Reagents and methods for detecting protein crotonylation

Abstract

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

What is claimed: 
     
         1 . An isolated peptide comprising a crotonylation site. 
     
     
         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 1 , wherein the peptide comprises a sequence selected from SEQ ID NOs: 11-43. 
     
     
         4 . The isolated peptide of  claim 1 , wherein the peptide is crotonylated at a lysine site. 
     
     
         5 . The isolated peptide of  claim 4 , wherein the crotonylated peptide is selected from the group consisting of QLATKcrAA, CQLATKcrAA, YQKcrST, CYQKcrSTELL, LLPKKcrTESHHKAK, CLLPKKcrTESHHKAKG, APAPKcrKGS, APAPKcrKGSC, APAPKKcrGS, CAPAPKKcrGS, GSKKcrA, GSKKcrAVTC, TKcrAQKKDG, AVTKcrAQKKDGC, ARTKcrQTAR, ARTKcrQTARC, APRKcrQLA, APRKcrQLATC, QLATKcrAARK, QLATKcrAARKC, AARKcrSAP, AARKcrSAPATGGC, CRLLRKcrGNYAER, CLLPKcrKTESHHKAKG, CLLPKKTESHHKcrAKG, CAVTKAQKcrKDG, CARTKQTARKcrSTG, CSGRGKcrGG and CGLGKGGAKcrRHR. 
     
     
         6 . The isolated peptide of  claim 1 , wherein the crotonylation site is selected from the group consisting of human H1.2K33, H1.2K63, H1.2K84, H1.2K89, H1.2K96, H1.2K158, H1.2K167, H2AK36, H2AK118, H2AK119, H2AK125, H2BK5, H2BK11, H2BK12, H2BK5, H2BK16, H2BK20, H2BK23, H2BK34, H3K4, H3K9, H3K18, H3K23, H3K27, H3K56, H4K5, H4K8, H4K12 and H4K16. 
     
     
         7 . The isolated peptide of  claim 1 , wherein the crotonylation site is selected from the group consisting of human H2AK119, H2BK11, H2BK12, H2BK16, H2BK20, H2BK34, H3K4, H3K18, H3K23, and H3K27. 
     
     
         8 . A method for producing a Kcr-specific affinity reagent that binds specifically to a protein or a fragment thereof comprising a crotonylation site, wherein the Kcr-specific affinity reagent is an antibody, comprising immunizing a host with the peptide of  claim 1 . 
     
     
         9 . The method of  claim 8 , wherein the peptide comprises a sequence selected from SEQ ID NOs: 11-43. 
     
     
         10 . A method for producing a Kcr-specific affinity reagent that binds specifically to a protein or a fragment thereof comprising a crotonylation site, wherein the Kcr-specific affinity reagent is a polypeptide, comprising screening a synthetic peptide library using the peptide of  claim 1 . 
     
     
         11 . The method of  claim 10 , wherein the synthetic peptide library is a phage display library or a yeast display library. 
     
     
         12 . The method of  claim 11 , wherein the peptide comprises a sequence selected from SEQ ID NOs: 11-43. 
     
     
         13 . An isolated Kcr-specific affinity reagent capable of binding specifically to the peptide of  claim 1 . 
     
     
         14 . The isolated Kcr-specific affinity reagent of  claim 13 , wherein the peptide comprises a sequence selected from SEQ ID NOs: 11-43. 
     
     
         15 . The isolated Kcr-specific affinity reagent of  claim 13 , wherein the binding of the Kcr-specific affinity reagent is dependent on the crotonylation site but not its surrounding peptide sequence. 
     
     
         16 . The isolated Kcr-specific affinity reagent of  claim 13 , wherein the binding of the Kcr-specific affinity reagent is dependent on the crotonylation site and its surrounding peptide sequence. 
     
     
         17 . The isolated Kcr-specific affinity reagent of  claim 13 , wherein the Kcr-specific affinity reagent binds specifically to the peptide when crotonylated at the crotonylation site, and wherein the Kcr-specific affinity reagent does not bind to the peptide when not crotonylated at the crotonylation site. 
     
     
         18 . The isolated Kcr-specific affinity reagent of  claim 13 , wherein the crotonylation site is selected from the group consisting of human H1.2K33, H1.2K63, H1.2K84, H1.2K89, H1.2K96, H1.2K158, H1.2K167, H2AK36, H2AK118, H2AK119, H2AK125, H2BK5, H2BK 11, H2BK12, H2BK15, H2BK16, H2BK20, H2BK23, H2BK34, H3K4, H3K9, H3K18, H3K23, H3K27, H3K56, H4K5, H4K8, H4K12 and H4K16. 
     
     
         19 . A method for detecting protein crotonylation in a sample, comprising:
 (a) contacting the sample with the isolated Kcr-specific affinity reagent of  claim 13  to form a binding complex, whereby the Kcr-specific affinity reagent binds specifically to a protein or a fragment thereof comprising a crotonylation site when the protein or a fragment thereof is crotonylated at the crotonylation site, and   (b) detecting the binding complex, wherein the presence of the binding complex indicates protein crotonylation in the sample.   
     
     
         20 . A kit for detecting protein crotonylation in a sample, comprising the isolated Kcr-specific affinity reagent of  claim 13 , wherein the Kcr-specific affinity reagent binds specifically to a protein or a fragment thereof comprising a crotonylation site when the protein or a fragment thereof is crotonylated at the crotonylation site.