Bioorthogonal Click Chemistry for Antibody-Free Profiling of Acetylation, Propionylation, and Butyrylation in <i>Pseudomonas aeruginosa</i> and Methicillin-Resistant <i>Staphylococcus aureus</i>

Lysine acylation is a posttranslational modification (PTM) conserved in all domains of life and is essential for regulating diverse biological processes. Traditional methods for investigating acylation rely on anti-acyl-lysine antibodies, which are costly and time-consuming and often exhibit variable affinity. To remedy these pitfalls, we developed an antibody-free method for bacterial acylome enrichment using bioorthogonal click chemistry coupled with tandem mass spectrometry. We applied this approach to the pathogens <i>Pseudomonas aeruginosa</i> and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) to explore the biological significance of acylation in each organism. We characterized the acetylome, propionylome, and butyrylome in <i>P. aeruginosa</i> UCBPP-PA14 and the acetylome and propionylome in MRSA. Comparative analyses revealed unique PTM dynamics showing that acylation regulated a wide range of cellular functions, including metabolism, antibiotic resistance, virulence, and stress response. This work establishes the first antibody-free enrichment method for defining bacterial acylomes and provides new insight into global lysine acylation networks in pathogenic bacteria.