Characterization of the mature
protein complement in cells is crucial for a better understanding of cellular processes on a systems-wide scale. Toward this end, we used single-dimension ultra-high-pressure liquid chromatography mass spectrometry to investigate the comprehensive "intact"
proteome of the Gram-negative bacterial pathogen Salmonella Typhimurium. Top-down proteomics analysis revealed 563 unique
proteins including 1,665 proteoforms generated by posttranslational modifications (PTMs), representing the largest microbial top-down dataset reported to date. We confirmed many previously recognized aspects of Salmonella biology and bacterial PTMs, and our analysis also revealed several additional
biological insights. Of particular interest was differential utilization of the
protein S-thiolation forms S-glutathionylation and S-cysteinylation in response to
infection-like conditions versus basal conditions. This finding of a S-glutathionylation-to-S-cysteinylation switch in a condition-specific manner was corroborated by bottom-up proteomics data and further by changes in corresponding biosynthetic pathways under
infection-like conditions and during actual
infection of host cells. This differential utilization highlights underlying metabolic mechanisms that modulate changes in cellular signaling, and represents a report of S-cysteinylation in Gram-negative bacteria. Additionally, the functional relevance of these PTMs was supported by
protein structure and gene deletion analyses. The demonstrated utility of our simple
proteome-wide intact
protein level measurement strategy for gaining
biological insight should promote broader adoption and applications of top-down proteomics approaches.