Detecting and Profiling Molecular Complexes

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.

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.   
   
   
       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 Her1, Her2, Her3, Her4, IGF-1R, VEGFR1, VERFR2, PDGFRα, and PDGFRβ. 
   
   
       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:
 providing a cleaving probe specific for an antigenic determinant of the first protein, the cleaving probe comprising a cleavage-inducing moiety;   providing one or more binding compounds each specific for an antigenic determinant of the second protein, 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 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 an 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.   
   
   
       12 . The method of  claim 11  wherein at least one of said second proteins is different than said first protein. 
   
   
       13 . The method of  claim 12  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. 
   
   
       14 . The method of  claim 13  wherein said cleavage-inducing moiety is a photosensitizer and said active species is singlet oxygen. 
   
   
       15 . The method of  claim 13  wherein said separation characteristic is electrophoretic mobility and wherein said separation profile is an electropherogram. 
   
   
       16 . The method according to  claims 11 ,  12 ,  13 ,  14  or  15  wherein said cleaving probe and said one or more binding compounds comprises an antibody binding composition. 
   
   
       17 . The method of  claim 16  wherein said complex comprises a Her3 receptor and a PI3 kinase. 
   
   
       18 . The method of  claim 17  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. 
   
   
       19 . The method of  claim 16  wherein said complex comprises said first protein and said one or more second proteins each selected from the group consisting of a receptor tyrosine kinase, a Shc protein, a Grb2 protein, an SOS protein, a Ras protein, and a Raf protein, with the proviso that said first protein is different from said one or more second proteins. 
   
   
       20 . The method of  claim 19  wherein said receptor tyrosine kinase is a human receptor tyrosine kinase selected from the group consisting of Her1, Her2, Her3, Her4, IGF-IR, VEGFR1, VERFR2, PDGFRα, and PDGFRβ. 
   
   
       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:
 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 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, 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 one 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.   
   
   
       22 . The method of  claim 21  further comprising, prior to said step of cleaving, a step of separating said unbound binding compounds from said binding compounds specifically bound to said complex. 
   
   
       23 . The method of  claim 22  wherein said separation characteristic is electrophoretic mobility and wherein said separation profile is an electropherogram. 
   
   
       24 . The method according to  claims 21 ,  22 , or  23  wherein each of said one or more binding compounds comprises an antibody binding composition. 
   
   
       25 . The method of  claim 24  wherein said complex comprises a Her3 receptor and a PI3 kinase. 
   
   
       26 . The method of  claim 25  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. 
   
   
       27 . The method of  claim 24  wherein said complex comprises said first protein and said one or more second proteins each selected from the group consisting of a receptor tyrosine kinase, a Shc protein, a Grb2 protein, an SOS protein, a Ras protein, and a Raf protein, with the proviso that said first protein is different from said one or more second proteins. 
   
   
       28 . The method of  claim 27  wherein said receptor tyrosine kinase is a human receptor tyrosine kinase selected from the group consisting of Her1, Her2, Her3, Her4, IGF-IR, VEGFR1, VERFR2, PDGFRα, and PDGFRβ.