Detecting and profiling molecular complexes
First Claim
1. A method of detecting one or more complexes of 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 having a cleavage-inducing moiety with an effective proximity;
providing one or more binding compounds specific for a second protein of each of the one or more complexes, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds 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 and such that cleavable linkages of the binding compounds are within the effective proximity of cleavage-inducing moieties of the cleaving probes so that molecular tags are released; and
separating and identifying the released molecular tags to determine the presence or absence or the amount of the 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.
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Citations
28 Claims
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1. A method of detecting one or more complexes of 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 having a cleavage-inducing moiety with an effective proximity;
providing one or more binding compounds specific for a second protein of each of the one or more complexes, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds 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 and such that cleavable linkages of the binding compounds are within the effective proximity of cleavage-inducing moieties of the cleaving probes so that molecular tags are released; and
separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more complexes of proteins. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. 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 with an effective proximity;
providing one or more binding compounds each specific for an antigenic determinant of a second protein, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds 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 the cleaving probe specifically binds to the first protein and the one or more binding compounds specifically bind to the one or more second proteins, and such that cleavable linkages of the binding compounds are within the effective proximity of cleavage-inducing moieties of the cleaving probes so that molecular tags are released; and
separating and identifying the released molecular tags to determine the presence or absence or the amount of the complex in the biological sample. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. 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 one or more binding compounds each specific for a different antigenic determinant of the first protein, and providing one or more binding compounds each specific for a different antigenic determinant of a second protein, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile, and such that at least one of the second proteins is different from the first protein;
combining the biological sample and the one or more binding compounds specific for the first protein and for the one or more second proteins of the complex such that the binding compounds specifically bind to their respective antigenic determinants whenever such antigenic determinants are available and are unbound whenever such antigenic determinants are unavailable;
cleaving the cleavable linkages of the on or more binding compounds specifically bound to the complex so that molecular tags are released; and
separating and identifying the released molecular tags to determine the presence or absence or the amount of the complex in the biological sample. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28)
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Specification