Phosphoprotein detection reagent and method of making and using the same

Abstract

A phosphoprotein detection reagent that selectively binds phosphoamino acids. Methods of generating and employing the reagent are also provided, as are methods of detecting modulation of protein phosphorylation are disclosed. Methods of detecting a change in state of a cell are also disclosed. Additionally, a kit for the detection of phosphoproteins is also disclosed.

1 . A phosphoprotein detection reagent (PPDR) comprising: 
 (a) a polydentate chelator that coordinates a Zn 2+  ion; and    (b) a detectable moiety conjugated to the polydentate chelator,    wherein when the Zn 2+  ion is coordinated to the chelator, a chelator-metal ion moiety is formed that can selectively bind to a phosphorylated amino acid residue in a phosphoprotein, if present, to create a chelator-metal-ion-phosphoprotein (CMPP) complex, and the detectable moiety allows the CMPP complex to be detected, if present.    
     
     
         2 . The phosphoprotein detection reagent (PPDR) of  claim 1 , wherein the PPDR is soluble in an aqueous medium.  
     
     
         3 . The phosphoprotein detection reagent (PPDR) of  claim 1 , wherein the chelator is nitriloacetic acid or iminodiacetic acid.  
     
     
         4 . The phosphoprotein detection reagent (PPDR) of  claim 1 , wherein the detectable moiety is biotin, a fluorescent molecule, or an enzyme.  
     
     
         5 . The phosphoprotein detection reagent (PPDR) of  claim 1 , wherein the polydentate chelator is bidentate, tridentate, tetradentate, or pentadentate.  
     
     
         6 . The reagent of  claim 1 , further comprising a spacer between the polydentate chelator and the detectable moiety.  
     
     
         7 . A method for synthesizing a phosphoprotein detection reagent (PPDR), the method comprising: 
 (a) reacting a polydentate chelator donor molecule with a detectable moiety donor under conditions wherein a detectable moiety is transferred to a polydentate chelator to form a chelator-detectable moiety complex; and    (b) mixing the chelator-detectable moiety complex and a Zn 2+  ion-containing solution under conditions wherein the chelator-detectable moiety complex coordinates the Zn 2+  ion, forming a PPDR that is soluble in aqueous medium.    
     
     
         8 . The method of  claim 7 , wherein the polydentate chelator is bidentate, tridentate, tetradentate, or pentadentate.  
     
     
         9 . The method of  claim 7 , wherein the detectable moiety is selected from the group consisting of biotin, a fluorescent molecule, and an enzyme.  
     
     
         10 . A method for detecting a phosphoprotein, the method comprising: 
 (a) obtaining a protein-containing solution;    (b) contacting the protein-containing solution with a reagent under conditions wherein the reagent selectively binds to a phosphorylated amino acid residue present in one or more of the proteins to form a reagent/amino acid complex, wherein: 
 (i) the reagent comprises a polydentate chelator and an detectable moiety conjugated to the polydentate chelator;  
 (ii) the polydentate chelator is coordinated to a Zn 2+  ion to form a polydentate chelator-Zn 2+  ion moiety:  
 (iii) the polydentate chelator-Zn 2+  ion moiety selectively binds to a phosphorylated amino acid residue; and  
 (iv) the detectable moiety allows the reagent/amino acid complex to be detected if present; and  
   (c) detecting the reagent/amino acid complex, wherein the detection of the reagent/amino acid complex detects a phosphoprotein.    
     
     
         11 . The method of  claim 10 , further comprising separating the proteins present in solution from each other by electrophoresis.  
     
     
         12 . The method of  claim 10 , further comprising immobilizing the separated proteins on a solid support.  
     
     
         13 . The method of  claim 10 , further comprising contacting the reagent and immobilized proteins at a pH between about 5.0 and 7.0, and washing at a pH between about 6.9 and 9.5.  
     
     
         14 . A method for detecting a change in phosphorylation status of a protein present within a target tissue in response to a change in state, the method comprising: 
 (a) obtaining a protein lysate from a cell from the target tissue prior to the change in state;    (b) separating the proteins present in the lysate from each other;    (c) contacting the proteins with a reagent under conditions wherein the reagent selectively binds to a phosphorylated amino acid residue present in at least one of the proteins to form a reagent/amino acid complex, wherein: 
 (i) the reagent comprises a polydentate chelator and an detectable moiety conjugated to the polydentate chelator;  
 (ii) the polydentate chelator is coordinated to a Zn 2+  ion to form a polydentate chelator-Zn 2+  ion moiety:  
 (iii) the polydentate chelator-Zn 2+  ion moiety selectively binds to a phosphorylated amino acid residue; and  
 (iv) the detectable moiety allows the reagent/amino acid complex to be detected if present;  
   (d) detecting the reagent/amino acid complex, wherein the reagent/amino acid complex is indicative of a phosphoprotein in the cell lysate;    (e) creating a profile indicative of the detected phosphoproteins of the lysate;    (f) obtaining a protein lysate from a cell from the target tissue after the change in state;    (g) repeating steps b) through e) for the lysate from a cell from the target tissue after the change in state; and    (h) comparing the profile from the lysate from the cell from the target tissue prior to the change in state to the profile from the lysate from the cell from the target tissue after the change in state, wherein a difference between the two profiles is indicative of a change in the phosphorylation status of a protein present within the target tissue in response to the change in state of the target tissue.    
     
     
         15 . A kit comprising: 
 (a) a phosphoprotein detection reagent (PPDR) comprising: 
 (i) a polydentate chelator that coordinates a Zn 2+  ion; and  
 (ii) a detectable moiety conjugated to the polydentate chelator,  
 wherein when the Zn 2+  ion is coordinated to the chelator, a chelator-metal ion moiety is formed that can selectively bind to a phosphorylated amino acid residue in a phosphoprotein, if present, to create a chelator-metal-ion-phosphoprotein (CMPP) complex, and the detectable moiety allows the CMPP complex to be detected, if present; and  
   (b) instructions for using the PPDR.    
     
     
         16 . The kit of  claim 15 , wherein the phosphoprotein detection reagent (PPDR) is soluble in aqueous medium.  
     
     
         17 . The kit of  claim 15 , wherein the detectable moiety is biotin, a fluorescent molecule, or an enzyme.  
     
     
         18 . The kit of  claim 15 , wherein the polydentate chelator is bidentate, tridentate, tetradentate, or pentadentate.  
     
     
         19 . A composition comprising: 
 (a) a Zn 2+  ion; and    (b) a phosphoprotein detection reagent (PPDR) comprising a chelator and a detectable moiety, wherein: 
 (i) the detectable moiety is conjugated to the chelator;  
 (ii) the chelator comprises a polydentate chelator coordinated to the Zn 2+  ion to form a chelator-metal ion moiety that can selectively bind to a phosphorylated amino acid residue in a phosphoprotein, if present, to create a chelator-metal ion-phosphoprotein (CMPP) complex; and  
 (iii) the detectable moiety allows the CMPP complex to be detected, if present.