Method for identifying acetyltransferase substrates
View Patent ↗The present invention is directed to a method of identifying an acetyltransferase substrate in a sample that includes the steps of: (a) contacting the sample with (i) a reagent comprising a thiol-containing compound, a halo-acetyl-CoA or a halo-acetyl-pantetheine, and (ii) an acetyltransferase, under conditions suitable for acetyltransferase enzyme activity, and (b) identifying a substrate that has formed a base-stable covalent bond to the reagent, wherein the reagent is labeled with a label and/or the acetyltransferase is labeled with an affinity tag, and the substrate is the acetyltransferase substrate.
1. A method of identifying an acetyltransferase substrate in a sample, the method comprising the steps of:
(a) contacting the sample with
(i) a reagent comprising an aminoethanethiol, a halo-acetyl-CoA or a halo-acetyl-pantetheine, and
(ii) an acetyltransferase,
under conditions suitable for acetyltransferase enzyme activity, and
(b) identifying a substrate that has formed a base-stable covalent bond to the reagent, wherein the reagent is labeled with a label and/or the acetyltransferase is labeled with an affinity tag, and the substrate is the acetyltransferase substrate.
2. The method of claim 1 , wherein the reagent is the halo-acetyl-CoA labeled with a label.
3. The method of claim 1 , wherein the reagent is the halo-acetyl-pantetheine labeled with a label.
4. The method of claim 1 , wherein the reagent is the aminoethanethiol labeled with a label.
5. The method of claim 1 , wherein the reagent comprises the halo-acetyl-CoA and the aminoethanethiol, wherein either the halo-acetyl-CoA or the aminoethanethiol is labeled with a label.
6. The method of claim 1 , wherein the reagent comprises the halo-pantetheine CoA and the aminoethanethiol, wherein either the halo-acetyl-CoA or the aminoethanethiol is labeled with a label.
7. The method of claim 2 , wherein the halo-acetyl-CoA is a chloroacetyl-CoA.
8. The method of claim 2 , wherein the halo-acetyl-CoA is a bromoacetyl-CoA.
9. The method of claim 2 , wherein the halo-acetyl-CoA is a fluoroacetyl-CoA or an iodoacetyl-CoA.
10. The method of claim 4 , wherein the aminoethanethiol is labeled with a fluorophore.
11. The method of claim 1 , wherein the label is radioactive.
12. The method of claim 11 , wherein the radioactive label is 32 P.
13. The method of claim 1 , wherein the label is fluorescent.
14. The method of claim 1 , wherein the label is an affinity label.
15. The method of claim 14 , wherein the affinity label is biotin.
16. The method of claim 1 , wherein the acetyltransferase is labeled with an affinity tag.
17. The method of claim 16 , wherein the affinity tag is an oligo-His tag.
18. The method of claim 1 , wherein the acetyltransferase is selected from the group consisting of a histone acetyltransferase, an N-terminal acetyltransferase, an arylamine N-acetyltransferase, an aminoglycoside acetyltransferase, chloramphenicol acetyltransferase, choline acetyltransferase, carnitine acetyltransferase, spermine acetyltransferase, and ornithine acetyltransferase.