Methods for detecting aggregations of proteins
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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.
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Citations
80 Claims
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1-48. -48. (canceled)
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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:
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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. - View Dependent Claims (50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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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:
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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. - View Dependent Claims (61, 62, 63, 64)
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65. A method of determining an aggregation of proteins in a sample, the method comprising the steps of:
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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. - View Dependent Claims (66, 67, 68, 69, 70, 71, 72, 73, 74, 75)
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76. A method of determining an aggregation of proteins in a sample, the method comprising the steps of:
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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. - View Dependent Claims (77, 78, 79, 80)
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Specification