A simplified method for comprehensive capture of the Staphylococcus aureus proteome: S. aureus proteome data table

Staphylococcus aureus is a major human pathogen causing myriad 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 employed streamlined liquid- and gas-phase fractionation with PASEF analysis on a TIMS-TOF instrument to expand coverage and explore the S. aureus dark proteome. In so doing, we captured the most comprehensive S. aureus proteome to date, totaling 2,231 proteins (85.6% coverage), using a significantly simplified process that demonstrated high reproducibility with minimal input material. We then showcase application of this library for differential expression profiling by investigating temporal dynamics of the S. aureus proteome. This revealed alterations in metabolic processes, ATP production, RNA processing, and stress-response proteins as cultures progressed to stationary growth. Notably, a significant portion of the library (94%) and proteome (80..., , # A simplified method for comprehensive capture of the Staphylococcus aureus proteome: S. aureus proteome data table

Dataset DOI: [10.5061/dryad.qbzkh18vz](10.5061/dryad.qbzkh18vz)

**Data set overview**: This dataset contains the data obtained from characterizing the *Staphylococcus aureus* LAC USA300 proteome using a simplified yet high-throughput methodology for MS/MS capture. This method uses TIMS fractionation as well as a simplified liquid-phase fractionation on a TIMS-TOF instrument designed to enhance proteome capture while minimizing sample processing and hands-on experimental time. We performed statistical analysis and quality assessment of this DDA-PASEF library of proteins and peptides. Following generation of this comprehensive bacterial proteome, we then utilized this resource to perform differential expression profiling of various growth phases of *S. aureus*, implementing a DIA-single shot approach. We characterized the secreted and cellular fractions of mid exponential...,