Method for analyzing formyl glycine residue

Patent metadata

Field	Value
Publication number	US-9896715-B2
Application number	US-201214366533-A
Country	US
Kind code	B2
Filing date	Dec 18, 2012
Priority date	Dec 20, 2011
Publication date	Feb 20, 2018
Grant date	Feb 20, 2018

Abstract

Disclosed is a method which enables semiquantitative or quantitative determination of the ratio between cysteine and formylglycine residues in a protein. The method includes (a) a step of labeling the protein (i) with a halogen-substituted carboxylic acid, (ii) with a halogen-substituted carboxylic acid amide, and (iii) with a halogen-substituted carboxylic acid and then with hydrazine, or with a halogen-substituted carboxylic acid and then by oximation, (b) a step of digesting each labeled protein to provide a corresponding mixture of peptide fragments, (c) a step of subjecting each mixture to reverse phase chromatography to separate the peptide fragments from each other to produce a chromatogram, (d) a step of comparing the produced chromatograms with each other to identify the peak corresponding to the peptide fragment that contained a cysteine residue and the peak corresponding to the peptide fragment that contained a formylglycine residue.

First claim

The invention claimed is:

1. A method of analysis for a formylglycine residue and a cysteine residue in the amino acid residues that makes up an analyte protein comprising,
(a) a step of converting the protein respectively into
(i) a halogen-substituted carboxylic acid-labeled protein by labeling the protein with a halogen-substituted carboxylic acid,
(ii) a halogen-substituted carboxylic acid amide-labeled protein by labeling the protein with a halogen-substituted carboxylic acid amide, and one of:
(iii)(a) a halogen-substituted carboxylic acid-hydrazine-labeled protein or a halogen-substituted carboxylic acid-oximation-labeled protein by labeling the protein with a halogen-substituted carboxylic acid and then with hydrazine or by labeling the protein with a halogen-substituted carboxylic acid and then by oximation, or
(iii)(b) a halogen-substituted carboxylic acid amide-hydrazine-labeled protein or a halogen-substituted carboxylic acid amide-oximation-labeled protein by labeling the protein with a halogen-substituted carboxylic acid amide and then with hydrazine or by labeling the protein with a halogen-substituted carboxylic acid amide and then by oximation;

wherein the sulfhydryl group of the cysteine residue in the analyte protein is alkylated with the halogen-substituted carboxylic acid or with the halogen-substituted carboxylic acid amide,
wherein the halogen-substituted carboxylic acid is represented by the following formula (I),
C m H 2m X—COOH  (I)

wherein X denotes halogen, and m denotes an integer of 1 to 5, and
the halogen-substituted carboxylic acid amide is represented by the following formula (II),
C m H 2m X—CONH 2   (II)

wherein X denotes halogen, and m denotes an integer of 1 to 5, and
wherein the labeling with hydrazine is performed using a hydrazine compound or a salt thereof that forms a hydrazone with the carbonyl group of a formylglycine residue, and
wherein the labeling by oximation is performed using a hydroxyl amine compound or a salt thereof that forms an oxime group with the carbonyl group of a formylglycine residue,
(b) a step of digesting each of the labeled proteins to provide a corresponding mixture of peptide fragments,
(c) a step of subjecting each mixture of peptide fragments to reverse phase chromatography to separate the peptide fragments from each other while monitoring the separated fragments with absorptiometer, producing a chromatogram of the mixture of peptide fragments,
(d) a step of comparing the produced chromatograms with each other to identify, on the chromatograms, the peak corresponding to a peptide fragment which contained a cysteine residue when in the analyte protein and the peak corresponding to a peptide fragment which contained a formylglycine residue when in the analyte protein.

2. The method according to claim 1 , wherein m is an integer of 1 to 3 in formula (I) and formula (II).

3. The method according to claim 1 , wherein the halogen is chlorine or iodine in formula (I) and formula (II).

4. The method according to claim 1 , wherein the halogen-substituted carboxylic acid is an iodo-substituted monocarboxylic acid or a chloro-substituted monocarboxylic acid; and the halogen-substituted carboxylic acid amide is an iodo-substituted monocarboxylic acid amide or a chloro-substituted monocarboxylic acid.

5. The method according to claim 4 , wherein the halogen-substituted monocarboxylic acid is one selected from the group consisting of iodoacetic acid, 2-iodopropionic acid, 3-iodopropionic acid, and chloroacetic acid, and the halogen-substituted carboxylic acid amide is one selected from the group consisting of iodoacetamide, 2-iodopropionic acid amide, 3-iodopropionic acid amide, and chloroacetamide.

6. The method according to claim 4 , wherein the halogen-substituted monocarboxylic acid is iodoacetic acid and the halogen-substituted carboxylic acid amide is iodoacetamide.

7. The method according to claim 4 , wherein the halogen-substituted monocarboxylic acid is 3-iodo-propionic acid and the halogen-substituted carboxylic acid amide is iodoacetamide.

8. The method according to claim 1 , wherein the hydrazine compound or the salt thereof is a 2,4-dinitrophenylhydrazine or a salt thereof, and wherein the hydroxylamine compound or the salt thereof is O-4-nitrobenzyl hydroxylamine or a salt thereof.

9. The method according to claim 1 , further comprising a step for determining, on the chromatogram, the ratio of the area of the peak corresponding to the peptide fragment that contained a cysteine residue when in the analyte protein and the area of the peak corresponding to the peptide fragment that contained a formylglycine residue when in the analyte protein.

10. The method according to claim 1 , wherein the analyte protein is a human sulfuric ester hydrolase.

11. The method according to claim 10 , wherein the sulfuric ester hydrolase is selected from the group consisting of iduronate-2-sulfatase, N-acetylgalactosamine-4-sulfatase, N-acetylgalactosamine-6-sulfatase, heparan-N-sulfatase, and N-acetyl glucosamine-6-sulphate sulfatase.

12. The method according to claim 11 , wherein the sulfuric ester hydrolase is iduronate-2-sulfatase.

13. The method according to claim 10 , wherein the cysteine residue is that cysteine residue which must have been converted into a formylglycine residue in order for the sulfuric ester hydrolase to exhibit its enzymatic activity, and the formylglycine residue is that formylglycine residue which has been generated by conversion of the cysteine residue.

14. The method according to claim 13 , wherein the sulfuric ester hydrolase is iduronate-2-sulfatase, and the cysteine residue and the formylglycine residue are those located at position 59 from the N-terminus of the mature iduronate-2-sulfatase.

15. The method according to claim 14 , wherein the iduronate-2-sulfatase is a recombinant iduronate-2-sulfatase.

Assignees

• Japan Chem Res

Inventors

• Yokoyama Tetsuo

Classifications

• C12Q1/44 Primary

  involving esterase · CPC title

• G01N2333/916

  acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4) · CPC title

• G01N2030/8831

  involving peptides or proteins · CPC title

• G01N33/6815

  containing sulfur, e.g. cysteine, cystine, methionine, homocysteine · CPC title

• B01D15/325

  Reversed phase · CPC title