# US 7,745,163 B2 · Method for identifying acetyltransferase substrates

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- Number: US 7,745,163 B2
- Document ID: US7745163B2
- Status: Granted
- Kind: B2
- Application number: 10/574,307
- Filed: 2004-10-01
- Published: 2007-11-22
- Granted: 2010-06-29
- Earliest claimed: 2003-10-03
- Assignees: Albert Einstein College of Medicine of Yeshiva University
- Inventors: John S. Blanchard
- Web page: https://patentgrep.com/patent/US7745163B2

## CPC

- C12Q 1/48: involving transferase
- G01N 2333/91051: Acyltransferases other than aminoacyltransferases (general) (2.3.1)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the reagent is the halo-acetyl-CoA labeled with a label.

### Claim 3 (depends on 1)

The method of claim 1, wherein the reagent is the halo-acetyl-pantetheine labeled with a label.

### Claim 4 (depends on 1)

The method of claim 1, wherein the reagent is the aminoethanethiol labeled with a label.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 2)

The method of claim 2, wherein the halo-acetyl-CoA is a chloroacetyl-CoA.

### Claim 8 (depends on 2)

The method of claim 2, wherein the halo-acetyl-CoA is a bromoacetyl-CoA.

### Claim 9 (depends on 2)

The method of claim 2, wherein the halo-acetyl-CoA is a fluoroacetyl-CoA or an iodoacetyl-CoA.

### Claim 10 (depends on 4)

The method of claim 4, wherein the aminoethanethiol is labeled with a fluorophore.

### Claim 11 (depends on 1)

The method of claim 1, wherein the label is radioactive.

### Claim 12 (depends on 11)

The method of claim 11, wherein the radioactive label is 32P.

### Claim 13 (depends on 1)

The method of claim 1, wherein the label is fluorescent.

### Claim 14 (depends on 1)

The method of claim 1, wherein the label is an affinity label.

### Claim 15 (depends on 14)

The method of claim 14, wherein the affinity label is biotin.

### Claim 16 (depends on 1)

The method of claim 1, wherein the acetyltransferase is labeled with an affinity tag.

### Claim 17 (depends on 16)

The method of claim 16, wherein the affinity tag is an oligo-His tag.

### Claim 18 (depends on 1)

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.