be utilized as biomarkers in diagnostics, for vaccine development, and as therapeutic targets due to their accessibility and role in pathogenicity. If utilized as diagnostic targets, surface biomarkers should be highly conserved across different strains of the pathogen, unique to the species to avoid cross-reactivity, abundantly expressed on the bacterial surface, and accessible to antibodies or detection reagents. Mass spectrometry-based proteomics methods have advanced the studies of surface proteins, often in combination with selective enrichment strategies such as tryptic "shaving". In this study, 11 clinical strains of underwent tryptic shaving to identify common surface proteins. Further bioinformatics analysis confirmed that these proteins are encoded in the core genome of strains and contain species-specific peptides. In silico analysis identified 26 k-mer peptides in 15 surface proteins with structural accessibility to detection agents, making them the ideal targets for molecular diagnostics or as linear epitope targets for vaccine development or therapeutics. Among the identified candidates were known virulence-associated proteins such as PbpA, Sbi, and Asp23─previously studied in the context of vaccines─as well as uncharacterized proteins encoded by the gene loci SAUSA300_1904 and SAUSA300_1685, whose unique and surface-exposed features suggest unexplored diagnostic potential.