Kit for assaying acetyl transferase or deacetylase activity
View Patent ↗The invention provides methods and kits for characterizing the activity of an acetyl transferase or deacetylase. The method involves enzymatically acetylating or deacetylating in vitro a substrate that is a peptide fragment of a full-length polypeptide, and then non-enzymatically acylating the peptide substrate with acyl groups that differ in molecular weight from the enzymatically added or removed acetyl groups. Typically, deuterated acetic anhydride is used to non-enzymatically acylate the substrate. The fully acylated substrate is then characterized by mass spectrometry to determine the amino acid positions of the substrate that are enzymatically acetylated or deacetylated.
1. A kit for characterizing the activity of an acyl transferase on a substrate, the kit comprising:
a polypeptide substrate for an acetyl transferase and/or a deacetylase, wherein the polypeptide substrate is a peptide fragment of a full-length protein; and
an acyl-containing substance that non-enzymatically reacts with the polypeptide substrate to add acyl groups to the polypeptide substrate to generate a fully acylated polypeptide; wherein the acyl group of the acyl-containing substance differs in molecular weight from a natural isotope composition acetyl group.
2. The kit of claim 1 wherein the polypeptide substrate is a fragment of a full-length histone.
3. The kit of claim 1 wherein the acyl-containing substance that non-enzymatically reacts with the polypeptide substrate is deuterated acetic anhydride.
4. The kit of claim 1 wherein the polypeptide substrate comprises a synthetic ligand and the kit further comprises a solid substrate coupled to a receptor for the ligand.
5. The kit of claim 1 wherein the polypeptide substrate is methylated.
6. The kit of claim 1 wherein the polypeptide substrate is acetylated.
7. The kit of claim 1 wherein the kit contains two or more polypeptide substrates that differ in post-translational modifications but are identical in their amino acid sequence.
8. The kit of claim 1 further comprising acetyl-coenzyme A.
9. The kit of claim 1 wherein the polypeptide substrate is a substrate for acetylation and/or deacetylation by the acetyl transferase or deacetylase at two or more amino acid residues.