Reagents And Methods for Detecting Protein Lysine 3-Hydroxybutyrylation

Abstract

The invention provides an isolated peptide comprising a lysine 3-hydroxybutyrylation site, a lysine 3-hydroxybutyrylation specific affinity reagent that specifically binds to the peptide, and a method for detecting protein lysine 3-hydroxybutyrylation in a sample using the reagent.

What is claimed: 
     
         1 . An isolated peptide comprising a 3-hydroxybutyrylated lysine. 
     
     
         2 . The isolated peptide of  claim 1 , wherein the peptide is derived from a histone protein or a fragment thereof. 
     
     
         3 . The isolated peptide of  claim 2 , wherein the histone protein is derived from an organism selected from the group consisting of human, mouse,  S. cerevisiae, Tetrahymena thermophila, D. melanogaster , and  C. elegans.    
     
     
         4 . The isolated peptide of  claim 1 , wherein the peptide comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-102. 
     
     
         5 . The isolated peptide of  claim 1 , wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 29-102. 
     
     
         6 . The isolated peptide of  claim 1 , wherein the peptide comprises at least 2 amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine. 
     
     
         7 . An isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to the peptide of  claim 1 . 
     
     
         8 . The isolated lysine 3-hydroxybutyrylation specific affinity reagent of  claim 7 , wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 29-102. 
     
     
         9 . The isolated lysine 3-hydroxybutyrylation specific affinity reagent of  claim 7 , wherein the binding is dependent on the presence of the 3-hydroxybutyrylated lysine but not a surrounding peptide sequence thereof in the peptide. 
     
     
         10 . The isolated lysine 3-hydroxybutyrylation specific affinity reagent of  claim 7 , wherein the binding is dependent on the presence of the 3-hydroxybutyrylated lysine and a surrounding peptide sequence thereof in the peptide. 
     
     
         11 . The isolated lysine 3-hydroxybutyrylation specific affinity reagent of  claim 7 , wherein the lysine 3-hydroxybutyrylation specific affinity reagent is a protein. 
     
     
         12 . A method for producing the lysine 3-hydroxybutyrylation specific affinity reagent of  claim 11 , comprising screening a protein library using a peptide comprising a 3-hydroxybutyrylated lysine and at least two amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine. 
     
     
         13 . The isolated lysine 3-hydroxybutyrylation specific affinity reagent of  claim 7 , wherein the lysine 3-hydroxybutyrylation specific affinity reagent is an antibody. 
     
     
         14 . A method for producing the lysine 3-hydroxybutyrylation specific affinity reagent of  claim 13 , comprising immunizing a host with a peptide comprising a 3-hydroxybutyrylated lysine and at least two amino acid residues on each of the N-terminal and C-terminal sides of the 3-hydroxybutyrylated lysine. 
     
     
         15 . A method for detecting a 3-hydroxybutyrylated lysine in a protein or a fragment thereof, comprising:
 (a) contacting the protein or a fragment thereof with the isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to the peptide of  claim 1 , whereby the lysine 3-hydroxybutyrylation specific affinity reagent and the protein or a fragment thereof forms a binding complex, and   (b) detecting the binding complex, wherein the presence of the binding complex indicates the presence of a 3-hydroxybutyrylated lysine in the protein or a fragment thereof.   
     
     
         16 . The method of  claim 15 , wherein the lysine 3-hydroxybutyrylation specific affinity reagent is a protein. 
     
     
         17 . The method of  claim 15 , wherein the lysine 3-hydroxybutyrylation specific affinity reagent is an antibody. 
     
     
         18 . A method for determining the level of protein lysine 3-hydroxybutyrylation in a sample, comprising detecting a 3-hydroxybutyrylated lysine in the sample. 
     
     
         19 . A kit for detecting a 3-hydroxybutyrylated lysine in a protein of a fragment thereof, comprising the isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to the peptide of  claim 1 . 
     
     
         20 . A kit for isolating a peptide containing a 3-hydroxybutyrylated lysine, comprising the isolated lysine 3-hydroxybutyrylation specific affinity reagent capable of binding specifically to the peptide of  claim 1 .