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 method for binding a phosphorylated target molecule in a sample, said method comprising the steps of:
 i) contacting said sample with a binding solution comprising:
 a) a BAPTA metal chelating moiety; 
 b) a salt comprising trivalent metal ions; and, 
 c) an acid; and, 
   ii) incubating said sample and said binding solution for sufficient time to allow said phosphate-binding compound to associate with said phosphorylated target molecule whereby said phosphorylated target molecule is bound.   
     
     
         2 . The method according to  claim 1 , wherein said method further comprises illuminating said phosphate-binding compound with a suitable light source whereby said labeled phosphorylated target molecule is detected. 
     
     
         3 . The method according to  claim 1 , wherein said phosphorylated target molecule is selected from the group consisting of proteins, peptides, nucleotides, carbohydrates, phosphatase substrates, kinase substrates, lipids and inorganic phosphate. 
     
     
         4 . The method according to  claim 3 , wherein said phosphorylated target molecules are immobilized on a solid or semi-solid matrix or are solubilized in solution. 
     
     
         5 . The method according to  claim 4 , wherein said solid or semi-solid matrix is a gel, a membrane, a polymeric particle, or an array. 
     
     
         6 . A method for detecting a phosphorylated target molecule in a sample immobilized on a membrane, said method comprising the steps of:
 i) optionally electrophoretically separating said sample on a gel;   ii) immobilizing said sample to a membrane;   iii) optionally contacting said membrane of step ii) with a fixing solution;   iv) contacting said membrane of step iii) with a binding solution, wherein said binding solution comprises:
 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; 
   v) incubating said membrane of step iv) and said binding solution for sufficient time to allow said phosphate-binding compound to associate with said phosphorylated target molecule; and,   vi) visualizing said compound whereby said phosphorylated target molecule is detected.   
     
     
         7 . The method according to  claim 6 , wherein step iii) further comprises contacting said membrane with a wash solution following contact with said fixing solution. 
     
     
         8 . The method according to  claim 7 , wherein said phosphorylated target molecule is selected from the group consisting of proteins, peptides, and nucleotides. 
     
     
         9 . The method according to  claim 8 , wherein said phosphate-binding compounds having said formula (A) m (L) n (B) wherein B comprises a compound selected from the group consisting of Compound 1, 2, 3, 4, 5, 9, 10, 11 and 12. 
     
     
         10 . The method according to  claim 9 , wherein said step v) further comprises contacting said membrane with a wash solution following contact with said binding solution. 
     
     
         11 . The method according to  claim 10 , further comprising adding an additional detection reagent to said membrane. 
     
     
         12 . The method according to  claim 11 , wherein said additional detection reagent is a staining solution specific for total proteins, a staining solution specific for glycoproteins or an antibody. 
     
     
         13 . A method for detecting a phosphorylated target molecule in a sample immobilized on a gel, said method comprising the steps of:
 i) immobilizing said sample on a gel;   ii) optionally contacting said gel of step i) with a fixing solution;   iii) contacting said gel of step ii) with a binding solution, wherein said binding solution comprises:
 a) a 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; 
   iv) incubating said gel of step iii) and said binding solution for sufficient time to allow said compound to associate with said phosphorylated target molecule; and,   v) visualizing said compound whereby said phosphorylated target molecule is detected.   
     
     
         14 . The method according to  claim 13 , wherein said phosphorylated target molecules are selected from the group consisting of proteins, peptides, and nucleotides. 
     
     
         15 . The method according to  claim 14 , wherein said phosphate-binding compounds having said formula (A) m (L) n (B) wherein B comprises a compound selected from the group consisting of Compound 1, 2, 3, 4, 5, 9, 10, 11 and 12. 
     
     
         16 . The method according to  claim 15 , further comprising adding an additional detection reagent to said gel. 
     
     
         17 . The method according to  claim 16 , wherein said additional detection reagent is a staining solution specific for total proteins or a staining solution specific for glycoproteins. 
     
     
         18 . The method according to  claim 15 , wherein step ii) further comprises contacting said gel with a wash solution following contact with said fixing solution. 
     
     
         19 . The method according to  claim 15 , wherein said method further comprises first electrofocusing said sample before step i. 
     
     
         20 . The method according to  claim 15 , wherein said step i of said method further comprise electrophoretically separating said sample. 
     
     
         21 . The method according to  claim 15 , wherein said step v) further comprises contacting said gel with a wash solution following contact with said binding solution. 
     
     
         22 . A method for detecting a phosphorylated target molecule in a sample immobilized on a matrix, said method comprising the steps of:
 i) immobilizing said sample on an matrix;   ii) contacting said matrix of step i) with a binding solution, wherein said binding solution comprises:
 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; 
   iii) incubating said matrix of step ii) and said binding solution for sufficient time to allow said compound to associate with said phosphorylated target molecule; and,   iv) visualizing said compound whereby said phosphorylated target molecule is detected.   
     
     
         23 . The method according to  claim 22 , wherein said sample is selected from the group consisting of proteins, peptides, nucleotides, lipids, kinase substrates, phosphatase substrates, phosphate binding proteins and carbohydrates. 
     
     
         24 . The method according to  claim 23 , wherein said matrix is a polymeric gel, glass, polymeric membrane, plastic, polymeric microparticle. 
     
     
         25 . The method according to  claim 24 , wherein said phosphate-binding compound having said formula (A) m (L) n (B) wherein B comprises a compound selected from the group consisting of Compound 1, 2, 3, 4, 5, 9, 10, 11 and 12. 
     
     
         26 . The method according to  claim 25 , wherein said step iii) further comprises contacting said matrix with a wash solution following contact with said binding solution. 
     
     
         27 . The method according to  claim 26 , further comprising adding an additional detection reagent to said matrix. 
     
     
         28 . The method according to  claim 26 , wherein said additional detection reagent is a staining solution specific for total proteins, a staining solution specific for glycoproteins or an antibody.