Detecting and profiling molecular complexes
First Claim
1. A method of detecting one or more complexes of proteins, wherein each complex comprises a first protein and one or more second proteins, the method comprising the steps of:
- providing for each of the one or more complexes a cleaving probe specific for a first protein in each of the one or more complexes, each cleaving probe comprises a cleavage-inducing moiety attached to a binding agent that specifically binds to said first protein in each of the one or more complexes;
providing one or more binding compounds specific for a second protein of each of the one or more complexes, such that each binding compound comprises one or more molecular tags each attached to a binding moiety through a cleavable linkage, wherein said binding moiety specifically binds to said second protein of each of the one or more complexes, and such that the one or more molecular tags attached to different binding moieties have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile;
mixing the cleaving probes, the binding compounds, and the one or more complexes such that cleaving probes specifically bind to first proteins of the complexes and binding compounds specifically bind to the second proteins of the complexes such that the cleavable linkages of the binding compounds are within the effective proximity of the cleavage-inducing moieties of the cleaving probes so that the molecular tags are released; and
separating and identifying the released molecular tags to determine the presence or absence or the amount of one or more complexes of proteins.
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Accused Products
Abstract
Methods are provided for detecting the formation of complexes of molecules, especially proteins, in a sample, such as a cell or tissue lysate. In one aspect, a cleaving probe specific for a first protein in a complex and one or more binding compounds specific for one or more second proteins in a complex are provided. Upon binding, the cleaving probe is induced to generate an active species, such as singlet oxygen, that cleaves molecular tags attached to the binding compounds only in the local region of the cleaving probe. The released molecular tags are separated from the assay mixture and from one another to provide a readout that is related to the number and types of proteins present in the complex.
138 Citations
28 Claims
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1. A method of detecting one or more complexes of proteins, wherein each complex comprises a first protein and one or more second proteins, the method comprising the steps of:
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providing for each of the one or more complexes a cleaving probe specific for a first protein in each of the one or more complexes, each cleaving probe comprises a cleavage-inducing moiety attached to a binding agent that specifically binds to said first protein in each of the one or more complexes; providing one or more binding compounds specific for a second protein of each of the one or more complexes, such that each binding compound comprises one or more molecular tags each attached to a binding moiety through a cleavable linkage, wherein said binding moiety specifically binds to said second protein of each of the one or more complexes, and such that the one or more molecular tags attached to different binding moieties have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile; mixing the cleaving probes, the binding compounds, and the one or more complexes such that cleaving probes specifically bind to first proteins of the complexes and binding compounds specifically bind to the second proteins of the complexes such that the cleavable linkages of the binding compounds are within the effective proximity of the cleavage-inducing moieties of the cleaving probes so that the molecular tags are released; and separating and identifying the released molecular tags to determine the presence or absence or the amount of one or more complexes of proteins. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 17, 18, 19, 20, 25, 26)
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9. A method of detecting in a biological sample a complex comprising a first protein and one or more second proteins, the method comprising the steps of:
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providing a cleaving probe specific for an antigenic determinant of the first protein, the cleaving probe having a cleavage-inducing moiety attached to a binding agent that specifically binds to said antigenic determinant of the first protein; providing one or more binding compounds each specific for an antigenic determinant of the second protein, such that each binding compound comprises one more molecular tags each attached to a binding moiety through a cleavable linkage, wherein said binding moiety specifically binds to said antigenic determinant of the second protein, and such that the one or more molecular tags attached to different binding moieties have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile; mixing the cleaving probe, the one or more binding compounds, and the biological sample such that cleaving probes specifically binds to first protein and the one or more binding compounds specifically bind to the one or more second proteins, and such that the cleavable linkages of the binding compounds are within the effective proximity of the cleavage-inducing moieties of the cleaving probes so that the molecular tags are released; and separating and identifying the released molecular tags to determine the presence or absence or the amount of complex in the biological sample. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 21, 22, 23, 24, 27, 28)
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Specification