Protein interaction reporter agents and methods for using same

Abstract

Particular aspects provide novel protein interaction reporter (PIR) compounds (e.g., formulas I and II), comprising at least two protein reactive moieties (e.g., N-hydroxysuccinamide), each linked to a reporter moiety (e.g., mass reporter) by a covalent labile bond that is differentially cleavable with respect to peptide bonds (e.g., by a method such as collisional activation in a mass spectrometer, activation by electron capture dissociation (ECD), photoactivation, etc.), wherein the reporter moiety is operatively releasable from the PIR agent upon cleavage of the labile bonds, the released reporter moiety having a characteristic identifying property or label (e.g., m/z value). Particular PIRs comprise a mass reporter moiety, and further comprise an affinity group, (e.g., biotin), linked to the PIR (e.g., to the mass reporter moiety) by a selectively cleavable bone (e.g. photo-labile bond)). Additional aspects provide methods for characterizing intermolecular or intramolecular protein interactions using one or more inventive PIR compounds.

1 .- 28 . (canceled) 
     
     
         29 . A method of identifying proteins participating in an intermolecular or intramolecular protein interaction within a mass spectrometry (MS) experiment comprising:
 a) obtaining a sample comprising at least one protein;   b) contacting the at least one protein with at least one multivalent protein interaction reporter (PIR) compound, under conditions suitable for cross-linking to provide a cross-linked protein sample product, wherein the PIR compound comprises at least two protein reactive moieties, each bound to a shared characteristic reporter moiety by a labile bond, wherein the two labile bonds can be differentially relative to peptide bonds cleaved under gas phase mass spectrometer (MS) conditions with respect to peptide bonds;   c) subjecting the cross-linked protein sample product to an MS analysis suitable to obtain a mass of the cross-linked protein sample product;   d) differentially cleaving the two labile bonds of the cross-linked protein sample in a gas phase within a mass spectrometer to provide for cleavage products;   e) subjecting the cleavage products to an MS analysis suitable to identify the mass of the cleavage products;   f) analyzing the mass of the cleavage products to identify the mass of the protein participating in an intermolecular or intramolecular protein interaction by identifying the mass of the reporter moiety and which masses in the mass spectrum of the cleavage products add thereto to yield the mass of the cross-linked protein sample;   g) determining a polypeptide sequence associated with the protein participating in an intermolecular or intramolecular protein interaction by comparing the polypeptide sequence to one or more known polypeptide or protein sequences.   
     
     
         30 . The method of  claim 29 , comprising, prior to c), digesting the cross-linked protein sample with at least one protease. 
     
     
         31 . The method of  claim 29  comprising, prior to c), concentrating the cross-linked protein sample through the use of an affinity or reactive group. 
     
     
         32 . The method of  claim 31  wherein the affinity or reactive group is a biotin. 
     
     
         33 . The method of  claim 31  wherein the affinity or reactive group is a photochemical labile group. 
     
     
         34 . The method of  claim 30  further comprising concentrating the cross-linked protein sample through the use of an affinity or reactive group after digestion and before c). 
     
     
         35 . The method of  claim 34  wherein the affinity or reactive group is a biotin. 
     
     
         36 . The method of  claim 34  wherein the affinity or reactive group is a photochemical labile group. 
     
     
         37 . The method of  claim 29 , wherein the differentially cleavable labile bond is cleaved by a method selected from the group consisting of collisional activation, activation by electron capture dissociation (ECD), photoactivation and combinations thereof. 
     
     
         38 . The method of  claim 29  comprising, prior to g), subjecting at least one cleavage product to a second, higher ms activation energy sufficient to fragment peptide bonds. 
     
     
         39 . The method of  claim 29  wherein the sample containing the at least one protein is a cell lysate. 
     
     
         40 . The method of  claim 29  wherein the sample containing the at least one protein is a cell. 
     
     
         41 . The method of  claim 40  wherein the unique characteristic reporter moieties have different solubility characteristics. 
     
     
         42 . The method of  claim 29  comprising, prior to g), determining the number of fragments in the mass spectrum of the cleavage products that are added to the mass of the reporter moiety to yield the mass of the cross-linked protein sample to determine if the cross-linked protein sample demonstrate an intermolecular or an intramolecular protein interaction. 
     
     
         43 . The method of  claim 42  further comprising identification of the mass of the of the reporter moiety to identify a dead-end. 
     
     
         44 . The method of  claim 29 , comprises subjecting the uncleaved and cleaved products to an analysis suitable to identify the cleavage products derived from the uncleaved products by virtue of being the reporter moiety or by inclusion of a protein reactive moiety or portion thereof. 
     
     
         45 . The method of  claim 29 , wherein the at least one multivalent protein interaction reporter (PIR) compound is a compound having the Formula I: 
       
         
           
           
               
               
           
         
         wherein: R 1  and R 2  are independently H, CH 3 , or CH 2 —CH 3 ; n=1-6; 
       
       
         
           
           
               
               
           
         
         wherein R 4  is hydroxyl, or 
         wherein A and B are independently selected from: 
       
       
         
           
           
               
               
           
         
         and wherein X is selected from the following: 
       
       
         
           
           
               
               
           
         
         wherein T 1 -T 12  are independently hydrogen, (C 1 -C 4 )-alkyl, or (C 1 -C 4 )-alkoxy. 
       
     
     
         46 . The method of  claim 29 , wherein the at least one multivalent protein interaction reporter (PIR) compound is a compound having the Formula II: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are independently selected from hydrogen, or (C 1 -C 4 )-alkyl; 
         wherein: n=1-6; 
         wherein A and B are independently selected from: 
       
       
         
           
           
               
               
           
         
         and W and X are independently selected from: 
       
       
         
           
           
               
               
           
         
         wherein T 1 -T 12  are independently hydrogen, (C 1 -C 4 )-alkyl, or (C 1 -C 4 )-alkoxy; 
         wherein Y is nothing or 
       
       
         
           
           
               
               
           
         
         wherein Z is: (—CH 2 —CH 2 —O—) m ; (—CH 2 —) q ; (—CO—CH 2 —) r ; (—CH 2 —CO 2 —) r ; (—CH 2 —CO—CH 2 —CO 2 —), 
         wherein m=1-5, q=5-15, r=3-7 and s=1-4; and wherein U is biotin, poly-histidine (6-10 residues), benzophenone, sulfhydryl, or aryl azide. 
       
     
     
         47 . The method of  claim 29 , wherein step b comprises contacting with a plurality of distinguishable protein interaction reporter (PIR) compounds. 
     
     
         48 . The method of  claim 47 , wherein at least one of the distinguishable protein interaction reporter (PIR) compounds is of formula I of  claim 45 . 
     
     
         49 . The method of  claim 47 , wherein at least one of the distinguishable protein interaction reporter (PIR) compounds is of formula II of  claim 46 . 
     
     
         50 . The method of  claim 29 , wherein the labile bonds, in each case, comprise a carbon-nitrogen bond that can be differentially cleaved with respect to peptide bonds. 
     
     
         51 . The method of  claim 50 , wherein the carbon atom of the differentially cleavable carbon-nitrogen bond, in each case, is adjacent to a moiety selected from the moiety group consisting of aromatic moieties, N-functionalized heterocyclic aromatic moieties, substituted biphenyl compounds, dithiols, phosphates, metal ligand complexes, and other moieties with significant π-delocalization. 
     
     
         52 . The method of  claim 51 , wherein the N-functionalized heterocyclic aromatic moiety is selected from the group consisting of substituted or unsubstituted indole moieties, pyridyl-based moieties, and combinations thereof. 
     
     
         53 . The method of  claim 50 , wherein the nitrogen atom of the differentially cleavable carbon-nitrogen bond, in each case, comprises a secondary or tertiary amine moiety.