title: The SNO/SOH TMT strategy for combinatorial analysis of reversible cysteine oxidations.
abstract: Redox homeostasis is essential for normal function of cells and redox imbalance has been recognised as a pathogenic factor of numerous human diseases. Oxidative modifications of cysteine thiols modulate function of many proteins, mediate signalling, and fine-tune transcriptional and metabolic processes. In this study we present the SNO/SOH TMT strategy, which enables simultaneous analysis of two different types of cysteine modification: S-nitrosylation (SNO) and S-sulfenylation (SOH). The method facilitates quantitation of modification changes corrected by changes in protein abundance levels and estimation of relative modification site occupancy in a single nLC-MSMS run. The approach was evaluated in vivo using an Escherichia coli based model of mild oxidative stress. Bacteria were grown anaerobically on fumarate or nitrate. Short-term treatment with sub-millimolar levels of hydrogen peroxide was used to induce SOH. We have identified and quantified 114 SNO and SOH modified peptides. In many instances SNO and SOH occupy the same site, suggesting an association between them. High site occupancy does not equate to a site of modification which responds to redox imbalance. The SNO/SOH TMT strategy is a viable alternative to existing methods for cysteine oxidation analysis and provides new features that will facilitate our understanding of the interplay between SNO and SOH.
  
  
  CONCLUSIONS
  SNO/SOH TMT strategy outperforms other available strategies for cysteine oxidation analysis. It provides quantitative profiling of S-nitrosylation and S-sulfenylation changes simultaneously in two experimental conditions. It allows correction of modification levels by protein abundance changes and determination of relative modification site occupancy - all in a single nLC-MSMS experiment based on commercially available reagents. The method has proven precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale.
authors.affiliation: Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Wojdyla
authors.affiliation: Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark.
authors.forename: James
authors.initials: J
authors.lastname: Williamson
authors.affiliation: Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff
authors.affiliation: Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark. Electronic address: adelinar@bmb.sdu.dk.
authors.forename: Adelina
authors.initials: A
authors.lastname: Rogowska-Wrzesinska
mesh_terms: D003545:Cysteine / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D010455:Peptides / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; D003545:Cysteine
keywords: Escherichia coli; Oxidative stress; Quantitative proteomics; S-nitrosylation; S-sulfenylation; Site occupancy