Linkers for protein interaction profiling and methods of making and using the same

Abstract

Crosslinking compounds for effective and efficient cross-linking and identification of intermolecular and intramolecular interactions of proteins, peptides and nucleic acids.

1 . A crosslinking compound having the chemical structure:
   Z-A n -Z   
       wherein:
 n is an integer between 1 and 5; 
 A is an atom or compound; and, 
 each Z is a Michael acceptor, and 
 A is optionally linked to at least one of an affinity handle (Q), and a molecular label (L). 
 
     
     
         2 . The crosslinking compound of  claim 1 , wherein A is phosphorus (P), sulfur (S), nitrogen (N), oxygen (O), C 1-10  alkyl optionally substituted with a reactive group or an affinity group, cycloalkyl, or heteroalkyl or heterocycloalkyl wherein the heteroatoms are selected from at least one of phosphorus (P), sulfur (S), nitrogen (N), or oxygen (O). 
     
     
         3 - 6 . (canceled) 
     
     
         7 . The crosslinking compound of  claim 2 , wherein A is pentane or hexane. 
     
     
         8 . The cross-linking compound of  claim 1 , wherein the Michael acceptor (Z) is selected from the group consisting of a chemical moiety conjugated to a protein, peptide or a nucleic acid, an electron-deficient alkyne, and a chemical moiety that forms a covalent bond with accessible ε-amino groups on lysine residues present in proteins or peptides. 
     
     
         9 - 12 . (canceled) 
     
     
         13 . The cross-linking compound of  claim 1 , comprising at least two (Z) and wherein at least one Michael acceptor (Z) is capable of forming a covalent bond with a protein or peptide and at least one Michael acceptor (Z) is capable of forming a covalent bond with a nucleic acid. 
     
     
         14 . The cross-linking compound of  claim 1 , comprising at least two (Z) and wherein each Michael acceptor (Z) is the same chemical moiety. 
     
     
         15 . The cross-linking compound of  claim 1 , comprising at least two (Z) and wherein each Michael acceptor (Z) is a unique chemical moiety. 
     
     
         16 . The cross-linking compound of  claim 1 , wherein each Michael acceptor (Z) is selected from the group consisting of propiolate, propiolamide, yn-one, ynethiolate, acrylate, vinylsulfone, and propargylamide. 
     
     
         17 . The cross-linking compound of  claim 1 , comprising an affinity handle (Q) that is a chemical moiety that facilitates enrichment of crosslinker-modified species from a sample selected from the group consisting of
 a chemical moiety that facilitates precipitation or separation of the cross-linking compound from a sample;   at least one of biotin, a histidine residue, and PEG; and,   at least one of an amino acid sequence, polyhistidine, an antibody fragment, and a nucleic acid sequence.   
     
     
         18 - 21 . (canceled) 
     
     
         22 . The cross-linking compound of  claim 1 , comprising a molecular label (L) that is at least one chemical entity that renders the crosslinking compound distinguishable by physical means due to the presence of the label, selected from the group consisting of phosphors, radioactive atoms, atomic isotopes, fluorescent dyes, electron-dense reagents, enzymes, biotin, digoxigenin, haptens or proteins made detectable by incorporating a metal, radiolabel or phosphor into the peptide, hydrolases, phosphatases, esterases and glycosidases, or oxidotases, peroxidases, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferin, 2,3-dihydrophthalazinediones, and a stable isotope. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . A cross-linking compound, having the chemical structure
   Z-Y-A n -Y-Z   wherein   each Z is a Michael acceptor,   A is an atom or compound;   n is an integer between 1 and 5; and,   each Y is independently an organic compound of C 1-10  alkyl, optionally substituted with one or more heteroatoms selected from O, N, S, or P.   
     
     
         26 . The cross-linking compound of  claim 25 , wherein each linking group (Y) independently comprises one or more cleavage sites cleaved by at least one of a chemical cleavage agent, an enzymatic cleavage agent, or both chemical cleavage agents and enzymatic cleavage agents. 
     
     
         27 . The cross-linking compound of  claim 25 , wherein each linking group (Y) comprises an affinity handle (Q), or a molecular label (L). 
     
     
         28 . The cross-linking compound of  claim 25 , wherein each linking group (Y) comprises one or more collision-induced dissociation groups which comprise signature ions formed upon collision-induced dissociation in mass spectrometric methods. 
     
     
         29 . (canceled) 
     
     
         30 . The cross-linking compound of  claim 28 , wherein the collision-induced dissociation groups comprise fixed-charge sulfonium ion derivatives of the amino acids methionine and cysteine, which fragment exclusively via neutral loss of the side chain CH 3 SR, where R is a substituted alkyl group. 
     
     
         31 . The cross-linking compound of  claim 25  having a molecular length of between about 1 angstrom and about 50 angstroms. 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . The cross-linking compound of  claim 1 , comprising a dipropiolate. 
     
     
         35 . The cross-linking compound of  claim 1 , comprising at least one of butane-1,4-diyl dipropiolate; oxybis(ethane-2,1-diyl) dipropiolate; and di(cylcohexyl-2,1-diyl) dipropriolate. 
     
     
         36 - 59 . (canceled) 
     
     
         60 . The crosslinking compound of  claim 25 , wherein A is phosphorus (P), sulfur (S), nitrogen (N), oxygen (O), C 1-10  alkyl optionally substituted with a reactive group or an affinity group, cycloalkyl, or heteroalkyl or heterocycloalkyl wherein the heteroatoms are selected from at least one of phosphorus (P), sulfur (S), nitrogen (N), or oxygen (O). 
     
     
         61 . The cross-linking compound of  claim 25 , wherein the Michael acceptor (Z) is selected from the group consisting of a chemical moiety conjugated to a protein, peptide or a nucleic acid, an electron-deficient alkyne, and a chemical moiety that forms a covalent bond with accessible ε-amino groups on lysine residues present in proteins or peptides.