Proteomic analysis
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 method for screening for the bloactivity 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 comprising a reactive functionality group specific for said group of target proteins and a ligand, 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 alkylene, oxyalkylene or polyoxyalkylene linking group, wherein said oxyalkylenes are of from 2 to 3 carbon atoms;
F is a phosphonate or sulfonyl functional group reactive at an active site of a target enzyme; and
R is bonded to F and a moiety of less than 1 kDal providing specific affinity for said enzymes, and when F is phosponate, R is fluorine and when F is sulfonyl, R is an aryl or heteroaryl group; said method comprising:
separating the proteomic mixture into two portions,
treating one of the two portions with the candidate compound to obtain an inactivated portion, wherein the candidate compound is a non-covalent agent,
combining at least one probe with the untreated portion of said mixture under conditions for reaction with said target proteins to obtain a first specimen,
combining at least one probe with the inactivated portion under conditions for reaction with said target proteins to obtain a second specimen;
sequestering proteins conjugated with said at least one probe from each of said specimens;
determining the proteins that are sequestered; and
comparing the amount of each of the proteins sequestered from the first specimen and the second specimen as indicative of the bioactivity of said candidate compound with said target proteins.
2. A method according to claim 1 , wherein said probe is a fluorophosphonyl and said enzymes are serine hydrolases.
3. A method according to claim 1 , wherein said probe is a sulfonate, R is a heteroaryl and said enzymes are aldehyde dehydrogenases.
4. A method according to claim 3 , wherein said heteroaryl is pyridyl.
5. A method according to claim 1 , wherein X is biotin.