Detecting and profiling molecular complexes
View Patent ↗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.
1. A method of detecting one or more complexes comprising 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 the first protein in each of the one or more complexes, each cleaving probe comprising a cleavage-inducing moiety;
providing one or more binding compounds specific for the second protein of each of the one or more complexes, such that each binding compound comprises 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.
2. The method of claim 1 wherein said step of mixing includes generating an active species by said cleavage-inducing moiety, the active species cleaving said cleavable linkages with said effective proximity.
3. The method of claim 2 wherein said cleavage-inducing moiety is a photosensitizer and said active species is singlet oxygen.
4. The method of claim 2 wherein said separation characteristic is electrophoretic mobility and wherein said separation profile is an electropherogram.
5. The method according to claims 1 , 2 , 3 , or 4 wherein said one or more complexes of proteins comprise up to three complexes, and wherein said one or more binding compounds comprise up to three binding compounds.
6. The method of claim 5 wherein each of said cleaving probes and said one or more binding compounds comprises an antibody binding composition.
7. The method of claim 6 wherein said one or more complexes includes a complex comprising a Her3 receptor and a PI3 kinase.
8. The method of claim 7 wherein said Her3 receptor is said first protein whenever said PI3 kinase is said second protein, and wherein said PI3 kinase is said first protein whenever said Her3 receptor is said second protein.
9. The method of claim 6 wherein said one or more complexes include a complex comprising said first protein and said one or more second proteins each selected from the group consisting of a receptor tyrosine kinase, Grb2, SOS, Ras, and Raf, with the proviso that said first protein is different from said one or more second proteins.
10. The method of claim 9 wherein said receptor tyrosine kinase is a human receptor tyrosine kinase selected from the group consisting of Hen, Her2, Her3, Her4, IGF-1R, VEGFR1, VERFR2, PDGFRα, and PDGFRβ.