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.

1 . A binding solution comprising:
 a) a phosphate-binding compound having formula (A)m(L)n(B) wherein A is a chemical moiety, L is a linker, B is a metal-chelating moiety, m is an integer from 1 to 4 and n is an integer from 0 to 4; and   b) a salt comprising metal ions.   
     
     
         2 . The binding solution of  claim 1 , further comprising an acid. 
     
     
         3 . The binding solution of  claim 1 , wherein the metal ions are a cationic transition metal. 
     
     
         4 . The binding solution of  claim 1 , wherein said linker comprises: —C(O)NH—. 
     
     
         5 . The binding solution of  claim 1 , wherein the chemical moiety is a label. 
     
     
         6 . The binding solution of  claim 5 , wherein the label is a dye selected from the group consisting of a benzofuran, a quinazolinone, a xanthene, an indole, a benzazole and a borapolyazaindacene. 
     
     
         7 . The binding solution of  claim 5 , wherein the label is selected from the group consisting of a dye, an enzyme and a hapten. 
     
     
         8 . The binding solution of  claim 1 , wherein m is 1 and n is 1. 
     
     
         9 . The binding solution of  claim 2 , wherein the acid is acetic acid. 
     
     
         10 . The binding solution of  claim 2 , wherein the acid is present at a concentration of 1%-20%. 
     
     
         11 . The binding solution of  claim 1 , wherein the binding solution has a pH about 3 to about pH 6. 
     
     
         12 . The binding solution of  claim 1 , further comprising a buffering agent. 
     
     
         13 . The binding solution of  claim 12 , wherein the buffering agent is selected from the group consisting of salts of formate, acetate, 2-(N-morpholino)ethanesulfonic acid, imidazole, N-(2-hydroxyethyl)piperazinylethanesulfonic acid, tris-(hydroxymethyl) aminomethane acetate, or tris(hydroxymethyl)aminomethane, and hydrochloride. 
     
     
         14 . The binding solution of  claim 1 , further comprising a water-miscible organic solvent. 
     
     
         15 . The binding solution of  claim 14 , wherein the water-miscible organic solvent is an alcohol. 
     
     
         16 . The binding solution of  claim 15 , wherein the alcohol is methanol. 
     
     
         17 . The binding solution of  claim 15 , wherein the alcohol is present in a concentration of less than about 20%. 
     
     
         18 . The binding solution of  claim 1 , wherein the phosphate-binding compound has the formula: (A)-[C(X)NH(CH 2 ) e ]—(B); wherein X is O and e is 0-6. 
     
     
         19 . The binding solution of  claim 1 , wherein the metal chelating moiety is immobilized on a solid or semi-solid matrix. 
     
     
         20 . The binding solution of  claim 1 , wherein the metal chelating moiety is capable of binding a trivalent metal ion. 
     
     
         21 . A method for detecting a phosphorylated target molecule immobilized on a gel comprising the following steps:
 immobilizing a sample on a gel;   contacting the gel of with a binding solution comprising:
 a) a phosphate-binding compound having formula (A)m(L)n(B) wherein A is a chemical moiety, L is a linker, B is a metal-chelating moiety, m is an integer from 1 to 4 and n is an integer from 0 to 4; 
 b) a salt comprising metal ions; and, 
 c) an acid; 
   incubating the gel and the binding solution for sufficient time to allow the phosphate-binding compound to associate with the phosphorylated target molecule; and,   visualizing the phosphate-binding compound whereby the phosphorylated target molecule is detected.   
     
     
         22 . The method of  claim 21 , further comprising contacting the gel with a fixing solution directly after the immobilizing step. 
     
     
         23 . The method of  claim 21 , further comprising adding a second (or third) stain to the gel to detect either total protein or proteins of another class, such as glycoproteins, or both. 
     
     
         24 . A kit for binding a phosphorylated target molecule in a sample, said kit comprising:
 i) a binding solution comprising:
 a) a phosphate-binding compound having formula (A)m(L)n(B) wherein A is a chemical moiety, L is a linker, B is a metal-chelating moiety, m is an integer from 1 to 4 and n is an integer from 0 to 4; and 
 b) a salt comprising metal ions; 
   ii) at least one of instructions, molecular weight markers that comprise phosphorylated and non-phosphorylated polypeptides, a fixing solution, a detection reagent, standards, a wash solution, a matrix, a gel, or a phosphatase or kinase substrate.   
     
     
         25 . The kit of  claim 24 , wherein the binding solution further comprises an acid.