A Simplified Method for Comprehensive Capture of the Staphylococcus aureus Proteome

Area of Science:

• Proteomics
• Microbiology
• Pathogen Biology

Background:

• Staphylococcus aureus is a significant human pathogen responsible for diverse infections.
• A complete understanding of the S. aureus dynamic and adaptive proteome remains incomplete.
• Existing proteomic studies have limitations in coverage and depth.

Purpose of the Study:

• To expand the coverage of the S. aureus proteome using advanced mass spectrometry techniques.
• To explore the 'dark proteome' of S. aureus, including low-abundance and uncharacterized proteins.
• To establish a comprehensive proteomic library for S. aureus for future research.

Main Methods:

• Streamlined liquid and gas-phase fractionation coupled with Parallel Accumulation-Serial Fragmentation (PASEF) analysis on a TIMS-TOF instrument.
• Data-independent acquisition (DIA) mass spectrometry for broad proteome coverage.
• Development of a simplified, reproducible workflow with minimal sample input.

Main Results:

• Identification of 2,231 S. aureus proteins, representing 85.6% proteome coverage, the most comprehensive to date.
• High reproducibility and minimal material input demonstrated in the proteomic profiling.
• Differential expression profiling revealed significant changes in metabolic processes, ATP production, RNA processing, and stress-response proteins during stationary growth.
• A substantial portion of the proteome (80.5%) was identified using a single-shot, DIA-based analysis.

Conclusions:

• The study presents a significantly expanded and comprehensive proteome map of Staphylococcus aureus.
• The developed methodology offers a simplified and highly reproducible approach for deep proteome exploration.
• This work generates a valuable resource for advancing the study of S. aureus pathogenesis and host-pathogen interactions.