- Compositions and methods for detecting S-nitrosylation and S-sulfinylation
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The present invention relates to methods for detecting protein S-sulfinylation and S-sulfinylation within thiol groups in proteins, metabolites, or materials.
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Page/Page column 5; 15; 16; 27; 28
(2019/09/23)
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- Harnessing Redox Cross-Reactivity to Profile Distinct Cysteine Modifications
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Cysteine S-nitrosation and S-sulfination are naturally occurring post-translational modifications (PTMs) on proteins induced by physiological signals and redox stress. Here we demonstrate that sulfinic acids and nitrosothiols react to form a stable thiosulfonate bond, and leverage this reactivity using sulfinate-linked probes to enrich and annotate hundreds of endogenous S-nitrosated proteins. In physiological buffers, sulfinic acids do not react with iodoacetamide or disulfides, enabling selective alkylation of free thiols and site-specific analysis of S-nitrosation. In parallel, S-nitrosothiol-linked probes enable enrichment and detection of endogenous S-sulfinated proteins, confirming that a single sulfinic acid can react with a nitrosothiol to form a thiosulfonate linkage. Using this approach, we find that hydrogen peroxide addition increases S-sulfination of human DJ-1 (PARK7) at Cys106, whereas Cys46 and Cys53 are fully oxidized to sulfonic acids. Comparative gel-based analysis of different mouse tissues reveals distinct profiles for both S-nitrosation and S-sulfination. Quantitative proteomic analysis demonstrates that both S-nitrosation and S-sulfination are widespread, yet exhibit enhanced occupancy on select proteins, including thioredoxin, peroxiredoxins, and other validated redox active proteins. Overall, we present a direct, bidirectional method to profile select redox cysteine modifications based on the unique nucleophilicity of sulfinic acids.
- Majmudar, Jaimeen D.,Konopko, Aaron M.,Labby, Kristin J.,Tom, Christopher T. M. B.,Crellin, John E.,Prakash, Ashesh,Martin, Brent R.
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supporting information
p. 1852 - 1859
(2016/03/01)
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- Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: Utility in protein thiol blocking and in protein-S-nitrosothiol detection
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A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.
- Reeves,Joshi,Campanello,Hilmer,Chetia,Vance,Reinschmidt,Miller,Giedroc,Dratz,Singel,Grieco
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p. 7942 - 7956
(2015/02/18)
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- Selective trapping of SNO-BSA and GSNO by benzenesulfinic acid sodium salt: Mechanistic study of thiosulfonate formation and feasibility as a protein S-nitrosothiol detection strategy
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The conversion of S-nitrosothiols to thiosulfonates by reaction with the sodium salt of benzenesulfinic acid (PhSO2Na) has been examined in detail with the exemplary substrates S-nitrosoglutathione (GSNO) and S-nitrosylated bovine serum albumin (SNO-BSA). The reaction stoichiometry (2:1, PhSO2Na:RSNO) and the rate law (first order in both PhSO 2Na and RSNO) have been determined under mild acidic conditions (pH 4.0). The products have been identified as the corresponding thiosulfonates (GSSO2Ph and BSA-SSO2Ph) along with PhSO2NHOH obtained in a 1:1 ratio. GSH, GSSG, and BSA were unreactive to PhSO 2Na.
- Reeves, Benjamin D.,Hilmer, Jonathan K.,Mellmann, Lisa,Hartzheim, Myra,Poffenberger, Kevin,Johnson, Keith,Joshi, Neelambari,Singel, David J.,Grieco, Paul A.
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supporting information
p. 5707 - 5710
(2013/09/24)
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