Proteomic analysis

Abstract

The present invention provides methods for analyzing proteomes, as cells or lysates. The analysis is based on the use of probes that have specificity to the active form of proteins, particularly enzymes and receptors. The probes can be identified in different ways. In accordance with the present invention, a method is provided for generating and screening compound libraries that are used for the identification of lead molecules, and for the parallel identification of their biological targets. By appending specific functionalities and/or groups to one or more binding moieties, the reactive functionalities gain binding affinity and specificity for particular proteins and classes of proteins. Such libraries of candidate compounds, referred to herein as activity-based probes, or ABPs, are used to screen for one or more desired biological activities or target proteins.

1 . A composition comprising at least one covalent product of a target protein member of a complex protein composition and at least one activity based probe (“ABP”) member of combinatorial chemical library comprising a plurality of members of the formula  
         R*(F-L)-X  
       wherein: 
 X is a ligand present prior to formation of said product or added to a reactive functionality to provide said ligand, said ligand having the same chemical structure for each of said members of said library;  
 L is a bond or linking group, which is the same in each of the members of said library;  
 F is a functional group reactive at an active site of a protein member, which functional group comprises the same reactive functionality in each of the members of said library; and  
 R is a group of less than 1 kdal, that is different in each of the members of the library;  
 the * intends that r is a part of F or L; and 
 wherein members of said library have different on rates with said protein member.  
 
 
     
     
         2 . The composition of  claim 1 , wherein F is an acyl group and R is a leaving group having at least one of different size, conformation and charge distribution in different members of said library.  
     
     
         3 . The composition of  claim 1 , wherein the library contains at least 10 different members differing in the R group.  
     
     
         4 . The composition of  claim 1 , wherein F is selected from the group consisting of an alkylating functionality, an acylating functionality, a ketone functionality, an epoxide functionality an aldehyde functionality, a sulphonyl functionality and a phosphoryl functionality.  
     
     
         5 . The composition of  claim 4 , wherein at least one of the ligand and the linking group for each member of the library comprises a different unnatural amount of at least one stable isotope.  
     
     
         6 . The composition of  claim 1  wherein the ligand produces an electromagnetic signal.  
     
     
         7 . The composition of  claim 1  wherein X is an oligonucleotide.  
     
     
         8 . The composition of  claim 1  wherein X is an encoding particle.  
     
     
         9 . The composition of  claim 1 , wherein said target protein is an enzyme and is a member of a group of enzymes having thc same catalytic activity.  
     
     
         10 . The composition of  claim 9 , wherein said enzymes are hydrolases.  
     
     
         11 . The composition of  claim 1 , wherein the linker is a cleavable linker.  
     
     
         12 . A composition comprising at least one covalent product of a target enzyme of a complex protein composition and at least one activity based probe member of combinatorial chemical library comprising a plurality of members of the formula  
         R(F-L)-X  
       wherein: 
 X is a ligand present prior to formation of said product or added to a reactive functionality to provide said ligand, said ligand having the same chemical structure for each of said members of said library;  
 L is a bond or linking group, which is the same in each of the members of said library;  
 F is a sulphonyl group reactive at an active site of a target enzyme; and  
 R is an organic group of less than 1 kDa1, that is different in each of the members of the library and is bonded to F; and  
 wherein members of the library have different on rates with said target enzyme.  
 
     
     
         13 . A composition according to  claim 12 , where in R is selected from the group consisting of alkyl, cycloalkyl, heterocycle and aryl and substituted members thereof.  
     
     
         14 . The composition of  claim 1 , wherein F is an epoxide.  
     
     
         15 . A combinatorial chemical library comprising a plurality of members of the formula  
         R(F-L)-X  
       wherein: 
 X is a ligand for binding to a reciprocal receptor;  
 L is a bond or linking group, which is the same in each of the members of the library;  
 F is a sulphonyl group reactive at an active site of a target enzyme; and  
 R is an organic group of less than 1 kDa1, that is different in each of the members of the library and is bonded to F; and  
 wherein members of the library have different on rates with said target enzyme.  
 
     
     
         16 . A combinatorial chemical library comprising a plurality of members of the formula  
         R(F-L)-X  
       wherein: 
 F is sulfonyl;  
 L is an alkylene linking group comprising from 1-3 amido groups or polyoxyalkylene;  
 X is biotin; and  
 R is bonded to F to form a sulphonate ester and is aryl or heteroaryl.  
 
     
     
         17 . A combinatorial library according to  claim 15 , wherein for one member R, said heteroaryl is pyridyl or thiophenyl.  
     
     
         18 . A combinatorial chemical library comprising a plurality of members of the formula  
         R(F-L)-X  
       wherein: 
 X is a ligand for binding to a reciprocal receptor;  
 L is a linking group;  
 F is a sulphonyl group reactive at an active site of a target enzyme; and  
 R is an organic group of less than 1 kdal, that is different in each of the members of the library and is in the chain of L; and  
 wherein members of the library have different on rates with said target enzyme.  
 
     
     
         19 - 28 . (canceled)  
     
     
         29 . A method of identifying activity-based probes (“ABPs”) having specific affinities for members of a protein genus having a common active site, wherein said ABPs comprise a functionality specific for said protein genus, a ligand for binding to a bound receptor, a linker and an affinity moiety, wherein said ABPs comprise a family of linkers, said method comprising: 
 combining candidate ABPs with at least one active member of said protein genus under conditions where activity-based probes bond to the active site of said member to form a conjugate;    combining candidate ABPs with said member, inactivated by other than covalent bonding, under the same conditions as with the active member;    isolating any conjugates formed with said active member and said inactived member by means of said ligand binding to said receptor;    determining the amount of conjugate formed with said inactived member and rejecting said candidate ABP if the amount of conjugate exceeds a predetermined level;    for an ABP candidate that is not rejected, identifying said probe, whereby an ABP having a specific affinity for said member is identified.    
     
     
         30 . A method according to  claim 29 , where said affinity moiety is an oligomer of at least one of natural and/or unnatural amino acids and nucleotides.  
     
     
         31 . A method according to  claim 29 , wherein said inactivated member is inactivated by thermal denaturation.  
     
     
         32 . A method according to  claim 29 , wherein said enzyme is a serine hydrolase and said  
       active functionality is fluorophosphorous acid derivative, or said enzyme is a cysteine hydrolase and said active functionality is an □-halocarbonyl, or said enzyme is an enzyme having cysteine, histidine, aspartate or glutamate at its active site and said active functionality is a sulfonate diester or an epoxide, or said enzyme is a redox enzyme and said active functionality is an alkyne, or said enzyme is a metalloenzyme and said functionality is an □-halohydroxamic acid.  
     
     
         33 . A system for identifying activity-based probes (“ABPs”) for target protein members of a proteome from a combinatorial library of candidate ABPs, said system comprising: 
 a combinatorial chemical library of candidate ABPs comprising a plurality of members of the formula      R*(F-L)-X    wherein:    X is a ligand present prior to formation of said product or added to a reactive functionality to provide said ligand, said ligand having the same chemical structure for each of said members of said library;    L is a bond or linking group, which is the same in each of the members of said library;    F is a functional group reactive at an active site of a protein member, which functional group comprises the same reactive functionality in each of the members of said library; and    R is a group of less than 1 kDa1, that is different in each of the members of the library;    the * intends that R is a part of F or L; and 
 wherein members of said library have different on rates with said protein member; at least one group of related target proteins;  
 a programmed data processor for receiving and transmitting values, comprising a program for evaluating results from the combining of said combinatorial library and said target proteins based on the formation of conjugates of said ABPs and said target proteins to determine the affinity of each of said ABPs for each of said target proteins, and providing a profile of the affinity of each of said ABPs for said target proteins of interest;  
   employing a method for obtaining said results, said method comprising:    combining under binding conditions said ABPs and said target proteins, whereby said ABPs bind to said target proteins in relation to the affinity of said ABPs to said target proteins;    determining the amount of conjugate of each ABP for each target protein as the results for said data processor;    feeding the results to said data processor; and    transmitting the values for said binding of each of said ABPs to identify ABPs forming conjugates.    
     
     
         34 . A system according to  claim 33 , wherein said values are transmitted as a dendrogram.  
     
     
         35 . A system according to  claim 34 , wherein said dendrogram is provided with a color scheme where the color and/or brightness is related to said binding.  
     
     
         36 . A system according to  claim 33 , wherein said values are transmitted as abundance ratios.  
     
     
         37 . A system for identifying activity-based probes (“ABPs”) from a combinatorial library of candidate compounds, employing the results from combining a combinatorial library of candidate ABPs comprising a plurality of members of the formula  
         R*(F-L)-X  
       wherein: 
 X is a ligand present prior to formation of said product or added to a reactive functionality to provide said ligand, said ligand having the same chemical structure for each of said members of said library;  
 L is a bond or linking group, which is the same in each of the members of said library;  
 F is a functional group reactive at an active site of a protein member, which functional group comprises the same reactive functionality in each of the members of said library; and  
 R is a group of less than 1 kdal, that is different in each of the members of the library;  
 the * intends that R is a part of F or L; and 
 wherein members of said library have different on rates with said protein member, and at least one group of related target proteins under conditions for reaction of candidate ABPs with target proteins in relation to the affinity of the candidate ABPs with a target protein to form conjugates and determining the amount of each conjugate and/or relative abundance of each conjugate, said system comprising:  
 a programmed data processor for receiving and transmitting data comprising a program for analyzing the amount of each conjugate and/or the relative abundance of each conjugate and determining the on rates of said candidate ABPs with said target proteins.  
 
 
     
     
         37 . A system according to  claim 36 , wherein said values are transmitted as a dendrogram.  
     
     
         38 . A system according to  claim 37 , wherein said dendrogram is provided with a color scheme where the color and/or brightness is related to said on rate.  
     
     
         39 . A system according to  claim 36 , wherein said values are transmitted as abundance ratios.