Enzyme coupled assay for quantification of protein and peptide binding by samdi mass spectrometry

Abstract

Disclosed herein are methods of using an immobilized substrate, immobilized ligand, and a fusion protein of an enzyme for the substrate and a receptor for the ligand, where the immobilized substrate can react to form an immobilized product that has a different mass than the immobilized substrate, and using this transformation to indirectly determine the binding of the receptor and the ligand. These methods can be used for high-throughput screening for possible modulators (e.g., inhibitors or activators) of the ligand-receptor interaction.

What is claimed is: 
     
         1 . A method comprising
 (a) contacting an immobilized substrate and an immobilized ligand with a fusion protein under conditions to transform the immobilized substrate to an immobilized product, wherein the immobilized product has a mass different from the immobilized substrate;   
       the fusion protein comprises (1) an enzyme capable of transforming the substrate to the product and (2) a receptor, and 
       the immobilized substrate for the enzyme and the immobilized ligand for the receptor are each bound to a self-assembled monolayer (SAM) on a surface;
 (b) subjecting the substrate and product to mass spectrometry to produce a mass spectrum having a product signal and a substrate signal; and 
 (c) detecting binding of the receptor and the ligand by correlating a signal intensity of the product to a signal intensity of the substrate to determine the extent of product formation and thereby detecting the binding of the receptor and the ligand, 
 wherein the total density of the ligand and the substrate on the surface is less than or equal to about 10%. 
 
     
     
         2 . The method of  claim 1 , wherein the total density of the ligand and the substrate on the surface is from about 3% to about 7%. 
     
     
         3 . The method of  claim 1  or  claim 2 , wherein the total density of the ligand and the substrate on the surface is from about 5% to about 6%. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the total density of the ligand and the substrate on the surface is at least about 0.1%. 
     
     
         5 . The method of any one of  claims 1  to  4 , wherein extent of product formation in the presence of the fusion protein is greater than extent of product formation in the presence of the enzyme not fused to the receptor. 
     
     
         6 . The method of any one of  claims 1  to  5 , wherein extent of product formation in the presence of the fusion protein is at least 2 times greater, at least 5 times greater, at least 10 times greater, at least 20 times greater, at least 30 times greater, or at least 50 times greater than extent of product formation in the presence of the enzyme not fused to the receptor. 
     
     
         7 . The method of any one of  claims 1  to  6 , wherein at least one of the substrate and ligand is bound to the surface via a linker. 
     
     
         8 . The method of  claim 7 , wherein the linker has a structure of formula I: 
       
         
           
           
               
               
           
         
       
       wherein L is the substrate or ligand. 
     
     
         9 . The method of  claim 8 , wherein L is attached to the pyrrolindinone of formula (I) via a thiol bond. 
     
     
         10 . The method of any one of  claims 7  to  9 , wherein the linker is attached to the SAM via an ethylene glycol moiety. 
     
     
         11 . The method of any one of  claims 1  to  10 , wherein the SAM is attached to the surface via a thiol bond. 
     
     
         12 . The method of any one of  claims 1  to  11 , wherein the surface comprises gold. 
     
     
         13 . The method of any one of  claims 1  to  11 , wherein the surface comprises silver. 
     
     
         14 . The method of any one of  claims 1  to  11 , wherein the surface comprises copper. 
     
     
         15 . The method of any one of  claims 1  to  14 , wherein at least one of the substrate and the ligand comprises a peptide. 
     
     
         16 . The method of  claim 15 , wherein the peptide is bound to the SAM via a cysteine residue. 
     
     
         17 . The method of any one of  claims 1  to  16 , wherein the enzyme is a deacetylase, acetyltransferase, esterase, phosphorylase/kinase, phosphatase, protease, methylase, demethylase, or a DNA or RNA modifying enzyme. 
     
     
         18 . The method of  claim 17 , wherein the deacetylase is KDAC8. 
     
     
         19 . The method of  claim 17 , wherein the esterase is cutinase or acetylcholine esterase. 
     
     
         20 . The method of  claim 17 , wherein the protease is TEV. 
     
     
         21 . The method of any one of  claims 17  to  20 , wherein the substrate comprises an acylated peptide and the product comprises a deacylated peptide. 
     
     
         22 . The method of any one of  claims 17  to  20 , wherein the substrate comprises a deacylated peptide and the product comprises an acylated peptide. 
     
     
         23 . The method of  claim 17 , wherein the substrate comprises a phosphorylated peptide and the product comprises a dephosphorylated peptide. 
     
     
         24 . The method of  claim 17 , wherein the substrate comprises a dephosphorylated peptide and the product comprises a phosphorylated peptide. 
     
     
         25 . The method of  claim 17 , wherein the substrate comprises a methylated peptide and the product comprises a demethylated peptide. 
     
     
         26 . The method of  claim 17 , wherein the substrate comprises a demethylated peptide and the product comprises a methylated peptide. 
     
     
         27 . The method of any one of  claims 1  to  24 , wherein the receptor is a chromodomain, a bromodomain, a PDZ domain, a Src homology 2 (SH2) domain SH2 domain, or a PDZ domain. 
     
     
         28 . The method of  claim 25 , wherein the chromodomain is a HP1 chromodomain, or a Pc chromodomain. 
     
     
         29 . The method of  claim 25  or  26 , wherein the ligand is a peptide and comprises a methylated lysine residue. 
     
     
         30 . The method of any one of  claims 1  to  27 , wherein the substrate density on the surface is greater than the ligand density on the surface. 
     
     
         31 . The method of any one of  claims 1  to  27 , wherein the ligand has a dissociation constant (K D ) of about 10 pM to about 100 μM. 
     
     
         32 . The method of  claim 29 , wherein the ligand has a dissociation constant (KD) of about 100 nM to about 10 μM. 
     
     
         33 . The method of  claim 29  or  claim 30 , wherein K D  is less than 10 μM. 
     
     
         34 . The method of any one of  claims 1  to  31 , further comprising contacting the immobilized substrate and the immobilized ligand with the fusion protein in the presence of one or more potential modulators of the receptor-ligand binding; subjecting the substrate and product to mass spectrometry to produce a mass spectrum having a product signal and a substrate signal; and detecting binding of the receptor and the ligand by correlating a signal intensity of the product to a signal intensity of the substrate to determine the extent of product formation and thereby detecting the binding of the receptor and the ligand in the presence of the one or more potential modulators. 
     
     
         35 . The method of  claim 32 , wherein the modulator is an inhibitor of the receptor-ligand binding. 
     
     
         36 . The method of  claim 32 , wherein the modulator is an activator of the receptor-ligand binding. 
     
     
         37 . The method of any one of  claims 1  to  34 , further comprising admixing a matrix to the substrate and product prior to subjecting the substrate and product to mass spectrometry.