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.

What is claimed is:  
     
         1 . A method for screening for the bioactivity of a candidate compound toward a group of related target proteins in a proteomic mixture of proteins from a cell, employing at least one probe, each probe characterized by comprising a reactive functionality group specific for said group of target proteins and a ligand and said probe, said method comprising: 
 combining at least one probe with an untreated portion of said mixture and with a portion inactivated with a non-covalent agent under conditions for reaction with said target proteins;    sequestering proteins conjugated with said at least one probe from each of said mixtures;    determining the proteins that are sequestered; and    comparing the amount of each of the proteins sequestered from the untreated portion and the inactivated portion as indicative of the bioactivity of said candidate compound with said target proteins.    
     
     
         2 . A method according to  claim 1 , wherein said activity-based probe(s) have a reciprocal receptor and said separating is by binding said probe(s) to said reciprocal receptor bound to a support.  
     
     
         3 . A method according to  claim 1 , wherein said activity-based probe is detectable as a result of an electromagnetic signal.  
     
     
         4 . A method according to  claim 1 , wherein said activity-based probe(s) are of the formula:  
       R*(F—L)—X  
       wherein: 
 X is a ligand for binding to a reciprocal receptor or a chemically reactive functionality for reacting with a reciprocal functionality to add a ligand;  
 L is a linking group;  
 F is a functional group reactive at an active site of a target enzyme; and  
 R is H or a moiety of less than 1 kDal providing specific affinity for said target enzymes;  
 the * intends that R is a part of F or L.  
 
     
     
         5 . A method according to  claim 4 , wherein F is a sulphonyl group and R is other than H and bonded to F.  
     
     
         6 . A method according to  claim 4 , wherein F is a fluorophosphonyl or fluorophosphoryl group.  
     
     
         7 . A method according to  claim 1 , wherein at least one of L and X comprise at least one isotope in unnatural amount, and including the additional step of: 
 releasing at least a portion of said probe from said conjugate and identifying said portion by means of isotopic difference.    
     
     
         8 . A method for screening for the bioactivity of a candidate compound toward a group of related target enzymes in a proteomic mixture of proteins employing at least one activity-based probe, each probe of the formula:  
       R*(F—L)—X  
       wherein: 
 X is a ligand for binding to a reciprocal receptor and/or providing a detectable signal;  
 L is an aliphatic linking group;  
 F is a functional group reactive at an active site of a target enzyme; and  
 R is H or a moiety of less than lkDal providing specific affinity for said enzymes;  
 the * intends that R is a part of F or L;  
 said method comprising: 
 combining at least one said probe with a first portion of said proteomic mixture, under conditions for reaction with said target enzymes;  
 combining at least one said probe with a second portion of said proteomic mixture and with a candidate compound under conditions for reaction with said target enzymes;  
 separating enzymes conjugated with said at least one said probe from each of said portions of said proteomic mixture; and  
 comparing the amount of each of the conjugates from the first portion with the amount of each of the conjugates from the second portionas indicative of the bioactivity of said candidate compound with said target enzymes.  
 
 
     
     
         9 . A method according to  claim 8 , wherein F is a sulphonyl group and R is other than H and bonded to F.  
     
     
         10 . A method according to  claim 8 , wherein F is a fluorophosphonyl or fluorophosphoryl group.  
     
     
         11 . A method according to any of claims  1 - 4 ,  6 - 8  or  10  wherein said activity-based probe(s) are FP-biotin.  
     
     
         12 . A method according to any of claims  1 - 4 ,  6 - 8  or  10  wherein said activity-based probe(s) are FP-peg-biotin.  
     
     
         13 . A method according to any of claims  5  or  9  wherein said activity-based probe(s) are selected from the group consisting of Sulfonate 1-Sulfonate 17.  
     
     
         14 . A method according to  claim 13  wherein said activity-based probe(s) are Sulfonate 15.