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 diagnostic 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; and   diagnosing the subject with an oxidative stress condition based on a result of the assay.   
     
     
         2 . The diagnostic method of  claim 1 , wherein the alkylating agent comprises a maleimide moiety. 
     
     
         3 . (canceled) 
     
     
         4 . The diagnostic 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 diagnostic method of  claim 1 , wherein the reducing agent comprises a reductase. 
     
     
         6 . The diagnostic method of  claim 1 , wherein the reducing agent comprises a glutaredoxin. 
     
     
         7 . The diagnostic 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 - 10 . (canceled) 
     
     
         11 . The diagnostic method of  claim 7 , wherein the second alkylating agent comprises a biotin moiety. 
     
     
         12 . The diagnostic method of  claim 7 , wherein the second alkylating agent comprises N-(3-maleimidylpropionyl)biocytin. 
     
     
         13 . The diagnostic method of  claim 7 , further comprising a signaling entity able to determine the second alkylthio moiety. 
     
     
         14 . The diagnostic method of  claim 7 , wherein the signaling entity comprises an avidin or a streptavidin moiety. 
     
     
         15 . (canceled) 
     
     
         16 . The diagnostic method of  claim 7 , wherein the signaling entity comprises at least one of streptavidin-HRP or streptavidin-FITC. 
     
     
         17 - 64 . (canceled) 
     
     
         65 . A kit, comprising:
 a container housing an alkylating agent and a reducing agent, wherein the alkylating agent is able to react a first thiol moiety on a protein to produce an alkylthio moiety, and the reducing agent is able to react a glutathionylated moiety on the protein to produce a second thiol moiety.   
     
     
         66 . The kit of  claim 65 , wherein the alkylating agent comprises a maleimide moiety. 
     
     
         67 . (canceled) 
     
     
         68 . The kit of  claim 65 , 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. 
     
     
         69 . (canceled) 
     
     
         70 . The kit of  claim 65 , wherein the reducing agent comprises a glutaredoxin. 
     
     
         71 . The kit of  claim 65 , further comprising a second alkylating agent able to react the second thiol moiety to form a second alkylthio moiety. 
     
     
         72 . (canceled) 
     
     
         73 . The kit of  claim 71 , wherein the second alkylating agent comprises a biotin moiety. 
     
     
         74 . The kit of  claim 71 , wherein the second alkylating agent comprises N-(3-maleimidylpropionyl)biocytin. 
     
     
         75 . The kit of  claim 71 , further comprising a signaling entity able to determine the second alkylthio moiety. 
     
     
         76 . The kit of  claim 75 , wherein the signaling entity comprises an avidin or a streptavidin moiety. 
     
     
         77 . (canceled) 
     
     
         78 . The kit of  claim 75 , wherein the signaling entity comprises at least one of streptavidin-HRP or streptavidin-FITC. 
     
     
         79 - 96 . (canceled) 
     
     
         97 . A method, comprising:
 spatially determining a glutathionylated protein in tissue.   
     
     
         98 . The method of  claim 97 , wherein the step of spatially determining comprises detecting a fluorescent signal associated with the glutathionylated protein. 
     
     
         99 - 105 . (canceled)