Methods for detecting aggregations of proteins

Abstract

Families of compositions are provided as labels, referred to as eTag reporters for attaching to polymeric compounds and assaying based on release of the eTag reporters from the polymeric compound and separation and detection. For oligonucleotides, the eTag reporters are synthesized at the end of the oligonucleotide by using phosphiste or phosphate chemistry, whereby mass-modifying regions, charge-modifying regions and detectable regions are added sequentially to produce the eTag labeled reporters. By using small building blocks and varying their combination large numbers of different eTag reporters can be readily produced attached to the oligonucleotide of interest for identification. Protocols are used that release the eTag reporter when the target sequence is present in the sample.

1 - 48 . (canceled)  
     
     
         49 . A method of performing a multiplexed assay for the determination of a plurality of target proteins in an aggregation of proteins, the method comprising the steps of: 
 providing a binding compound for each of the plurality of target proteins, each binding compound having one or more eTag reporters attached thereto by a cleavable linkage, the one or more eTag reporters of each different binding compound having a different electrophoretic mobility so that eTag reporters of each different binding compound form distinct peaks upon electrophoretic separation;    combining with the aggregation a binding compound for each of the plurality of target proteins such that in the presence of a target protein a complex is formed between each target protein and the binding compound specific therefor;    cleaving the cleavable linkage of each binding compound forming such complex so that eTag reporters are released; and    electrophoretically separating and identifying the released eTag reporters to determine the plurality of target proteins in the aggregation.    
     
     
         50 . The method of  claim 49  further including a step prior to said step of cleaving, the step comprising separating said complexes from unbound said binding compounds.  
     
     
         51 . The method of  claim 50  wherein said step of cleaving includes treating said cleavage linkage with an enzyme to release said eTag reporters.  
     
     
         52 . The method of  claim 50  wherein each of said eTag reporters has a fluorescent label or an electrochemical label.  
     
     
         53 . The method of  claim 49  wherein said binding compound is an antibody or fragment thereof.  
     
     
         54 . The method of  claim 53  wherein said released eTag reporters have a charge opposite that of said complexes and said binding compounds, wherein said cleavable linkage is cleaved by oxidation, and wherein said step of cleaving includes providing an active species for oxidizing said cleavable linkage.  
     
     
         55 . The method of  claim 53  wherein said cleavable linkage is cleaved by oxidation and wherein said step of cleaving includes providing an active species for oxidizing said cleavable linkage.  
     
     
         56 . The method of  claim 55  wherein said active species is selected from the group consisting of singlet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals.  
     
     
         57 . The method of  claim 56  wherein said step of cleaving further includes providing a second binding compound specific for a protein of said aggregation of proteins, the second binding compound being conjugated with an active species producing moiety for generating said active species for oxidizing said cleavable linkage.  
     
     
         58 . The method according to  claim 54 ,  55 ,  56 , or  57  wherein said active species is singlet oxygen, wherein said second binding compound is an antibody or fragment thereof, and wherein said cleavable linkage is an olefin, a thioether, a sulfoxide, or a selenium analog of the thioether or sulfoxide.  
     
     
         59 . The method of  claim 58  wherein said binding compound and said second binding compound are each antibodies.  
     
     
         60 . A method of performing a multiplexed assay for the determination of a plurality of target proteins in an aggregation of proteins, the method comprising the steps of: 
 providing a binding compound for each of a plurality of target proteins in the aggregation, each binding compound having one or more eTag reporters attached thereto by a cleavable linkage, the one or more eTag reporters of each different binding compound having a different mass/charge ratio or adsorption so that eTag reporters of each different binding compound form distinct peaks upon electrophoretic or chromatographic separation;    providing a second binding compound for a target protein in the aggregation, the second binding compound being conjugated with an active species producing moiety;    combining with the aggregation a binding compound for each of the plurality of target proteins and the second binding compound such that in the presence of a target protein in the aggregation a complex is formed between each target protein and the binding compound or the second binding compound specific therefor, and such that the active species producing moiety of the second binding compound causes the generation of an active species and the cleavage of one or more cleavable linkages to release one or more eTag reporters; and    electrophoretically or chromatographically separating and identifying the released eTag reporters to determine the plurality of target proteins in the aggregation.    
     
     
         61 . The method of  claim 60  wherein said cleavable linkage is cleaved by oxidation and wherein said active species is singlet oxygen and wherein said active species producing moiety is a sensitizer.  
     
     
         62 . The method of  claim 61  wherein said cleavable linkage is an olefin, a thioether, a sulfoxide, or a selenium analog of the thioether or sulfoxide.  
     
     
         63 . The method of  claim 62  wherein each of said one or more eTag reporters of each said different binding compound has a different mass/charge ratio so that said eTag reporters of each said different binding compound forms a distinct peak upon electrophoretic separation.  
     
     
         64 . The method according to  claim 60 ,  61 ,  62 , or  63  wherein said binding compound and said second binding compound are each an antibody or fragment thereof.  
     
     
         65 . A method of determining an aggregation of proteins in a sample, the method comprising the steps of: 
 providing a binding compound for a protein of the aggregation, the binding compound having one or more eTag reporters attached thereto by a cleavable linkage, the eTag reporters having a unique electrophoretic mobility so that a distinct peak is formed upon electrophoretic separation;    combining with the sample the binding compound such that in the presence of the aggregation a complex is formed between a protein of the aggregation and the binding compound specific therefor;    cleaving the cleavable linkage of the binding compound forming such complex so that eTag reporters are released; and    electrophoretically separating and identifying the released eTag reporters to determine the presence of the aggregation in the sample.    
     
     
         66 . The method of  claim 65  further including a step prior to said step of cleaving, the step comprising separating said complexes from unbound said binding compounds.  
     
     
         67 . The method of  claim 66  wherein said step of cleaving includes treating said cleavage linkage with an enzyme to release said eTag reporters.  
     
     
         68 . The method of  claim 66  wherein each of said eTag reporters has a fluorescent label or an electrochemical label.  
     
     
         69 . The method of  claim 65  wherein said binding compound is an antibody or fragment thereof.  
     
     
         70 . The method of  claim 69  wherein said released eTag reporters have a charge opposite that of said complexes and said binding compound, wherein said cleavable linkage is cleaved by oxidation, and wherein said step of cleaving includes providing an active species for oxidizing said cleavable linkage.  
     
     
         71 . The method of  claim 69  wherein said cleavable linkage is cleaved by oxidation and wherein said step of cleaving includes providing an active species for oxidizing said cleavable linkage.  
     
     
         72 . The method of  claim 71  wherein said active species is selected from the group consisting of singlet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals.  
     
     
         73 . The method of  claim 71  wherein said step of cleaving further includes providing a second binding compound specific for a protein of said aggregation of proteins, the second binding compound being conjugated with an active species producing moiety for generating said active species for oxidizing said cleavable linkage.  
     
     
         74 . The method according to  claim 70 ,  71 ,  72 , or  73  wherein said active species is singlet oxygen, wherein said second binding compound is an antibody or fragment thereof, and wherein said cleavable linkage is an olefin, a thioether, a sulfoxide, or a selenium analog of the thioether or sulfoxide.  
     
     
         75 . The method of  claim 74  wherein said binding compound and said second binding compound are each antibodies.  
     
     
         76 . A method of determining an aggregation of proteins in a sample, the method comprising the steps of: 
 providing a binding compound for a protein of the aggregation, the binding compound having one or more eTag reporters attached thereto by a cleavable linkage, the eTag reporters having a unique electrophoretic mobility or adsorption so that a distinct peak is formed upon electrophoretic or chromatographic separation;    providing a second binding compound for a protein of the aggregation, the second binding compound being conjugated with an active species producing moiety;    combining with the sample the binding compound and the second binding compound such that in the presence of the aggregation a complex is formed between proteins of the aggregation and the binding compound and the second binding compound, and such that the active species producing moiety of the second binding compound causes the generation of an active species and the cleavage of one or more cleavable linkages to release one or more eTag reporters; and    electrophoretically or chromatographically separating and identifying the released eTag reporters to detect the aggregation of proteins in the sample.    
     
     
         77 . The method of  claim 76  wherein said cleavable linkage is cleaved by oxidation and wherein said active species is singlet oxygen and wherein said active species producing moiety is a sensitizer.  
     
     
         78 . The method of  claim 77  wherein said cleavable linkage is an olefin, a thioether, a sulfoxide, or a selenium analog of the thioether or sulfoxide.  
     
     
         79 . The method of  claim 76  wherein each of said at least one eTag reporter has a distinct mass/charge ratio so that said eTag reporter forms a distinct peak upon electrophoretic separation.  
     
     
         80 . The method according to  claim 76 ,  77 ,  78 , or  79  wherein said binding compound and said second binding compound are each an antibody or fragment thereof.