Method for isolating subpopulations of proteins that engage in protein-protein interactions
The invention provides a method for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a chemically reactive supporting matrix to isolate proteins that in turn non-covalently bind other proteins. The supporting matrix is isolated, and the non-covalently bound proteins are subsequently released for analysis. Because the proteins are accessible to chemical manipulation at both the binding and release steps, identification of the non-covalently bound proteins yields information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The method has the advantage of screening the entire proteome simultaneously, unlike two-hybrid systems or phage display methods which can only detect proteins binding to a single bait protein at a time. The method is applicable to the study of protein-protein interactions in biopsy and autopsy specimens, to the study of protein-protein interactions in the presence of signalling molecules, pharmacological agents or toxins, and for comparison of diseased and normal tissues or cancerous and untransformed cells.
1 . A method of isolating, from a mixture of proteins, a subpopulation consisting essentially of proteins that engage in protein-protein interactions, comprising:
(a) contacting the mixture with a chemically reactive support under conditions that permit
(i) covalent binding of proteins to the support, and
(ii) protein-protein interactions;
(b) permitting proteins in the mixture to become covalently bound to the support;
(c) separation of the support from any proteins not bound thereto;
(d) subjecting the support to conditions that disrupt protein-protein interactions; and
(e) separating the support from any proteins not bound thereto.
2 . The method of claim 1 , wherein the chemically reactive support comprises chemically reactive moieties selected from the group consisting of: cyanate groups, isocyanate groups, isothiocyanate groups, activated carboxyl groups, activated sulfonyl groups, aldehyde groups, epoxide groups, and thiol groups.
3 . The method of claim 2 , wherein the chemically reactive support comprises cyanate groups.
4 . The method according to any one of claims 1 - 3 , wherein the support comprises an optionally cross-linked polymer or gel.
5 . The method of claim 4 , wherein the support comprises a material selected from the group consisting of polystyrene, agar, agarose, polyacrylamide, dextran, hydroxylated vinyl polymers, and carboxylated vinyl polymers.
6 . The method of claim 5 , wherein the support comprises agarose.
7 . In a method for analyzing a mixture of proteins, which comprises contacting said mixture with an array of immobilized proteins, the improvement which consists of isolating, from said mixture of proteins, a subpopulation consisting essentially of proteins that engage in protein-protein interactions, and subsequently contacting said subpopulation with said array, wherein the method of isolating the subpopulation comprises:
(a) contacting the mixture with a chemically reactive support under conditions that permit
(i) covalent binding of proteins to the support, and
(ii) protein-protein interactions;
(b) permitting proteins in the mixture to become covalently bound to the support;
(c) separation of the support from any proteins not bound thereto;
(d) subjecting the support to conditions that disrupt protein-protein interactions; and
(e) separating the support from any proteins not bound thereto.