Method for liberating sugar chain from glycoprotein

• US 11,325,935 B2
• Filed: 09/26/2017
• Issued: 05/10/2022
• Est. Priority Date: 09/27/2016
• Status: Active Grant

• Current Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan), Sumitomo Bakelite Company Limited
• Original Assignee: National Institute of Advanced Industrial Science and Technology (Government of Japan)
• Inventors: Kameyama, Akihiko, Toyoda, Masaaki, Sakaguchi, Midori
• Primary Examiner(s): Gordon, Brian R
• Assistant Examiner(s): Gerido, Dwan A
• Application Number:
  US16/335,939
• Publication Number:
  US 20190309003A1
• Time in Patent Office: 1,687 Days
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 1/08 from natural products
• CPC Class Codes: C07H 5/06 Aminosugars
• CPC Class Codes: C08B 37/00 Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof cellulose D21; microbiological processes C12P
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Provided is a method for liberating sugar chains from a glycoprotein, including: a step of brining a reaction solution which contains hydroxylamines (a) and a basic reagent (b) into contact with the glycoprotein to obtain a mixed solution of sugar chains liberated from the glycoprotein and the reaction solution.

Claims

1. A method for liberating sugar chains from a glycoprotein having proteins and at least one of N-binding sugar chains and O-binding sugar chains, the at least one of N-binding sugar chains and O-binding sugar chains being bound to the proteins through an N-glycosidic bond or an O-glycosidic bond, respectively, the method comprising:

a step of bringing a reaction solution which contains hydroxylamines (a) and a basic reagent (b) into contact with the glycoprotein and cleaving the N-glycosidic bond or the O-glycosidic bond, to obtain a mixed solution of the sugar chains liberated from the glycoprotein and the reaction solution,

wherein, in the step of bringing the reaction solution into contact with the glycoprotein, a pH of the reaction solution is in a range of 8 to 14.

2. The method according to claim 1,

wherein the reaction solution contains 2% to 70% (w/w) of the hydroxylamines (a).

3. The method according to claim 1,

wherein the reaction solution further contains amines (c), and

wherein the amines (c) are at least one compound selected from the group consisting of ammonia water, a methylamine aqueous solution, a dimethylamine aqueous solution, ethylamine, diethylamine, ethanolamine, ethylenediamine, butylamine, morpholine, DABCO, and anthranilic acid.

4. The method according to claim 1,

wherein the sugar chains liberated from the glycoprotein contain a sugar chain oxime.

5. The method according to claim 1,

wherein the hydroxylamines (a) are at least one compound selected from the group consisting of hydroxylamine, a salt of hydroxylamine, O-substituted hydroxylamine, and a salt of O-substituted hydroxylamine.

6. The method according to claim 1,

wherein the basic reagent (b) is at least one selected from the group consisting of a hydroxide of an alkali metal, a weak acid salt of an alkali metal, a hydroxide of an alkaline earth metal, a salt of an alkaline earth metal dissolved in an ammonia aqueous solution, and an organic base.

7. The method according to claim 6,

wherein the hydroxide of an alkali metal is lithium hydroxide, sodium hydroxide, or potassium hydroxide,

the weak acid salt of an alkali metal is sodium bicarbonate or sodium carbonate,

the hydroxide of an alkaline earth metal is calcium hydroxide, barium hydroxide, or strontium hydroxide,

the salt of an alkaline earth metal is calcium acetate, calcium chloride, barium acetate, or magnesium acetate, and

the organic base is 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5,7-triazabicyclo[4.4.0]dec-5-ene, 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 1,1,3,3-tetramethylguanidine, 2-tert-butyl-1,1,3,3-tetramethylguanidine, or cetyltrimethylammonium hydroxide.

8. The method according to claim 1, further comprising:

a step of adding a ketone, an aldehyde, or an acid anhydride to the mixed solution and converting the hydroxylamines (a) remaining in the mixed solution to a ketoxime, an aldoxime, or an amide;

a step of bringing the mixed solution into contact with a solid phase having an affinity for sugar chains so that the sugar chains liberated from the glycoprotein are adsorbed on the solid phase; and

a step of eluting the sugar chains from the solid phase.

9. A method for analyzing sugar chains of a glycoprotein, comprising:

a step of liberating sugar chains from a glycoprotein using the method according to claim 1;

a step of labeling the liberated sugar chains, which includes labeling of a sugar chain oxime; and

a step of analyzing the labelled sugar chain.

10. A method for analyzing sugar chains of a glycoprotein, comprising:

a step of liberating sugar chains from a glycoprotein using the method according to claim 1; and

a step of fluorescent labeling the liberated sugar chains, which includes labeling of a sugar chain oxime.

11. The method according to claim 1,

wherein the hydroxylamines (a) are at least one compound selected from a hydroxylamine aqueous solution, hydroxylamine sulfate, hydroxylamine phosphate, O-methylhydroxylamine hydrochloride, O-methylhydroxylamine hydrochloride, O-ethylhydroxylamine hydrochloride, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, nitrobenzylhydroxylamine hydrochloride, O-tert-butyldimethylsilylhydroxylamine, and O-trimethylsilylhydroxylamine.