Compositions and methods for detection and isolation of phosphorylated molecules

Abstract

The present invention relates to phosphate-binding compounds that find use in binding, detecting and isolating phosphorylated target molecules including the subsequent identification of target molecules that interact with phosphorylated target molecules or molecules capable of being phosphorylated. A binding solution is provide that comprises a phosphate-binding compound, an acid and a metal ion wherein the metal ion simultaneously interacts with an exposed phosphate group on a target molecule and the metal chelating moiety of the phosphate-binding compound forming a bridge between the phosphate-binding compound and a phosphorylated target molecule resulting in a ternary complex. The binding solution of the present invention finds use in binding and detecting immobilized and solubilized phosphorylated target molecules, isolation of phosphorylated target molecules from a complex mixture and aiding in proteomic analysis wherein kinase and phosphatase substrates and enzymes can be identified.

What is claimed is:  
     
         1 . A binding solution comprising: 
 a) a metal chelating moiety;    b) a salt comprising trivalent metal ions, wherein said metal ion is capable of simultaneously binding said metal chelating moiety and a phosphorylated target molecule; and,    c) an acid.    
     
     
         2 . The binding solution according to  claim 1 , wherein said metal chelating moiety is independently covalently bonded to a label and a chemically reactive group.  
     
     
         3 . The binding solution according to  2 , wherein said metal chelating moiety is covalently bonded to both a label and chemically reactive group.  
     
     
         4 . The binding solution according to  claim 2 , wherein said metal chelating moiety is independently covalently bonded to a label or a chemically reactive group.  
     
     
         5 . The binding solution according to  claim 2 , wherein said chemically reactive group is a photoactivatable group.  
     
     
         6 . The binding solution according to  claim 5 , wherein said label is selected from the group consisting of a dye, an enzyme and a hapten.  
     
     
         7 . The binding solution according to  claim 6 , wherein said dye is selected from the group consisting of a benzofuran, a quinazolinone, a xanthene, an indole, a benzazole and a borapolyazaindacene provided that said dyes are not sulfonated.  
     
     
         8 . The binding solution according to  claim 2 , wherein said metal chelating moiety is selected from the group consisting of BAPTA, IDA, DTPA and phenanthrolines.  
     
     
         9 . The binding solution according to  claim 8 , wherein said binding solution has a pH about 3 to about pH 6.  
     
     
         10 . The binding solution according to  claim 9 , wherein said metal ion is selected from the group consisting of Ga 3+ , Fe 3+  and Al 3+ .  
     
     
         11 . The binding solution according to  claim 10 , wherein said salt is gallium chloride.  
     
     
         12 . The binding solution according to  claim 9 , wherein said metal chelating moiety is in solution or immobilized on a solid or semi-solid matrix.  
     
     
         13 . A method for binding a phosphorylated target molecule in a sample, said method comprising the steps of: 
 i) contacting said sample with a binding solution according to any one of claims  1 - 12 ; and,    ii) incubating said sample and said binding solution for sufficient time to allow said metal chelating moiety and said metal ion to associate with said phosphorylated target molecule whereby said phosphorylated target molecule is bound.    
     
     
         14 . The method according to  claim 13 , wherein said metal chelating moiety is covalently bonded to a label and said method further comprises illuminating said label with a suitable light source whereby said bound phosphorylated target molecule is detected.  
     
     
         15 . The method according to  claim 13 , wherein metal chelating moiety is covalently bonded to a photoactivatable reactive group and said method further comprises illuminating said reactive group with an appropriate wavelength where said reactive group specifically forms a covalent bond with said phosphorylated target molecule.  
     
     
         16 . The method according to  claim 13 , wherein said phosphorylated target molecule is selected from the group consisting of proteins, peptides, nucleotides, carbohydrates, phosphatase substrates, kinase substrates, lipids and inorganic phosphate.  
     
     
         17 . The method according to  claim 16 , wherein said phosphorylated target molecules are immobilized on a solid or semi-solid matrix or are solubilized in solution.  
     
     
         18 . The method according to  claim 17 , wherein said solid or semi-solid matrix is a gel, a membrane, a polymeric particle, or an array.  
     
     
         19 . A method of specifically covalently labeling phosphorylated target molecules in a sample, wherein said method comprises: 
 i) contacting said sample with a binding solution comprising a phosphate binding compound that is covalently bonded to a label and a photoactivatable chemically reactive group; a salt comprising trivalent metal ions and an acid; 
 a. incubating said sample and said binding solution for sufficient time to allow said phosphate binding compound and said metal salt to associate with said phosphorylated target molecule;  
 b. illuminating said phosphate binding compound with an appropriate wavelength where said photoactivatable reactive group specifically forms a covalent bond with said phosphorylated target molecule whereby said phosphorylated target molecule is labeled.  
   
     
     
         20 . The method according to  claim 19 , wherein said sample is selected from the group consisting of proteins, peptides, nucleotides, lipids, kinase substrates, phosphatase substrates, phosphate binding proteins and carbohydrates.  
     
     
         21 . The method according to  claim 20 , wherein said method further comprises immobilizing said sample on a solid or semi-solid matrix.  
     
     
         22 . The method according to  claim 21 , wherein said matrix is a polymeric gel, glass, polymeric membrane, plastic, polymeric microparticle.  
     
     
         23 . The method according to  claim 22 , wherein said method further comprises electrophoretically separating said sample on a gel and optionally transferring said immobilized sample to a membrane.  
     
     
         24 . The method according to  claim 23 , wherein said phosphorylated target molecule is selected from the group consisting of proteins, peptides, and nucleotides.  
     
     
         25 . The method according to  claim 24 , wherein said phosphate-binding compounds are selected from the group consisting of Compounds 34, 36, 39, 42 and 44  
     
     
         26 . The method according to  claim 24 , wherein said step ii) further comprises contacting said immobilized sample with a wash solution following incubating said sample with said binding solution.  
     
     
         27 . The method according to  claim 26 , wherein said method further comprises adding an additional detection reagent to said immobilized sample.  
     
     
         28 . The method according to  claim 27 , wherein said additional detection reagent is a staining solution specific for total proteins, a staining solution specific for glycoproteins or an antibody.  
     
     
         29 . A kit for binding a phosphorylated target molecule in a sample, said kit comprising: 
 i) a binding solution comprising a metal chelating moiety; a salt comprising trivalent metal ions, wherein said metal ion is capable of simultaneously binding said metal chelating moiety and a phosphorylated target molecule; and, an acid;    ii) wherein said kit optionally includes molecular weight markers that comprise phosphorylated and non-phosphorylated polypeptides.    
     
     
         30 . The kit according to  claim 29 , wherein said kit independently further comprises a fixing solution, additional detection reagent, standards or a wash solution.  
     
     
         31 . The kit according to  claim 30 , wherein said metal chelating moiety is covalently bonded to a photoactivatable chemically reactive group.  
     
     
         32 . The kit according to  claim 31 , wherein said kit further comprises a matrix.  
     
     
         33 . The kit according to  claim 32 , wherein said kit further comprises a phosphatase or kinase substrate.  
     
     
         34 . The kit according to  claim 30 , wherein said additional detection reagent is independently a staining solution specific for total proteins, a staining solution specific for glycoproteins or antibodies.  
     
     
         35 . The kit according to  claim 30 , wherein said phosphate-binding compounds are selected from the group consisting of Compounds 34, 36, 39, 42 and 44.  
     
     
         36 . A matrix comprising an immobilized phosphate-binding compound comprising a metal chelating moiety that is capable of specifically binding to a phosphorylated target molecule.  
     
     
         37 . The matrix according to  claim 36 , wherein said matrix is a member selected from the group consisting of an amine microarray substrate, an aldehyde microarray substrate, an epoxy microarray substrate, a poly-L-lysine coated slides and polyacrylamide coated slide.  
     
     
         38 . A phosphate-binding compound, wherein said compound comprises a metal chelating moiety and a covalently bonded photoactivatable chemically reactive group, where said metal chelating moiety is capable of specifically binding to a phosphorylated target molecule.  
     
     
         39 . The phosphate-binding compounds according to  claim 38 , wherein said compounds are selected from the group consisting of Compounds 34, 36, 39, 42 and 44  
     
     
         40 . A ternary complex comprising a trivalent metal ion, a phosphorylated target molecule and a phosphate-binding compound comprising a metal chelating moiety wherein said metal ion is capable of simultaneously binding said phosphorylated target molecule and said metal chelating moiety.  
     
     
         41 . The ternary complex according to  claim 40 , wherein said chelating moiety is selected from the group consisting of a BAPTA, IDA, DTPA and phenanthrolines.  
     
     
         42 . The ternary complex according to  claim 41 , wherein said metal ion is selected from the group consisting of Ga 3 , Fe 3+  and Al 3+ .  
     
     
         43 . A phosphorylated target molecule determined by combining a sample comprising a phosphorylated target molecule with a binding solution according to any one of claims  1 - 10 .  
     
     
         44 . A method for detecting a phosphorylated target molecule in a sample wherein said sample is in an aqueous solution, wherein said method comprises: 
 i) contacting said sample with a binding solution to form a combined mixture, wherein said binding solution comprises a phosphate-binding compound, a salt comprising trivalent metal ions and an acid, wherein said combined mixture comprises a first dye label that has a first absorption and emission spectra and a second dye label that has a second absorption and emission spectra;    ii) incubating said phosphate binding compound and said sample for a sufficient amount of time for said phosphate binding compound to bind a phosphorylated target molecule; and,    iii) illuminating said sample with an appropriate wavelength whereby said phosphorylated target molecule is detected by a change in fluorescence signal.    
     
     
         45 . The method according to  claim 44 , wherein said change in fluorescence signal is due to an energy transfer between said first and said second dye label.  
     
     
         46 . The method according to  claim 44 , wherein said change in fluorescence signal is a quenching of said first dye label.  
     
     
         47 . The method according to  claim 44 , wherein said change in fluorescence signal is a quenching of said second dye label.  
     
     
         48 . The method according to  claim 44 , wherein said phosphorylated target molecule is selected from the group consisting of proteins, peptides, nucleotides, carbohydrates, phosphatase substrates, kinase substrates, lipids and inorganic phosphate.  
     
     
         49 . The method according to  claim 48 , wherein said sample further comprises ATP and a kinase enzyme.  
     
     
         50 . The method according to  claim 48 , wherein said sample further comprises a phosphatase enzyme.  
     
     
         51 . A method for detecting kinase enzymes in a sample, wherein said method comprises; 
 a) contacting a sample with a kinase substrate and a source of phosphate;    b) incubating said sample of step a) for a sufficient amount of time for said kinase substrate to be phosphorylated;    c) contacting said phosphorylated kinase substrate with a binding solution, wherein said binding solution comprises a metal chelating moiety, a salt comprising trivalent metal ions, wherein said metal ion is capable of simultaneously binding said metal chelating moiety and a phosphorylated target molecule; and, an acid for a sufficient amount of time for said metal chelating moiety to bind said phosphorylated kinase substrate.    d) illuminating said sample with an appropriate wavelength whereby said kinase substrate is detected.    
     
     
         52 . The method according to  claim 51 , wherein said metal chelating moiety comprises a dye label that is a member selected from the group consisting of benzofuran, a quinazolinone, a xanthene, an indole, a benzazole and a borapolyazaindacene provided that said dyes are not sulfonated.  
     
     
         53 . The method according to  claim 54 , wherein said metal chelating moiety further comprises a photoactivatable reactive group.  
     
     
         54 . The method according to  claim 52 , wherein said kinase substrate is in solution or immobilized to a solid or semi-solid matrix.  
     
     
         55 . The method according to  claim 54 , wherein said kinase substrate is in solution.  
     
     
         56 . The method according to  claim 55 , wherein said kinase substrate comprises a dye label.