Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella Typhimurium in response to infection-like conditions

Abstract

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.

Fig. 1.

Analysis of distinct proteoforms. The counts of total proteoforms per unique protein are shown. A majority of entries have only one form identified.

Fig. 2.

Switch in modification from glutathionylation to cysteinylation in response to environmental conditions. (A) Representative zero charge state spectra from top-down proteomics data showing switch in S-thiolation forms in the hypothetical protein YifE (STM14_4694). Fragmentation maps show specific cysteine residue on which switch occurs. (B) Estimated stoichiometry from intact protein mass spectra summed across corresponding LC peak.

Fig. 3.

Structures of E. coli homologs of Salmonella proteins found to be S-thiolated. (A) DnaK (1dkg). (B) Enolase dimer (1e9i). Modified Cys residues are shown as spheres and the residue number and chain ID indicated. Relative accessibilities of the modified Cys residues calculated with Naccess are 1.3 and 2.1, respectively.

Fig. 4.

Quantification of glutathionylated and cysteinylated peptides. Heat map representation of peak intensity-based quantification of glutathionylated (Upper) and cysteinylated (Lower) peptides identified from bottom-up proteomics data under LB and LPM growth conditions.

Fig. 5.

Working model of differential thiol utilization in Salmonella during infection. C, cysteine; CSSC, cysteine disulfide (cystine); GSH, glutathione; GSSG, glutathione disulfide.