A Simplified Method for Comprehensive Capture of the Staphylococcus aureus Proteome

Staphylococcus aureus is major human pathogen causing severe and invasive infections in both community and healthcare settings. Although well studied, a comprehensive exploration of its dynamic and adaptive proteome is still somewhat lacking. Herein we employ streamlined liquid- and gas-phase fractionation with PASEF analysis on a TIMS-TOF instrument to expand coverage and explore the dark proteome of S. aureus. In so doing, we captured the most comprehensive S. aureus proteome to date, totaling 2,231 proteins (85.6% proteome coverage), and did so using a significantly simplified process, requiring less samples and input material. Furthermore, we demonstrated the utility of this library within the context of differential expression profiling by investigating the temporal dynamics of the S. aureus proteome. Notably, a significant portion of the library (94%) and related proteome (80.5%) was characterized by DIA-based proteomic analysis, where we noted alterations in metabolic/biosynthetic processes, ATP production, RNA processing, and an increase in stress response proteins such as chaperones and repair enzymes as cultures progressed into stationary phase. Collectively, our study shines new light on the hidden S. aureus proteome, generating a valuable new resource to the scientific community to facilitate further study of this dangerous human pathogen.