Method for assaying protein nitrosylation
AssigneesThe Johns Hopkins University, Memorial Sloan Kettering Cancer Center
InventorsSolomon H. Snyder, Samie R. Jaffrey, Christopher D. Ferris, Hediye Erdjument-Bromage, Paul Tempst
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Abstract
Many of the effects of nitric oxide are mediated by the direct modification of cysteine residues resulting in an adduct called a nitrosothiol. A method to detect proteins which contain nitrosothiols involves several steps. Nitrosylated cysteines are converted to tagged cysteines. Tagged proteins can then be detected, for example, by immunoblotting and/or can be purified by affinity chromatography. The method is applicable to the detection of S-nitrosylated proteins in cell lysates following in vitro S-nitrosylation, as well as to the detection of endogenous S-nitrosothiols in selected protein substrates.
Claims36
A method for screening for potential drugs useful in modulating protein nitrosylation, comprising:
- contacting a test sample comprising at least one protein substrate with a test compound;
- treating the test sample with an alkylthiolating agent to block free thiol groups on the protein substrate;
- reducing nitrosothiol bonds on the protein substrate to form new free thiol groups;
- removing the alkylthiolating agent from the test sample;
- reacting the new free thiol groups on the protein substrate with a detectably tagged, activated mixed disulfide, thereby forming detectably tagged protein substrate, wherein the detectably tagged protein substrate indicates that the protein substrate in the test sample was nitrosylated;
- detecting the detectably tagged protein substrate;
- and
- comparing the amount of detectably tagged protein substrate in the test sample to an amount of detectably tagged protein substrate in a control sample similarly treated but not contacted with the test compound, wherein a test compound which increases or decreases the amount of detectably tagged protein substrate in the test sample relative to the control sample is identified as a modulator of protein nitrosylation.
Dependencies are inferred from the claim text.