Methods for conducting assays for enzyme activity on protein microarrays

Abstract

The present invention relates to methods of conducting assays for enzymatic activity on microarrays useful for the large-scale study of protein function, screening assays, and high-throughput analysis of enzymatic reactions. The invention relates to methods of using protein chips to assay the presence, amount, activity and/or function of enzymes present in a protein sample on a protein chip. In particular, the methods of the invention relate to conducting enzymatic assays using a microarray wherein a protein and a substance are immobilized on the surface of a solid support and wherein the protein and the substance are in proximity to each other sufficient for the occurrence of an enzymatic reaction between the substance and the protein. The invention also relates to microarrays that have an enzyme and a substrate immobilized on their surface wherein the enzyme and the substrate are in proximity to each other sufficient for the occurrence of an enzymatic reaction between the enzyme and the substrate.

We claim:  
     
         1 . A method for assaying an enzymatic reaction, the method comprising: 
 (a) incubating at least one protein and at least one substance under conditions conducive to the occurrence of an enzymatic reaction between the protein and the substance, wherein (i) the protein and the substance are immobilized on the surface of a solid support; (ii) the protein and the substance are in proximity sufficient for the occurrence of said enzymatic reaction; and (iii) the protein and the substance are not identical; and    (b) determining whether said enzymatic reaction occurs.    
     
     
         2 . The method of  claim 1 , wherein the protein, the substance, or the protein and the substance, are purified.  
     
     
         3 . The method of  claim 1 , wherein the substance is a mixture of different substances.  
     
     
         4 . The method of  claim 1 , wherein the method comprises immobilizing a protein and a substance on the surface of the solid support.  
     
     
         5 . The method of  claim 4 , wherein the protein is immobilized prior to immobilizing the substance.  
     
     
         6 . The method of  claim 4 , wherein the substance is immobilized prior to immobilizing the protein.  
     
     
         7 . The method of  claim 4 , wherein the substance and the protein are immobilized simultaneously.  
     
     
         8 . The method of  claim 1 , wherein the substance is a known substrate for the type of enzymatic activity assayed in said enzymatic reaction, and said determining step determines whether said protein is an enzyme having said type of enzymatic activity.  
     
     
         9 . The method of  claim 1 , wherein the protein is an enzyme known to have the type of enzymatic activity assayed in said enzymatic reaction, and said determining step determines whether said substrate is a substrate for said type of enzymatic activity.  
     
     
         10 . The method of  claim 8 , wherein the protein comprises a region that is homologous to the catalytic domain of an enzyme that is known to have the type of enzymatic activity assayed in said enzymatic reaction.  
     
     
         11 . The method of  claim 9 , wherein the enzyme is an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase.  
     
     
         12 . The method of  claim 11 , wherein the enzyme is a kinase.  
     
     
         13 . The method of  claim 1 , wherein (i) the substance is a known substrate for the type of enzymatic activity assayed in said enzymatic reaction; and (ii) the protein is known to catalyze the type of enzymatic activity assayed in said enzymatic reaction.  
     
     
         14 . The method of  claim 13 , wherein said incubating step is done in the presence of one or more test molecules so as to determine whether said test molecules modulate said enzymatic reaction; and said determining step comprises detecting whether a change in the amount of said enzymatic reaction occurs relative to the amount of said enzymatic reaction in the absence of the test molecules.  
     
     
         15 . The method of  claim 14 , wherein said determining step comprises detecting a decrease in the amount of said enzymatic reaction relative to the amount of said enzymatic reaction in the absence of the test molecules, thereby identifying the test molecules as an inhibitor of said enzymatic reaction.  
     
     
         16 . The method of  claim 14 , wherein said determining step comprises detecting an increase in the amount of said enzymatic reaction relative to the amount of said enzymatic reaction in the absence of the test molecules, thereby identifying the test molecules as an activator of said enzymatic reaction.  
     
     
         17 . The method of  claim 13 , wherein the substance is known to be a substrate of said enzyme.  
     
     
         18 . The method of  claim 1 , wherein said at least one protein is one of a plurality of different proteins organized on the surface of the solid support in a positionally addressable array.  
     
     
         19 . The method of  claim 18 , wherein the substance is coated onto the surface of the solid support.  
     
     
         20 . The method of  claim 18 , wherein each protein of the plurality of proteins is in proximity with the substance sufficient for the occurrence of an enzymatic reaction between the protein and the substance, and said determining step comprises determining for at least a portion of said plurality of proteins whether said enzymatic reaction occurs.  
     
     
         21 . The method of  claim 1 , wherein said at least one substance is one of a plurality of different substances organized on the surface of the solid support in a positionally addressable array.  
     
     
         22 . The method of  claim 21 , wherein the protein is coated onto the surface of the solid support.  
     
     
         23 . The method of  claim 21 , wherein each substance of the plurality of different substances is in proximity with the protein sufficient for the occurrence of an enzymatic reaction between the protein and the substance, and said determining step comprises determining for at least a portion of said plurality of different substances whether said enzymatic reaction occurs.  
     
     
         24 . The method of  claim 18 , wherein said plurality consists of between 2 different proteins and 100 different proteins.  
     
     
         25 . The method of  claim 18 , wherein said plurality consists of between 100 different proteins and 1,000 different proteins.  
     
     
         26 . The method of  claim 18 , wherein said plurality consists of between 1,000 different proteins and 10,000 different proteins.  
     
     
         27 . The method of  claim 21 , wherein said plurality consists of between 2 different substances and 100 different substances.  
     
     
         28 . The method of  claim 21 , wherein said plurality consists of between 100 different substances and 1,000 different substances.  
     
     
         29 . The method of  claim 21 , wherein said plurality consists of between 1,000 different substances and 10,000 different substances.  
     
     
         30 . The method of  claim 1 , wherein (i) a first plurality of different substances are organized on the surface of the solid support in a positionally addressable array, and (ii) a second plurality of different proteins are organized on the surface of the solid support in a positionally addressable array.  
     
     
         31 . The method of  claim 30 , wherein said first plurality consists of between 2 different substances and 100 different substances, and said second plurality consists of between 2 different proteins and 100 different proteins.  
     
     
         32 . The method of  claim 30 , wherein said first plurality consists of between 100 different substances and 1,000 different substances, and said second plurality consists of between 100 different proteins and 1,000 different proteins.  
     
     
         33 . The method of  claim 30 , wherein said first plurality consists of between 1,000 different substances and 10,000 different substances, and said second plurality consists of between 100 different proteins and 10,000 different proteins.  
     
     
         34 . The method of  claim 1 , wherein the substance, the protein, or the substance and the protein, are covalently bound to the surface of the solid support.  
     
     
         35 . The method of  claim 1 , wherein the substance, the protein, or the substance and the protein, are non-covalently immobilized to the surface of the solid support.  
     
     
         36 . The method of  claim 1 , wherein the substance, the protein, or the substrate and the protein, are immobilized to the surface of the solid support via a linker.  
     
     
         37 . The method of  claim 1 , wherein the method further comprises quantifying the enzymatic reaction.  
     
     
         38 . The method of  claim 1 , wherein the solid support has at least one well and wherein the well comprises one or more different proteins immobilized on the surface of the solid support within the well.  
     
     
         39 . The method of  claim 38 , wherein the solid support has at least two wells and wherein each well comprises the same one or more different proteins immobilized on the surface of the solid support within the well.  
     
     
         40 . The method of  claim 1 , wherein the solid support has at least one well and wherein each well comprises one or more different substances immobilized on the surface of the solid support within the well.  
     
     
         41 . The method of  claim 40 , wherein the solid support has at least two wells and wherein each well comprises the same set of one or more different substances immobilized on the surface of the solid support within the well.  
     
     
         42 . The method of  claim 1 , wherein said determining step comprises measuring a change in a detectable signal resulting from said enzymatic reaction.  
     
     
         43 . A positionally addressable array comprising at least one known enzyme and at least one candidate substrate of the enzyme, wherein (i) the enzyme and the substrate are immobilized on the surface of a solid support; (ii) the enzyme and the substrate are in proximity sufficient for the occurrence of the enzymatic reaction catalyzed by the enzyme between the enzyme and the substrate; and (iii) the enzyme and the substrate are not identical to each other.  
     
     
         44 . A positionally addressable array comprising at least one known substrate of an enzymatic reaction and at least one candidate enzyme for the catalysis of the enzymatic reaction, wherein (i) the enzyme and the substrate are immobilized on the surface of a solid support; (ii) the enzyme and the substrate are in proximity sufficient for the occurrence of the enzymatic reaction between the enzyme and the substrate; and (iii) the enzyme and the substrate are not identical to each other.  
     
     
         45 . A positionally addressable array comprising at least one known substrate of an enzymatic reaction and at least one enzyme that is known to catalyze the enzymatic reaction, wherein (i) the enzyme and the substrate are immobilized on the surface of a solid support; (ii) the enzyme and the substrate are in proximity sufficient for the occurrence of the enzymatic reaction between the enzyme and the substrate; and (iii) the enzyme and the substrate are not identical to each other.  
     
     
         46 . The positionally addressable array of  claim 43 ,  44 , or  45 , further comprising a reaction mixture, said reaction mixture being (i) in contact with the enzyme and the substrate, and (ii) conducive to the occurrence of the enzymatic reaction.  
     
     
         47 . The positionally addressable array of  claim 43 ,  44 , or  45 , wherein the enzyme, the substrate, or the enzyme and the substrate, are purified.  
     
     
         48 . The positionally addressable array of  claim 43 ,  44 , or  45 , wherein the surface of the solid support is coated with the enzyme or with the substrate.  
     
     
         49 . The positionally addressable array of  claim 43 ,  44 , or  45 , wherein said substrate is one of a plurality of different substrates.  
     
     
         50 . The positionally addressable array of  claim 49 , wherein each different substrate is in proximity with an enzyme sufficient for the occurrence of the enzymatic reaction between the substrate and the enzyme.  
     
     
         51 . The positionally addressable array of  claim 43 ,  44 , or  45 , wherein said enzyme is one of a plurality of different enzymes.  
     
     
         52 . The positionally addressable array of  claim 51 , wherein each different enzyme is in proximity with a substrate sufficient for the occurrence of the enzymatic reaction between the substrate and the enzyme.  
     
     
         53 . The positionally addressable array of  claim 43 ,  44 , or  45 , wherein said enzyme is one of a plurality of different enzymes and said substrate is one of a plurality of different substrates.  
     
     
         54 . A positionally addressable array comprising at least one known enzyme immobilized on the surface of a solid support, wherein the surface is modified to allow for the immobilization of a candidate substrate of the enzymatic reaction catalyzed by said enzyme in proximity with the enzyme sufficient for the occurrence of the enzymatic reaction between the enzyme and the substrate.  
     
     
         55 . A positionally addressable array comprising at least one substrate of an enzymatic reaction immobilized on the surface of a solid support, wherein the surface is modified to allow for the immobilization of a candidate enzyme capable of catalyzing said enzymatic reaction in proximity with the substrate sufficient for the occurrence of the enzymatic reaction between the enzyme and the substrate.  
     
     
         56 . A kit comprising the positionally addressable array of  claim 43 ,  44 , or  45 , and a container comprising a reaction mixture conducive to the occurrence of the enzymatic reaction.