Label-Free Proteomic Approach to Characterize Protease-Dependent and -Independent Effects of sarAInactivation on the Staphylococcus aureusExoproteome

The staphylococcal accessory regulator A (sarA) impacts the extracellular accumulation of Staphylococcus aureusvirulence factors at the level of intracellular production and extracellular protease-mediated degradation. We previously used a proteomics approach that measures protein abundance of all proteoforms to demonstrate that mutation of sarAresults in increased levels of extracellular proteases and assesses the impact of this on the accumulation of S. aureusexoproteins. Our previous approach was limited as it did not take into account that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, our goal was to use an expanded proteomics approach utilizing a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels to alleviate these false negatives and thereby provide for characterization of protease-dependent and -independent effects of sarAmutation on the S. aureusexoproteome. Proteins present in conditioned medium from overnight, stationary phase cultures of the USA300strain LAC, an isogenic sarAmutant, and a sarAmutant unable to produce any of the known extracellular proteases (sarA/protease) were resolved using one-dimensional gel electrophoresis. Quantitative proteomic comparisons of sarAversus sarA/proteasemutants identified proteins that were cleaved in a protease-dependent manner owing to mutation of sarA, and comparisons of sarA/proteasemutant versus the LAC parent strain identified proteins in which abundance was altered in a sarAmutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This expanded approach provided for a more comprehensive analysis of the impact of mutating sarAon the S. aureusexoproteome.