Compositions and methods for detecting s-nitrosylation and s-sulfinylation

Abstract

The present invention relates to methods for detecting protein S-sulfinylation and S-sulfinylation within thiol groups in proteins, metabolites, or materials.

1 - 28 . (canceled) 
     
     
         29 . A method for detecting protein S-sulfinylation within cysteine residues of a protein, comprising:
 providing a biological sample comprising one or more proteins having cysteine residues and a composition comprising a labeled sulfinic acid moiety,   exposing the composition to the biological sample such that an interaction between the labeled sulfinic acid moiety and a S-nitrosothiol side chain of a cysteine residue results in generation of a labeled sulfonothioate moiety, and   characterizing the cysteine residues of the one or more proteins having labeled sulfonothioate moieties as having undergone S-sulfinylation.   
     
     
         30 . The method of  claim 29 , further comprising identifying the protein having been characterized as having undergone S-sulfinylation and/or identifying the exact amino acid sites on the protein having been characterized as having undergone S-sulfinylation. 
     
     
         31 . The method of  claim 29 , wherein the labeled sulfinic acid moiety comprises an imaging agent and/or an enrichment tag. 
     
     
         32 . The method of  claim 31 , wherein said imaging agent is a fluorescent dye. 
     
     
         33 . The method of  claim 32 , wherein in-gel fluorescence is used to characterize the cysteine residues of a protein having a labeled sulfonothioate moiety as having undergone S-sulfinylation. 
     
     
         34 . The method of  claim 31 , wherein said enrichment agent is biotin or desthiobiotin. 
     
     
         35 . The method of  claim 31 , wherein flash chromatography followed by NMR and/or mass spectrometry is used to structurally identify the product of the reaction between a labeled sulfinic acid moiety and a S-nitrosothiol side chain of a cysteine residue. 
     
     
         36 . The method of  claim 31 , wherein high performance liquid chromatography is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-sulfinylation. 
     
     
         37 . The method of  claim 31 , wherein mass spectrometry is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-sulfinylation. 
     
     
         38 . The method of  claim 29 , wherein the one or more proteins having cysteine residues is selected from one or more of the proteins recited in  FIG. 16 . 
     
     
         39 . A method of identifying pharmaceutical agents capable of inhibiting S-sulfinylation, comprising:
 providing a pharmaceutical agent and a biological sample comprising one or more proteins having cysteine residues known to undergo S-sulfinylation,   exposing the pharmaceutical agent to the biological sample,   characterizing the cysteine residues of the one or more proteins as having or not having undergone S-sulfinylation with the method of claim  1 , and   identifying the pharmaceutical agent as a S-sulfinylation inhibitor if the characterizing indicates an absence of S-sulfinylation.   
     
     
         40 . The method of  claim 39 , wherein the one or more proteins having cysteine residues known to undergo S-sulfinylation are selected from one or more of the proteins recited in  FIG. 16 . 
     
     
         41 . A method for detecting protein S-sulfinylation within cysteine residues of a protein, comprising:
 providing a biological sample comprising one or more proteins having cysteine residues and a composition comprising a labeled nitrosothiol moiety,   exposing the composition to the biological sample such that an interaction between the labeled nitrosothiol moiety and a sulfinic acid side chain of a cysteine residue results in generation of a labeled sulfonothioate moiety, and   characterizing the cysteine residues of the one or more proteins having labeled sulfonothioate moieties as having undergone S-sulfinylation.   
     
     
         42 . The method of  claim 41 , further comprising identifying the protein having been characterized as having undergone S-sulfinylation and/or identifying the exact amino acid sites on the protein having been characterized as having undergone S-sulfinylation. 
     
     
         43 . The method of  claim 41 , wherein the labeled nitrosothiol moiety comprises an imaging agent and/or an enrichment tag. 
     
     
         44 . The method of  claim 43 , wherein said imaging agent is a fluorescent dye. 
     
     
         45 . The method of  claim 44 , wherein in-gel fluorescence is used to characterize the cysteine residues of a protein having a labeled sulfonothioate moiety as having undergone S-sulfinylation. 
     
     
         46 . The method of  claim 43 , wherein said enrichment agent is biotin or desthiobiotin. 
     
     
         47 . The method of  claim 43 , wherein flash chromatography followed by NMR and/or mass spectrometry is used to structurally identify the product of the reaction between a labeled nitrosothiol moiety and a sulfinic acid side chain of a cysteine residue. 
     
     
         48 . The method of  claim 43 , wherein high performance liquid chromatography is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-sulfinylation. 
     
     
         49 . The method of  claim 43 , wherein mass spectrometry is used to characterize the cysteine residues of a protein having labeled sulfonothioate moieties as having undergone S-sulfinylation.