title: 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.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA. Charles.ansong@pnnl.gov
authors.forename: Charles
authors.initials: C
authors.lastname: Ansong
authors.forename: Si
authors.initials: S
authors.lastname: Wu
authors.forename: Da
authors.initials: D
authors.lastname: Meng
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Liu
authors.forename: Heather M
authors.initials: HM
authors.lastname: Brewer
authors.forename: Brooke L
authors.initials: BL
authors.lastname: Deatherage Kaiser
authors.forename: Ernesto S
authors.initials: ES
authors.lastname: Nakayasu
authors.forename: John R
authors.initials: JR
authors.lastname: Cort
authors.forename: Pavel
authors.initials: P
authors.lastname: Pevzner
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Fred
authors.initials: F
authors.lastname: Heffron
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Adkins
authors.forename: Ljiljana
authors.initials: L
authors.lastname: Pasa-Tolic
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D002853:Chromatography, Liquid; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D013058:Mass Spectrometry; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012480:Salmonella Infections / Q000382:microbiology*; D012486:Salmonella typhimurium / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D013455:Sulfur / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013455:Sulfur; D003545:Cysteine