Detection of glutathionylated proteins

Abstract

The present invention, in some aspects, relates to systems and methods for determining oxidized proteins, including glutathionylated proteins such as S-glutathionylated proteins. The systems and methods of the invention can be used in vitro (e.g., in cell or tissue culture) or in vivo, for example, to diagnose a person having an oxidative stress condition. For instance, in some cases, the invention can be used to spatially determine the location and/or concentration of oxidized proteins within cells and/or tissues (e.g., through visual detection). In one set of embodiments, a glutathionylated or otherwise oxidized moiety on a protein may be reacted with a detection entity, which may be, for example, fluorescent, radioactive, electron-dense, able to bind to a signaling entity or a binding partner in order to produce a signal, etc. As a specific example, a glutathionylated moiety on a glutathionylated protein may be reacted with an alkylating agent to form an alkylthio moiety; the alkylthio moiety may include a detection entity or otherwise be able to interact with a signaling entity. In some embodiments, other moieties on the protein may be altered or blocked before reaction of the protein with the detection entity. Such moieties on the protein may be, for instance, non-oxidized or non-glutathionylated moieties able to react with the detection entity. As a particular example, in a protein containing a glutathionylated moiety and non-glutathionylated thiol moieties, the thiol moieties may first be altered or blocked prior to reaction of the protein with the detection entity. Also provided in certain aspects of the present invention are kits for determining oxidized proteins, which may include components such as detection entities, alkylating agents, blocking agents, reducing agents, signaling entities, binding partners, antibodies, instructions, and the like.

What is claimed is: 
     
         1 . A method, comprising:
 providing a sample taken from a subject;   exposing the sample to an alkylating agent able to react a first thiol moiety on a protein to produce an alkylthio moiety;   exposing the sample to a reducing agent able to react a glutathionylated moiety on the protein to produce a second thiol moiety;   diagnosing the subject with an oxidative stress condition based on a result of the assay; and   administering, to the subject, a medicine for treating the oxidative stress condition.   
     
     
         2 . The method of  claim 1 , wherein the alkylating agent comprises a maleimide moiety. 
     
     
         3 . The method of  claim 2 , wherein the maleimide moiety comprises N-ethylmaleimide. 
     
     
         4 . The method of  claim 1 , wherein the alkylating agent comprises at least one of 2-iodoacetamide, 2-iodoacetate, p-chloromercuriphenylsulfonate, p-chloromercuribenzoate, dithiobis(2-nitro)benzoic acid, N-tosyllysyl chloromethyl ketone, 6-acryloyl-2-dimethylaminonaphthalene, dansyl aziridine, acrylodan, a benzylic halide, or a bromomethylketone. 
     
     
         5 . The method of  claim 1 , wherein the reducing agent comprises a reductase. 
     
     
         6 . The method of  claim 1 , wherein the reducing agent comprises a glutaredoxin. 
     
     
         7 . The method of  claim 1 , further comprising exposing the sample to a second alkylating agent able to react the second thiol moiety to form a second alkylthio moiety. 
     
     
         8 . The method of  claim 7 , wherein the second alkylthio moiety comprises a detection entity. 
     
     
         9 . The method of  claim 8 , further comprising determining the detection entity, the determination being a result of the assay. 
     
     
         10 . The method of  claim 9 , wherein determining the detection entity comprises fluorescently detecting the detection entity. 
     
     
         11 . The method of  claim 7 , wherein the second alkylating agent comprises a biotin moiety. 
     
     
         12 . The method of  claim 7 , wherein the second alkylating agent comprises N-(3-maleimidylpropionyl)biocytin. 
     
     
         13 . The method of  claim 7 , further comprising providing a signaling entity able to determine the second alkylthio moiety. 
     
     
         14 . The method of  claim 7 , wherein the signaling entity comprises an avidin or a streptavidin moiety. 
     
     
         15 . The method of  claim 7 , wherein the signaling entity comprises a fluorescent moiety. 
     
     
         16 . The method of  claim 7 , wherein the signaling entity comprises at least one of streptavidin-HRP or streptavidin-FITC. 
     
     
         17 - 96 . (canceled) 
     
     
         97 . A method, comprising:
 spatially determining a glutathionylated protein in tissue in a subject by detecting a fluorescent signal within the tissue associated with the glutathionylated protein.   
     
     
         98 . (canceled) 
     
     
         99 . A method, comprising:
 determining a glutathionylated state of a protein within a subject;   diagnosing the subject with a medical condition based on the glutathionylated state of the protein; and   administering, to the subject, a medicine for treating the oxidative stress condition based on the diagnosis.   
     
     
         100 . The method of  claim 99 , wherein the act of determining the glutathionylated state of the protein comprises:
 providing a sample taken from the subject;   exposing the sample to an alkylating agent able to react a first thiol moiety on a protein to produce an alkylthio moiety; and   exposing the sample to a reducing agent able to react a glutathionylated moiety on the protein to produce a second thiol moiety.   
     
     
         101 . (canceled) 
     
     
         102 . The method of  claim 99 , wherein the medical condition is an inflammatory condition. 
     
     
         103 . The method of  claim 99 , wherein the medical condition is an allergic airway inflammation disease. 
     
     
         104 . The method of  claim 99 , wherein the medical condition is asthma. 
     
     
         105 . (canceled)