WO2018062167A1 - 糖タンパク質の糖鎖遊離法 - Google Patents
糖タンパク質の糖鎖遊離法 Download PDFInfo
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- WO2018062167A1 WO2018062167A1 PCT/JP2017/034717 JP2017034717W WO2018062167A1 WO 2018062167 A1 WO2018062167 A1 WO 2018062167A1 JP 2017034717 W JP2017034717 W JP 2017034717W WO 2018062167 A1 WO2018062167 A1 WO 2018062167A1
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
- C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
- C07H5/06—Aminosugars
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
Definitions
- the present invention relates to a method for releasing a glycoprotein sugar chain.
- sugar chains are bound to many proteins, including biopharmaceuticals such as antibody drugs.
- biopharmaceuticals it has been suggested that sugar chains affect not only the activity of the pharmaceuticals but also antigenicity and dynamics, and analysis of sugar chains is an important issue in biopharmaceutical development.
- sugar chains of glycoproteins can be used as disease-related biomarkers, it is necessary to analyze the sugar chains of glycoproteins contained in multiple biological samples in order to find sugar chains that can serve as markers.
- the sugar chain is released from the glycoprotein, and after post-treatment such as desalting or protein removal, the sugar chain is analyzed with a mass spectrometer, or the released sugar chain In general, the analysis is performed by high-performance liquid chromatography, capillary electrophoresis, or a combination thereof, which is provided with a fluorescent label and equipped with a fluorescence detector. In either case, the first step is to release the sugar chain from the glycoprotein.
- the linkage between the sugar chain and the protein consists of an N-linked sugar chain in which N-acetylglucosamine is linked to the amino group of the asparagine residue of the protein by an N-glycosidic bond, and an N-acetylgalactosamine to the hydroxyl group of serine and threonine residues. It is divided into O-linked sugar chains to be linked. In order to release the sugar chain from the protein, a method suitable for each is used.
- sugar chains can be released using enzymes such as PNGase F and glycopeptidase A.
- enzymes such as PNGase F and glycopeptidase A.
- the enzyme depends on the species to be analyzed. It is necessary to use properly.
- pretreatment such as protein denaturation or protease degradation is generally performed in advance.
- Patent Document 1 discloses a hydrazine decomposition method in which a sufficiently dried glycoprotein is dissolved in anhydrous hydrazine and heat-treated at 100 ° C. for 10 hours or more, and this method mainly uses an N-type sugar chain. It is used as a method of releasing. After the reaction, anhydrous hydrazine is distilled off under reduced pressure, and acetylation is further performed using sodium hydrogen carbonate and acetic anhydride.
- the released alditol cannot be given a fluorescent label because the aldehyde group, which is a functional group on the sugar chain side for giving a fluorescent label, is reduced to alcohol. Therefore, it is common to analyze with a mass spectrometer after methylating all hydroxyl groups of alditol (complete methylation).
- Patent Document 3 discloses that in ⁇ -elimination by strong alkali, in order to obtain an O-linked sugar chain in a state where the aldehyde group is preserved, the alkali solution and the sample solution are allowed to react for a short time while flowing in the flow path.
- a system for immediate neutralization is disclosed.
- hydrazine decomposition method As another method for chemically releasing an O-linked sugar chain while preserving the aldehyde group, the above-described hydrazine decomposition method is known. After the reaction, hydrazine is distilled off and reacetylated, and after fluorescent labeling, analysis is performed using HPLC or the like.
- JP-A-3-228000 JP 2007-45889 A Japanese Patent No. 4283272 JP2015-107969A
- releasing the sugar chain from the protein is the first step, but conventionally, it took a long time to include the pretreatment to release the sugar chain.
- the protein is previously treated with a reducing agent such as dithiothreitol, then denatured by treatment with an alkylating agent such as iodoacetamide, and further a protease such as trypsin. Since the sugar chain was released by reacting with a sugar chain-releasing enzyme after being decomposed into a peptide by 1), it was not possible to analyze the sugar chain on the day when the sample was taken at the pharmaceutical production site.
- a reducing agent such as dithiothreitol
- an alkylating agent such as iodoacetamide
- a protease such as trypsin
- PNGase F and glycopeptidase A which are enzymes that liberate sugar chains, are very expensive, and have caused considerable cost burden for analyzing many samples.
- the hydrazine decomposition method requires complete anhydrous conditions, and if a small amount of water is mixed in the reaction system, the yield is significantly reduced. Therefore, it was necessary to react the sample after drying it under reduced pressure in advance. Furthermore, the reaction time is 10 hours or longer, and when including hydrazine distillation and reacetylation after the reaction, it takes at least 2 days to complete the entire process. Furthermore, since hydrazine is a carcinogenic poison and an easily explosive compound, it must be handled with great care.
- N glycolylneuraminic acid was decomposed, so that there was a problem that N glycolylneuraminic acid could not be analyzed.
- N-glycolylneuraminic acid which is a type of sialic acid, can be problematic, and the hydrazine degradation method cannot be used for this purpose.
- sugar chains can be released from proteins using a basic catalyst in an aqueous solution in the presence of hydroxylamine, and have completed the present invention.
- the present invention includes the following aspects.
- a method of releasing a sugar chain from a glycoprotein wherein the glycoprotein is released from the glycoprotein by contacting a reaction solution containing (a) hydroxylamines and (b) a basic reagent.
- a method comprising a step of obtaining a mixed solution of the sugar chain and the reaction solution.
- the reaction solution contains 2 to 70% (w / w) of the (a) hydroxylamine.
- the reaction solution further contains (c) an amine.
- the (c) amine is at least one compound selected from the group consisting of aqueous ammonia, aqueous methylamine, aqueous dimethylamine, ethylamine, diethylamine, ethanolamine, ethylenediamine, butylamine, morpholine, DABCO, and anthranilic acid.
- the method according to [3], wherein [5] The method according to any one of [1] to [4], wherein the sugar chain released from the glycoprotein contains a sugar chain oxime.
- [6] The method according to any one of [1] to [5], wherein in the step of bringing the reaction solution into contact with the glycoprotein, the pH of the reaction solution is in the range of 8 to 14.
- the (a) hydroxylamine is at least one compound selected from the group consisting of hydroxylamine, a hydroxylamine salt, an O-substituted hydroxylamine, and an O-substituted hydroxylamine salt. 6] The method according to any one of the above. [8]
- the basic reagent (b) comprises an alkali metal hydroxide, an alkali metal weak acid salt, an alkaline earth metal hydroxide, an alkaline earth metal salt dissolved in an aqueous ammonia solution, and an organic base.
- the alkali metal hydroxide is lithium hydroxide, sodium hydroxide or potassium hydroxide
- the alkali metal weak acid salt is sodium bicarbonate or sodium carbonate
- the alkaline earth metal water is calcium hydroxide, barium hydroxide or strontium hydroxide
- the alkaline earth metal salt is calcium acetate, calcium chloride, barium acetate or magnesium acetate
- 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 A tilt trimethyl ammonium hydroxide The method of claim 8.
- a method for analyzing a sugar chain of a glycoprotein, the step of releasing the sugar chain from the glycoprotein by the method according to any one of [1] to [10] and the step of labeling the released sugar chain A method of analyzing a sugar chain, comprising a step of including a label of a sugar chain oxime.
- a kit for releasing a sugar chain from a glycoprotein comprising (a) a basic reagent and (c) an amine for use in combination with hydroxylamines.
- the kit according to [16] further comprising a ketone, an aldehyde or an acid anhydride, and a solid phase having affinity for a sugar chain.
- the kit according to [16] or [17] further comprising instructions for performing the method according to any one of [1] to [10].
- a container holding unit that holds a container in which a sample containing a glycoprotein is stored, and a reagent introducing unit that introduces a reagent into the container, wherein the reagent introducing unit includes (a) hydroxylamine in the container And (b) a device for releasing a sugar chain from a glycoprotein, comprising a reaction solution introduction part for introducing a reaction solution containing a basic reagent.
- the reagent introduction unit further includes a ketone introduction unit for introducing ketone into the container, an aldehyde introduction unit for introducing aldehyde, or an acid anhydride introduction unit for introducing acid anhydride. .
- the apparatus according to [19] or [20] further comprising a solid phase holding unit that holds a container including a solid phase having affinity for sugar chains.
- the present invention includes the following aspects.
- a method for releasing a sugar chain from a glycoprotein Contacting the glycoprotein with a reaction solution containing (a) hydroxylamines and (b) a basic reagent, wherein the (a) hydroxylamines are 2 to 50% (w / w) And a step included in the reaction solution.
- the method for releasing a sugar chain from the glycoprotein of the present invention is [2] the method described in [1] above,
- the reaction solution further includes (c) an amine.
- the method for releasing a sugar chain from the glycoprotein of the present invention is, in one embodiment, [3] the method described in [1] or [2] above,
- the sugar chain released from the glycoprotein contains a sugar chain oxime.
- the method for releasing a sugar chain from the glycoprotein of the present invention is, in one embodiment, [4] the method according to any one of [1] to [3], In the step of bringing the reaction solution into contact with the glycoprotein, the pH of the reaction solution is in the range of 8 to 14.
- the method for releasing a sugar chain from the glycoprotein of the present invention in one embodiment, [5]
- the (a) hydroxylamine is at least one compound selected from the group consisting of hydroxylamine and salts thereof, and O-substituted hydroxylamine and salts thereof.
- the basic reagent is at least selected from the group consisting of an alkali metal hydroxide, an alkali metal weak acid salt, an alkaline earth hydroxide, an alkaline earth salt dissolved in an aqueous ammonia solution, and an organic base It is characterized by being one.
- the method for releasing a sugar chain from the glycoprotein of the present invention is [7] the method described in [6] above,
- the alkali metal hydroxide, alkali metal weak acid salt, or alkaline earth hydroxide is lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, calcium hydroxide, barium hydroxide, water.
- the alkaline earth salt dissolved in the aqueous ammonia solution is at least one selected from the group consisting of calcium acetate, calcium chloride, barium acetate, and magnesium acetate.
- the method for releasing a sugar chain from the glycoprotein of the present invention is, in one embodiment, [8] the method described in [6] above,
- 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 And at least one selected from the group consisting of cetyltrimethylammonium hydroxide.
- the method for releasing a sugar chain from the glycoprotein of the present invention in one embodiment, [9]
- the (c) amine is at least one compound selected from the group consisting of ammonia, methylamine, dimethylamine, ethylamine, diethylamine, ethanolamine, ethylenediamine, butylamine, morpholine, DABCO, and anthranilic acid. To do.
- the present invention provides: [10] A method for analyzing a sugar chain of a glycoprotein, Releasing sugar chains from glycoproteins by the method according to any one of claims 1 to 9,
- the present invention relates to a method for analyzing a sugar chain, comprising a step of labeling a released sugar chain and a step of labeling a sugar chain oxime.
- the present invention provides: [11] A kit for releasing a sugar chain from a glycoprotein, A kit containing (a) hydroxylamines and (b) a basic reagent.
- a kit for releasing a sugar chain from the glycoprotein of the present invention is, in one embodiment, [12] The kit according to [11] above, (A) It further comprises amines.
- a kit for releasing a sugar chain from the glycoprotein of the present invention is, in one embodiment, [13] A kit for releasing a sugar chain from a glycoprotein, (A) A kit containing (b) a basic reagent and (c) amines for use in combination with hydroxylamines.
- sugar chains can be released from glycoproteins in a short processing time while suppressing the degradation of sugar chains, and the free sugar chains are also released. Can be recovered as a mixture containing a sugar chain oxime.
- the N-glycolylneuraminic acid could not be analyzed in the conventional hydrazine decomposition method because the N-glycolyl group was decomposed.
- the sugar chain can be liberated while inhibiting the decomposition of the N glycolyl group.
- FIG. 2 shows an HPLC chromatogram of an N-linked sugar chain released from a monoclonal antibody (IgG) in Example 1.
- FIG. 2 shows an HPLC chromatogram of an N-linked sugar chain released from a monoclonal antibody (IgG) by a method using an enzyme that releases an N-linked sugar chain (PNGase F) in Example 1.
- FIG. 2 shows an HPLC chromatogram of an N-linked sugar chain released from human serum-derived IgG in Example 2.
- Example 3 the HPLC chromatogram of the O-linked sugar chain released from bovine fetzin is shown.
- Example 4 the HPLC chromatogram of the N-linked sugar chain liberated from siapotransferrin is shown.
- Example 5 the HPLC chromatogram of the N-linked sugar chain liberated from horseradish peroxidase is shown.
- Example 6 the HPLC chromatogram of the O-linked sugar chain released from bovine submandibular gland mucin is shown.
- (A) to (d) show HPLC chromatograms of O-linked sugar chains released from bovine fetuin in Examples 9 to 11.
- (A) is the result of Example 10
- (b) is the result of Example 11
- (c) is the result of Example 9 (acetone treatment)
- (d) is Example 9 (salicylaldehyde). Result of processing).
- Example 12 the HPLC chromatogram of the O-linked sugar chain released from bovine fetuin is shown. It is a mimetic diagram explaining one embodiment of a device which releases a sugar chain from glycoprotein.
- glycoprotein refers to a protein in which at least one O-linked sugar chain or N-linked sugar chain is bound in the amino acid sequence of the protein.
- the glycoprotein to which the present invention is applicable is not particularly limited, and may be naturally derived or synthesized.
- “Sugar chains” include O-linked sugar chains and N-linked sugar chains.
- the O-linked sugar chain has a structure in which a sugar chain is bound to the amino acid residue of serine (Ser) or threonine (Thr) of a protein via an —OH group contained in each amino acid side chain.
- O-linked sugar chains are classified into 1 to 8 types according to the structure of the core.
- the N-linked sugar chain refers to a sugar chain that binds to the nitrogen atom of the amide group on the side chain of the protein asparagine residue.
- N-linked sugar chains include those that form a branch with mannose as a base point, and include, for example, two branched chains, three branched chains, and four branched chains.
- N-linked sugar chains can be classified into basic types, high mannose types, hybrid types, complex types, and the like according to their structures.
- both O-linked sugar chains and N-linked sugar chains can be released from glycoproteins.
- the present invention is a method for releasing a sugar chain from a glycoprotein, wherein the glycoprotein is contacted with a reaction solution containing (a) hydroxylamines and (b) a basic reagent. And a method comprising a step of obtaining a mixed solution of a sugar chain liberated from the glycoprotein and the reaction solution.
- a reaction solution containing (a) hydroxylamines and (b) a basic reagent
- contacting a glycoprotein with a reaction solution containing (a) hydroxylamines and (b) a basic reagent finally means glycoprotein, (a) hydroxylamines, and ( b) What is necessary is just to be in the state which the basic reagent contacted, and the order of the mixing is not ask
- a hydroxylamines may be added to the glycoprotein, and then (b) a basic reagent may be added.
- a basic reagent may be added to the glycoprotein, and then (a) hydroxylamines may be added.
- first, (a) hydroxylamines and (b) a basic reagent may be mixed and glycoprotein may be added thereto.
- examples of “(a) hydroxylamines” that can be used in the present embodiment include hydroxylamine, a hydroxylamine salt, an O-substituted hydroxylamine, and an O-substituted hydroxylamine salt.
- hydroxylamine hydrochloride hydroxylamine aqueous solution, hydroxylamine sulfate, hydroxylamine phosphate, O-methylhydroxylamine hydrochloride, O-ethylhydroxylamine hydrochloride, O-
- “(a) hydroxylamine” is an aqueous hydroxylamine solution.
- the final concentration of “(a) hydroxylamine” in the reaction solution can be in a concentration range of, for example, 2 to 70% (w / w), for example, 2 to 50% (w / w). However, it is not limited to the above-mentioned concentration range, and those skilled in the art will know the type of glycoprotein of interest, other components in the reaction solution (amines, basic reagents, other additives), reaction conditions (time) , Temperature, etc.) and the like.
- the final concentration of “(a) hydroxylamines” is preferably as high as possible, particularly when an O-linked sugar chain is liberated. Therefore, when releasing the O-linked sugar chain, the final concentration of “(a) hydroxylamines” is, for example, a concentration range of 10 to 70% (w / w), for example, 30 to 60% (w / w). In particular, about 50% (w / w) is preferred.
- the final concentration of “(a) hydroxylamine” can be 2 to 50% (w / w), preferably 10 to 20% ( w / w). In a more preferred embodiment, it is 10% (w / w).
- hydroxylamine When hydroxylamine is used as “(a) hydroxylamines”, a sufficient sugar chain recovery amount is obtained when the concentration of hydroxylamine is more than 2% (w / w) and 50% (w / w) or less. Hydroxylamine also tends to be stable.
- (b) basic reagent that can be used in the present embodiment includes alkali metal hydroxides, alkali metal weak acid salts, alkaline earth metal hydroxides, alkaline earth metal dissolved in aqueous ammonia. Mention may be made of at least one compound selected from the group consisting of salts and organic bases.
- alkali metal hydroxide examples include, but are not limited to, lithium hydroxide, sodium hydroxide, and potassium hydroxide.
- the alkali metal weak acid salt is not limited to the following, and examples thereof include sodium bicarbonate and sodium carbonate.
- alkaline earth metal hydroxide examples include, but are not limited to, calcium hydroxide, barium hydroxide, and strontium hydroxide.
- examples of the alkaline earth salt dissolved in aqueous ammonia include, but are not limited to, calcium acetate, calcium chloride, barium acetate, and magnesium acetate.
- lithium hydroxide is particularly preferable.
- organic base examples include, but are not limited to, for example, DBU: 1,8-diazabicyclo [5.4.0] undec-7-ene (1,8-diazabicyclo [5.4.0] undec-7 -Ene), TBD: 1,5,7-triazabicyclo [4.4.0] decan-5-ene (1,5,7-Triazabicyclo [4.4.0] dec-5-ene), MTBD 7-methyl-1,5,7-triazabicyclo [4.4.0] dec-5-ene (7-Methyl-1,5,7-triazabicyclo [4.4.0] dec-5-ene ), TMG: 1,1,3,3-tetramethylguanidine (1,1,3,3-tetramethylguanidine), t-BuTMG: 2-tert-butyl-1,1,3,3 Examples include tetramethylguanidine (2-tert-Butyl-1,1,3,3-tetramethylguanidine), DBN: 1,5-diazabiccyclo [4.3.0] non-5
- the “organic base” is a strong organic base (pKa12 or higher), and specific examples include DBU, TMG, TBD, MTBD, and CTAH. Using these DBU, TMG, TBD, MTBD, and CTAH as “organic bases” is preferable in that the base after the reaction can be removed by washing with an organic solvent.
- the final concentration of “(b) basic reagent” in the reaction solution can be in a concentration range of 2 mM to 2 M, for example.
- concentration range 2 mM to 2 M
- “(b) basic reagent” when lithium hydroxide is used, it can be 6 mM to 1 M, and preferably 100 mM to 500 mM. In a more preferred embodiment, it is 100 mM to 200 mM. If the concentration of the basic reagent is less than 2 mM, it is not preferable in that the reaction time becomes long, and if it exceeds 2 M, it is not preferable in that the sugar chain recovery rate is reduced.
- a sugar chain is released from a glycoprotein by bringing a reaction solution containing (a) hydroxylamines and (b) a basic reagent into contact with the glycoprotein. To release. At this time, (a) hydroxylamines are contained in the range of 2 to 70% (w / w) with respect to the reaction solution.
- the molar ratio of (a) hydroxylamines and (b) basic reagent in the reaction solution is preferably 1: 1 to 300: 1, and preferably 3: 1 to 200: 1. More preferred.
- the pH of the reaction solution can be in the range of 8 to 14, and more preferably in the range of 10 to 13.
- the conditions of temperature and reaction time are not particularly limited as long as the sugar chain can be released from the target protein, and the target glycoprotein and hydroxylamines are not limited.
- the temperature can be, for example, room temperature to 80 ° C.
- N glycolyl group etc. are easily decomposed
- the reaction time can be about 5 minutes to 16 hours depending on the conditions.
- “(c) amines” can be further added to the reaction solution in one embodiment.
- “(c) amines” that can be used in this embodiment include, but are not limited to, ammonia water, methylamine aqueous solution, dimethylamine aqueous solution, ethylamine, diethylamine, ethanolamine, ethylenediamine, butylamine, morpholine, DABCO, Mention may be made of at least one compound selected from the group consisting of anthranilic acid. In addition, two or more of the aforementioned compounds may be used in combination.
- “(c) amines” is aqueous ammonia, morpholine, DABCO, anthranilic acid.
- Ammonia water, morpholine, DABCO, anthranilic acid and the like are preferably used as “(c) amines” in that peeling, isomerization, and amide decomposition are suppressed.
- isomerization which is a side reaction when releasing an N-linked sugar chain, can be suppressed.
- the final concentration of “(c) amines” in the reaction solution can be in a concentration range of 40 mM to 15 M, for example. However, it is not limited to the above-mentioned concentration range, and those skilled in the art will know the type of target glycoprotein, other components in the reaction solution (hydroxylamines, basic reagents, other additives), reaction conditions ( Time, temperature, etc.) etc. can adjust suitably.
- the final concentration of ammonia in the reaction solution can be 2 to 25% (w / w), preferably 10 to It can be 20% (w / w). In a more preferred embodiment, it is 20% (w / w).
- the present invention is a method for analyzing a sugar chain possessed by a glycoprotein, and includes a step of releasing a sugar chain from the glycoprotein by the method described above, and a step of labeling the released sugar chain.
- a method for analyzing a sugar chain is provided.
- the sugar chain oxime is also labeled in the step of labeling the released sugar chain. Details will be described later.
- the reaction solution after releasing the sugar chain from the glycoprotein is desalted using a known method (for example, a solid-phase extraction cartridge filled with graphite carbon), and the sugar chain contained in the reaction solution is removed. Can be used for analysis.
- a known method can be appropriately employed.
- a fluorescent labeling method using picoline borane and 2-aminobenzamide can be used.
- the sugar chain liberated by the above-described method partly becomes an aldehyde type to form a sugar chain oxime. That is, in the conventional method, when an aldehyde group, which is a functional group of a sugar chain after being released, is reduced and converted into alditol, it cannot be directly fluorescently labeled. In addition, in the conventional method, when an aldehyde group, which is a functional group of a sugar chain, is converted to hydrazone by hydrazine, it is necessary to return to the original free sugar chain by a reacetylation operation in order to give a fluorescent label. It was.
- a free sugar chain can be obtained as a sugar chain oxime that can be directly labeled. That is, the sugar chain released from the glycoprotein by the method of this embodiment contains a sugar chain oxime. The sugar chain oxime can be directly labeled.
- the free sugar chain obtained by the above-mentioned method can be obtained in solution as a mixture of glycosylamine, sugar chain oxime, and normal sugar chain having a hemiacetal hydroxyl group at the reducing end, and these can be labeled together. can do.
- the inventors have clarified that (a) the final concentration of hydroxylamines is preferably as high as possible, particularly when an O-linked sugar chain is liberated.
- unreacted (a) hydroxylamines may remain in the mixed solution after releasing the sugar chain.
- Unreacted (a) hydroxylamines are preferably removed because they inhibit the fluorescent labeling reaction of sugar chains.
- the above-described method may further include an additional step of removing unreacted (a) hydroxylamines. That is, in the above-described method, a ketone, an aldehyde, or an acid anhydride is added to a mixed solution of a glycoprotein and a reaction solution, and (a) hydroxylamines remaining in the mixed solution are converted into ketoxime, aldoxime, or amide. A step of bringing the mixed solution into contact with a solid phase having affinity for sugar chains to adsorb sugar chains released from the glycoprotein to the solid phase; and eluting the sugar chains from the solid phase And a process.
- ketone acetone, methyl ethyl ketone, methyl isobutyl ketone, 4-hydroxybutanone, or the like can be used.
- aldehyde salicylaldehyde, benzaldehyde, 4-hydroxybenzaldehyde and the like can be used.
- acid anhydride acetic anhydride, succinic anhydride, or the like can be used.
- the sugar chain is recovered from the above mixture.
- the mixed solution is brought into contact with a solid phase having affinity for sugar chains, and sugar chains released from the glycoprotein are adsorbed on the solid phase, and then the sugar chains are released from the solid phase. Can be recovered from the above mixed solution.
- the solid phase having affinity for the sugar chain is not particularly limited, and examples thereof include hydrophilic carriers such as graphite carbon, crystalline cellulose, silica, and monolithic silica, and monolithic silica is particularly preferable.
- Monolithic silica is a filter-like porous continuous silica having a three-dimensional network structure, and has advantages such as better liquid permeability and less dead volume compared to conventional particulate silica.
- Monolithic silica may be fixed to a columnar container, for example, may be fixed to a multiwell plate.
- the pore size of the monolithic silica is such that the pores (through pores) continuous with each other are preferably 1 to 100 ⁇ m, more preferably 1 to 50 ⁇ m, still more preferably 1 to 30 ⁇ m, and more preferably 1 to 20 ⁇ m. It is particularly preferred.
- the mixed solution to which a ketone, aldehyde or acid anhydride is added, which is applied to the hydrophilic carrier preferably contains 90% by volume or more of an organic solvent, and more preferably contains 95% by volume or more of an organic solvent.
- an organic solvent acetonitrile, methanol, ethanol, 2-propanol, hexane, ethyl acetate, methylene chloride, tetrahydrofuran and the like can be used, and acetonitrile is particularly preferable.
- the cleaning liquid preferably contains 90% by volume or more of an organic solvent and 10% by volume or less of water, and more preferably contains 95% by volume or more of an organic solvent and 5% by volume or less of water.
- organic solvent contained in the cleaning liquid acetonitrile, methanol, ethanol, 2-propanol, hexane, ethyl acetate, methylene chloride, tetrahydrofuran, and the like can be used, and acetonitrile is particularly preferable.
- the eluate preferably contains 10% by volume or more of water, more preferably contains 50% by volume or more of water, and water is particularly preferred.
- the components other than water contained in the eluate are preferably acetonitrile and an organic solvent selected from alcohols typified by methanol, ethanol, propanol, and butanol.
- the eluted sugar chain can be subjected to a fluorescent labeling reaction.
- the present invention provides a kit for releasing a sugar chain from a glycoprotein, comprising (a) hydroxylamines and (b) a basic reagent.
- the kit of this embodiment is a kit which contains (c) amines in addition to (a) hydroxylamines and (b) basic reagents in one form of form.
- the kit for releasing a sugar chain from the glycoprotein of this embodiment comprises (a) a basic reagent for use in combination with hydroxylamines and (c).
- a kit containing amines is provided.
- the kit of this embodiment includes other reagents (such as (a) hydroxylamines, (c) amines, and other additives), ketones, aldehydes, or acid anhydrides used in the step of releasing sugar chains from glycoproteins.
- reagents such as (a) hydroxylamines, (c) amines, and other additives
- ketones such as (a) hydroxylamines, (c) amines, and other additives
- ketones such as ketones, aldehydes, or acid anhydrides used in the step of releasing sugar chains from glycoproteins.
- the present invention includes a container holding unit that holds a container in which a sample containing a glycoprotein is stored, and a reagent introduction unit that introduces a reagent into the container, and the reagent introduction unit is attached to the container.
- a device for releasing a sugar chain from a glycoprotein comprising a reaction liquid introduction part for introducing a reaction liquid containing (a) hydroxylamines and (b) a basic reagent. Note that the configuration of the apparatus described below is merely an example, and the apparatus of the present embodiment is not limited to this configuration.
- FIG. 10 is a schematic diagram for explaining the apparatus of this embodiment.
- the apparatus 100 includes a container holding unit 130 that holds a container 120 in which a sample 110 containing glycoprotein is stored, and a reagent introduction unit 140 that introduces a reagent into the container 120, and the reagent introduction unit 140 is attached to the container 120.
- a reaction liquid introduction unit 141 for introducing a reaction liquid 151 containing (a) hydroxylamines and (b) a basic reagent is included.
- the container holding part 130 is for holding the container 120 in which the sample 110 containing glycoprotein is accommodated.
- the aspect in which the container holding part 130 holds the container 120 is not particularly limited. For example, an aspect in which most of the container is fitted and held in the holding hole or the holding hole of the container holding part 130 may be mentioned. In addition to this, the container holding portion is held by the holding portion of the container holding portion. And the like.
- the reagent introduction unit 140 is for introducing a reagent into the inside of the container 120 held by the container holding unit 130 or inside the container 170 held by the solid phase holding unit 160 described later.
- the reagent introduction unit 140 includes at least a reaction solution introduction unit 141 that introduces a reaction solution 151 containing (a) hydroxylamines and (b) a basic reagent into the container 120.
- the reagent introduction unit 140 may further include a ketone introduction unit that introduces a ketone into the container 120, an aldehyde introduction unit that introduces an aldehyde, or an acid anhydride introduction unit that introduces an acid anhydride.
- the reagent introduction unit 140 includes a reaction liquid 151, a ketone, an aldehyde, or an acid anhydride 152, a tank 150 that stores a labeling reagent 153, and a liquid supply pipe that supplies each reagent stored in the tank 150. (142, 143, 144), valves (145, 146, 147) for controlling the feeding of each reagent, and an introduction part 141 for introducing each reagent into the container 120 or the container 170.
- the introduction part 141 serves also as a reaction liquid introduction part, a ketone introduction part, an aldehyde introduction part or an acid anhydride introduction part, and a labeling reagent introduction part.
- the reaction solution 151 contains (a) hydroxylamines and (b) a basic reagent.
- the aspect in which the reagent introduction part 140 introduces the reagent into the container 120 or the container 170 is not particularly limited.
- the tubular member is removed from the liquid supply source (151, 152, 153) in which the liquid to be supplied is stored.
- a mode in which the liquid is fed into the container 120 or the container 170 through the medium is mentioned.
- a mode in which the liquid collected in the tubular member is injected into the reaction container, and the like can be mentioned.
- reaction liquid introduction section may be configured as separate and independent structural members, or may be configured as the same structural member.
- the apparatus 100 may further include a solid phase holding unit 160 that holds a container 170 including a solid phase 171 having affinity for sugar chains.
- the structure of the solid phase holder 160 may be the same as the structure of the container holder 130.
- the apparatus 100 may further include a solid-liquid separation unit 180 that separates the contents of the container 170 into a solid-liquid separation.
- the solid-liquid separator 180 separates the solid and the liquid from the contents contained in the container 170.
- the solid is substantially the solid phase 10 and sugar chains adsorbed thereto.
- the specific separation format of the solid-liquid separation unit 180 is not particularly limited, and may be any of centrifugation, reduced pressure, and pressurization.
- the separation type of the solid-liquid separation unit 180 is centrifugation.
- the solid-liquid separation unit 180 includes a rack 181 that holds the container 170, a drive shaft 182, and a motor 183.
- the solid-liquid separation unit 180 may be configured as a constituent member independent of the solid phase holding unit 160.
- the apparatus 100 may include a container transfer unit 190 that automatically transfers the container 170 from the solid phase holding unit 160 to the solid-liquid separation unit 180.
- the container transfer unit 190 may include an arm that operates to grip and open and move the container 170, and an arm control unit that controls the operation of the arm.
- the liquid is collected in the lower part of the container 170 by operating the solid-liquid separator 180. Therefore, for example, the sugar chain adsorbed on the solid phase 171 can be eluted and recovered in the lower part of the container 170.
- the apparatus 100 may further include a temperature adjusting unit 195 that adjusts the temperature of the container 120 or 170.
- a temperature adjusting unit 195 that adjusts the temperature of the container 120 or 170.
- the temperature adjustment unit 195 only needs to have at least a heater function.
- the temperature adjustment unit 195 can heat the container 120 or 170 to a necessary temperature.
- the apparatus 100 may be automatically controlled by at least any one of preferably operable components (for example, the introduction unit 141, the arm 190, the solid-liquid separation unit 180, and the temperature adjustment unit 195). This makes it possible to prepare sugar chains from glycoproteins more rapidly.
- preferably operable components for example, the introduction unit 141, the arm 190, the solid-liquid separation unit 180, and the temperature adjustment unit 195.
- Example 1 ⁇ Sugar chain analysis of monoclonal antibody (IgG1)> (N-linked sugar chain release reaction) A monoclonal antibody (IgG1, 40 ⁇ g) was dissolved in 30 ⁇ L of a 25% aqueous ammonia solution saturated with calcium acetate, 20 ⁇ L of a 50% aqueous hydroxylamine solution was added and mixed, and then at 80 ° C. in a draft using a heat block. Heated for 1 hour. Then, it immediately cooled in the ice bath and neutralized the reaction liquid with 1N hydrochloric acid.
- FIG. 1 shows the chromatogram.
- the horizontal axis represents the elution time
- the vertical axis represents the fluorescence intensity (relative value).
- Example 2 ⁇ Sugar chain analysis of human serum-derived IgG> (N-linked sugar chain release reaction) Put 20 ⁇ L of human serum IgG (200 ⁇ g) in a sample tube, add 10 ⁇ L of 50% aqueous hydroxylamine to the tube, then add 25 ⁇ L of 25% aqueous ammonia and 5 ⁇ L of 1.2 M lithium hydroxide and mix. Then, it was heated at 50 ° C. for 1 hour in a draft using a heat block. Then, it immediately cooled in the ice bath and neutralized the reaction liquid with 1N hydrochloric acid.
- FIG. 3 shows the chromatogram.
- the horizontal axis indicates the elution time
- the vertical axis indicates the fluorescence intensity (relative value).
- Example 3 ⁇ Sugar chain analysis of bovine fetuin> (O-linked sugar chain release reaction) Beef fetuin (20 ⁇ g) was dissolved in 38.5 ⁇ L of distilled water, 10 ⁇ L of 50% hydroxylamine and 1.5 ⁇ L of diazabicycloundecene were added and mixed, and then heated at 60 ° C. for 5 minutes in a draft using a heat block. did. Then, it immediately cooled in the ice bath and neutralized the reaction liquid with 1N hydrochloric acid.
- Example 4 ⁇ Sugar chain analysis of usiapotransferrin> (N-linked sugar chain release reaction) Add 20 ⁇ L of an aqueous solution of usiapotransferrin (200 ⁇ g) to a sample tube, add 20 ⁇ L of 50% hydroxylamine aqueous solution to the tube, add 10 ⁇ L of 1.0 M lithium hydroxide, mix, and then use a heat block in the draft. And heated at 50 ° C. for 1 hour. Then, it immediately cooled in the ice bath and neutralized the reaction liquid with 1N hydrochloric acid.
- FIG. 5 shows the chromatogram.
- the horizontal axis indicates the elution time
- the vertical axis indicates the fluorescence intensity (relative value).
- Example 5 ⁇ Analysis of sugar chains of horseradish peroxidase> (N-linked sugar chain release reaction) Add 20 ⁇ L of an aqueous solution containing horseradish peroxidase (200 ⁇ g) to a sample tube, add 20 ⁇ L of 50% hydroxylamine aqueous solution to the tube, add 10 ⁇ L of 1.0 M lithium hydroxide, mix, and then heat block in the draft. And heated at 50 ° C. for 1 hour. Then, it immediately cooled in the ice bath and neutralized the reaction liquid with 1N hydrochloric acid.
- Example 6 ⁇ Analysis of sugar chain of bovine submandibular gland mucin> (O-linked sugar chain release reaction) After mixing 20 ⁇ L of an aqueous solution of bovine submaxillary gland mucin (50 ⁇ g), 20 ⁇ L of 50% hydroxylamine and 10 ⁇ L of diazabicycloundecene, the mixture was heated in a fume hood at 60 ° C. for 5 minutes. Then, it immediately cooled in the ice bath and neutralized the reaction liquid with 1N hydrochloric acid.
- FIG. 7 shows the chromatogram.
- the horizontal axis indicates the elution time
- the vertical axis indicates the fluorescence intensity (relative value).
- Example 7 ⁇ N-linked sugar chain release reaction under various conditions> (A) Hydroxylamines, (b) Basic reagents, and / or (c) Various tests with various conditions such as reagent types and concentrations, temperature conditions, reaction times, etc., changed for amines. went. The conditions and yield of each test example are shown in Table 1A and Table 1B. In Test Examples 1 to 6, 200 ⁇ g of human serum IgG was used as the glycoprotein, and in Test Examples 7 to 80, 40 ⁇ g of monoclonal antibody M-L001 was used. The concentration of each reagent represents the final concentration, and the yield represents the total area value of all sugar chain peaks in HPLC.
- the result “A” indicates that a result almost the same as the conventional method using PNGase F was obtained, and “B” showed a result that the yield was less than half that of the conventional method using PNGase F.
- “C” indicates the result of detection of the peak of the decomposed sugar chain in addition to the original sugar chain, and “D” indicates that the sugar chain could hardly be analyzed.
- Test examples 1 and 2 were performed in order to confirm the influence of the combined use of lithium hydroxide and ammonia. As a result, the yield was significantly increased in Test Example 1 in which lithium hydroxide and ammonia were used in combination, compared with Test Example 2 in which ammonia was not added.
- Test Examples 4 to 6 the use of alkaline earth metal calcium salt or calcium hydroxide as a basic reagent was compared. As a result, any calcium compound was able to obtain good results, and in particular, calcium hydroxide had the highest yield.
- the yield itself fell compared with lithium hydroxide the isomerization (epimerization) which is a kind of decomposition reaction could be suppressed.
- Test Examples 8 to 14 a comparison was made by changing the concentration conditions of hydroxylamines. As a result, hydroxylamine was unable to obtain an effect at 0.5% and 150 mM. On the other hand, the effect of stabilizing the sugar chain released from 2% and 600 mM could be obtained.
- Test Examples 15 to 20 comparison was made by changing the reaction time conditions. As a result, under the condition of 80 ° C. using ammonia and calcium acetate, the most preferable result was obtained when the reaction time was about 2 hours, but when the reaction time exceeded 2 hours, the decomposition of the released sugar chain proceeded. I understood.
- (5) In Test Examples 21 to 26 comparison was made by changing the basic reagent (alkali catalyst).
- Test Examples 62 and 63 the use of hydroxylamine hydrochloride as a hydroxylamine was compared and examined in DMSO (DMSO is not indicated in the table). As a result, it was found that the reaction hardly progressed when DMSO was used as a solvent.
- Test Examples 64-67 the use of various organic bases other than DBU was compared. As a result, except for the combination of DBU and ammonia, very good yields and results were obtained.
- Test Examples 68 to 71 comparison was made regarding changes in DBU concentration. As a result, DBU was able to obtain a favorable yield and result at higher concentrations. Specifically, an excellent effect could be obtained by using 100 mM or more of DBU.
- Test Examples 72 to 75 the use of various organic bases other than DBU was compared. As a result, Proton sponge was unable to obtain a favorable yield. With organic bases other than Proton sponge, favorable yields and results could be obtained.
- Test Examples 76 to 79 changes in the concentration of DBU in the absence of amines were compared. As a result, it was possible to obtain an excellent effect by using 100 mM or more of DBU.
- Test Example 80 4% cetyltrimethylammonium hydroxide was examined. As a result, good yields and results could be obtained.
- Example 8 ⁇ O-linked sugar chain release reaction under various conditions> (A) Hydroxylamines, (b) Basic reagents, and / or (c) Various tests with various conditions such as reagent types and concentrations, temperature conditions, reaction times, etc., changed for amines. went. The conditions and yield of each test example are shown in Table 2A and Table 2B. In Test Examples 1 to 52, 20 ⁇ g of bovine fetuin was used as the glycoprotein. The concentration of each reagent represents the final concentration, and the yield represents the total area value of all sugar chain peaks in HPLC.
- the result “A” indicates that the sugar chain yield was 2000 or more and the peeling product was 20% or less, and “B” indicates that the peeling product was 20% or more or the sugar chain yield was 1000 or more and 2000 or less. “C” indicates that the yield of sugar chains was 200 or more and 1000 or less, and “D” indicates that the yield of sugar chains was 200 or less.
- Test Examples 3 and 4 compared the presence or absence of calcium when ammonia was used as a basic reagent. As a result, the yield increased due to the presence of calcium, which was a favorable result.
- Test examples 5 and 6 were compared using no base or a base weaker than ammonia. As a result, it was found that basicity stronger than that of ammonia is indispensable for the release of sugar chains.
- Test Examples 7 to 10 were compared by differences in alkali strength. As a result, favorable results were obtained in any of the test examples. Moreover, the yield increased by using strong alkali conditions. (4) In Test Examples 11 to 14, changes in temperature under strong alkali conditions were compared.
- Test Examples 40 to 44 the use of other organic bases was examined. As a result, all of TMG, t-butyl TMG, TBD, and MTBD were able to release O-linked sugar chains. In particular, TMD, TBD, and MTBD were able to obtain more favorable yields and results. .
- Test Examples 45 to 47 the concentration of DBU was examined. As a result, the higher the DBU concentration, the higher the yield.
- Test Examples 48 to 51 the concentration of hydroxylamine was examined. As a result, it was possible to suppress peeling by increasing the amount of hydroxylamine relative to DBU. (16) Test Example 52 shows test results using 4% cetyltrimethylammonium hydroxide as a basic reagent. Good yield and results could be obtained.
- Example 9 ⁇ O-linked glycan analysis of bovine fetuin combined with reagent removal by monolithic silica> (O-type sugar chain release reaction using 50% hydroxylamine) Beef fetuin (20 ⁇ g) was dissolved in 50 ⁇ L of 50% hydroxylamine, mixed with 10 ⁇ L of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), and then mixed in a draft using a heat block. And heated at 60 ° C. for 20 minutes. Then, it immediately cooled in the ice bath and neutralized the reaction liquid with 1N hydrochloric acid.
- DBU 1,8-diazabicyclo [5.4.0] undec-7-ene
- the sugar chain was eluted from the monolithic silica using 100 ⁇ L of 10% acetic acid, mixed with 900 ⁇ L of pure water, and then desalted with a graphite carbon cartridge in the same manner as in Example 1 to obtain 0.1% triglyceride.
- the sugar chain was eluted with 1 mL of 50% acetonitrile containing fluoroacetic acid (TFA) and dried under reduced pressure.
- TFA fluoroacetic acid
- Example 10 ⁇ O-linked sugar chain release reaction using 10% hydroxylamine> Bovine fetuin (20 ⁇ g) was dissolved in 50 ⁇ L of 10% hydroxylamine, 10 ⁇ L of DBU was added and mixed, and then heated in a fume hood at 60 ° C. for 20 minutes. Then, it immediately cooled in the ice bath and neutralized the reaction liquid with 1N hydrochloric acid.
- Example 11 ⁇ O-linked sugar chain release reaction using 50% hydroxylamine> Bovine fetuin (20 ⁇ g) was dissolved in 50 ⁇ L of 50% hydroxylamine, 10 ⁇ L of DBU was added and mixed, and then heated in a draft at 60 ° C. for 20 minutes using a heat block. Then, it immediately cooled in the ice bath and neutralized the reaction liquid with 1N hydrochloric acid.
- FIGS. 8A to 8D are chromatograms showing the results of HPLC analysis of the sugar chains fluorescently labeled in Examples 9 to 11.
- FIG. 8 (a) shows the results of Example 10
- FIG. 8 (b) shows the results of Example 11
- FIG. 8 (c) shows the results of Example 9 (acetone treatment)
- FIG. ) Is the result of Example 9 (salicylaldehyde treatment).
- Example 12 ⁇ Examination of sugar chain recovery from monolithic silica> (O-linked sugar chain release reaction using 50% hydroxylamine) Bovine fetuin (20 ⁇ g) was dissolved in 50 ⁇ L of 50% hydroxylamine, 10 ⁇ L of DBU was added and mixed, and then heated in a fume hood at 60 ° C. for 20 minutes. Then, it immediately cooled in the ice bath and neutralized the reaction liquid with 1N hydrochloric acid.
- FIG. 9 is a chromatogram showing the result of HPLC analysis of the sugar chain fluorescently labeled in Example 12.
- the horizontal axis indicates the elution time
- the vertical axis indicates the fluorescence intensity (relative value).
- sugar chains can be released from glycoproteins in a short processing time while suppressing the degradation of sugar chains, and the free sugar chains are also released. Can be recovered as a mixture containing a sugar chain oxime.
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Abstract
Description
[1]糖タンパク質から糖鎖を遊離させる方法であって、前記糖タンパク質に、(a)ヒドロキシルアミン類と、(b)塩基性試薬とを含む反応液を接触させて、前記糖タンパク質から遊離した糖鎖と前記反応液との混合液を得る工程を含む方法。
[2]前記反応液が前記(a)ヒドロキシルアミン類を2~70%(w/w)含む、[1]に記載の方法。
[3]前記反応液が(c)アミン類を更に含む、[1]又は[2]に記載の方法。
[4]前記(c)アミン類が、アンモニア水、メチルアミン水溶液、ジメチルアミン水溶液、エチルアミン、ジエチルアミン、エタノールアミン、エチレンジアミン、ブチルアミン、モルホリン、DABCO、アントラニル酸からなる群より選択される少なくとも一つの化合物である、[3]に記載の方法。
[5]前記糖タンパク質から遊離した糖鎖が糖鎖オキシムを含む、[1]~[4]のいずれかに記載の方法。
[6]前記糖タンパク質に前記反応液を接触させる工程において、前記反応液のpHが、8~14の範囲内である、[1]~[5]のいずれかに記載の方法。
[7]前記(a)ヒドロキシルアミン類が、ヒドロキシルアミン、ヒドロキシルアミンの塩、O置換ヒドロキシルアミン及びO置換ヒドロキシルアミンの塩からなる群より選択される少なくとも一つの化合物である、[1]~[6]のいずれかに記載の方法。
[8]前記(b)塩基性試薬が、アルカリ金属の水酸化物、アルカリ金属の弱酸塩、アルカリ土類金属の水酸化物、アンモニア水溶液に溶解したアルカリ土類金属の塩及び有機塩基からなる群より選択される少なくとも一つである、[1]~[7]のいずれかに記載の方法。
[9]前記アルカリ金属の水酸化物が、水酸化リチウム、水酸化ナトリウム又は水酸化カリウムであり、前記アルカリ金属の弱酸塩が、重炭酸ナトリウム又は炭酸ナトリウムであり、前記アルカリ土類金属の水酸化物が、水酸化カルシウム、水酸化バリウム又は水酸化ストロンチウムであり、前記アルカリ土類金属の塩が、酢酸カルシウム、塩化カルシウム、酢酸バリウム又は酢酸マグネシウムであり、前記有機塩基が、1,8-ジアザビシクロ[5.4.0]ウンデカ-7-エン、1,5,7-トリアザビシクロ[4.4.0]デカ-5-エン、7-メチル-1,5,7-トリアザビシクロ[4.4.0]デカ-5-エン、1,1,3,3-テトラメチルグアニジン、2-tert-ブチル-1,1,3,3-テトラメチルグアニジン又はセチルトリメチルアンモニウムヒドロキシドである、請求項8に記載の方法。
[10]前記混合液にケトン、アルデヒド又は酸無水物を添加し、前記混合液中に残存する(a)ヒドロキシルアミン類をケトキシム、アルドキシム又はアミドに変換する工程と、前記混合液を、糖鎖に親和性を有する固相と接触させて、前記固相に前記糖タンパク質から遊離した糖鎖を吸着させる工程と、前記固相から前記糖鎖を溶出させる工程と、を更に含む、[1]~[9]のいずれかに記載の方法。
[11]糖タンパク質が有する糖鎖の分析方法であって、[1]~[10]のいずれかに記載の方法により糖タンパク質から糖鎖を遊離する工程と、遊離した糖鎖を標識する工程であって、糖鎖オキシムの標識を含む工程と、を含む、糖鎖の分析方法。
[12]糖タンパク質から糖鎖を遊離するためのキットであって、(a)ヒドロキシルアミン類と(b)塩基性試薬とを含むキット。
[13](c)アミン類を更に含む、[12]に記載のキット。
[14]ケトン、アルデヒド又は酸無水物と、糖鎖に親和性を有する固相とを更に含む、[12]又は[13]に記載のキット。
[15][1]~[10]のいずれかに記載の方法を行うための説明書を更に含む、[12]~[14]のいずれかに記載のキット。
[16]糖タンパク質から糖鎖を遊離するためのキットであって、(a)ヒドロキシルアミン類と併用して使用するための(b)塩基性試薬と(c)アミン類とを含むキット。
[17]ケトン、アルデヒド又は酸無水物と、糖鎖に親和性を有する固相とを更に含む、[16]に記載のキット。
[18][1]~[10]のいずれかに記載の方法を行うための説明書を更に含む、[16]又は[17]に記載のキット。
[19]糖タンパク質を含む試料が収容される容器を保持する容器保持部と、前記容器に試薬を導入する試薬導入部と、を備え、前記試薬導入部が、前記容器に(a)ヒドロキシルアミン類と、(b)塩基性試薬とを含む反応液を導入する反応液導入部を含む、糖タンパク質から糖鎖を遊離させる装置。
[20]前記試薬導入部が、前記容器にケトンを導入するケトン導入部、アルデヒドを導入するアルデヒド導入部又は酸無水物を導入する酸無水物導入部を更に含む、[19]に記載の装置。
[21]糖鎖に親和性を有する固相を含む容器を保持する固相保持部を更に備える、[19]又は[20]に記載の装置。
〔1〕糖タンパク質から糖鎖を遊離させる方法であって、
前記糖タンパク質に、(a)ヒドロキシルアミン類と、(b)塩基性試薬とを含む反応液を接触させる工程であって、前記(a)ヒドロキシルアミン類が、2~50%(w/w)で反応液中に含まれる工程、を含む方法に関する。
ここで、本発明の糖タンパク質から糖鎖を遊離させる方法は、一実施の形態において、〔2〕上記〔1〕に記載の方法であって、
前記反応液がさらに(c)アミン類を含むことを特徴とする。
また、本発明の糖タンパク質から糖鎖を遊離させる方法は、一実施の形態において、〔3〕上記〔1〕または〔2〕に記載の方法であって、
前記糖タンパク質から遊離した糖鎖が糖鎖オキシムを含むことを特徴とする。
また、本発明の糖タンパク質から糖鎖を遊離させる方法は、一実施の形態において、〔4〕上記〔1〕~〔3〕のいずれかに記載の方法であって、
前記糖タンパク質に前記反応液を接触させる工程において、前記反応液のpHが、8~14の範囲内であることを特徴とする。
また、本発明の糖タンパク質から糖鎖を遊離させる方法は、一実施の形態において、
〔5〕上記〔1〕~〔4〕のいずれかに記載の方法であって、
前記(a)ヒドロキシルアミン類が、ヒドロキシルアミンおよびその塩類、ならびに、O置換ヒドロキシルアミンおよびその塩類からなる群より選択される少なくとも一つの化合物であることを特徴とする。
また、本発明の糖タンパク質から糖鎖を遊離させる方法は、一実施の形態において、
〔6〕上記〔1〕~〔5〕のいずれかに記載の方法であって、
前記(b)塩基性試薬が、アルカリ金属水酸化物、アルカリ金属弱酸塩、アルカリ土類水酸化物、アンモニア水溶液に溶解したアルカリ土類の塩、および、有機塩基からなる群より選択される少なくとも一つであることを特徴とする。
また、本発明の糖タンパク質から糖鎖を遊離させる方法は、一実施の形態において、〔7〕上記〔6〕に記載の方法であって、
前記アルカリ金属水酸化物、アルカリ金属弱酸塩、または、アルカリ土類水酸化物が、水酸化リチウム、水酸化ナトリウム、水酸化カリウム、重炭酸ナトリウム、炭酸ナトリウム、水酸化カルシウム、水酸化バリウム、水酸化ストロンチウム、からなる群より選択される少なくとも一つであり、
前記アンモニア水溶液に溶解したアルカリ土類の塩が、酢酸カルシウム、塩化カルシウム、酢酸バリウム、酢酸マグネシウムからなる群より選択される少なくとも一つであることを特徴とする。
また、本発明の糖タンパク質から糖鎖を遊離させる方法は、一実施の形態において、〔8〕上記〔6〕に記載の方法であって、
前記有機塩基が、1,8-ジアザビシクロ[5.4.0]ウンデカ-7-エン、1,5,7-トリアザビシクロ[4.4.0]デカ-5-エン、7-メチル-1,5,7-トリアザビシクロ[4.4.0]デカ-5-エン、1,1,3,3-テトラメチルグアニジン、2-tert-ブチル-1,1,3,3-テトラメチルグアニジン、セチルトリメチルアンモニウムヒドロキシドからなる群より選択される少なくとも一つであることを特徴とする。
また、本発明の糖タンパク質から糖鎖を遊離させる方法は、一実施の形態において、
〔9〕上記〔1〕~〔6〕のいずれかに記載の方法であって、
前記(c)アミン類が、アンモニア、メチルアミン、ジメチルアミン、エチルアミン、ジエチルアミン、エタノールアミン、エチレンジアミン、ブチルアミン、モルホリン、DABCO、アントラニル酸からなる群より選択される少なくとも一つの化合物であることを特徴とする。
また、本発明は、別の態様において、
〔10〕糖タンパク質が有する糖鎖の分析方法であって、
請求項1~9のいずれか一項に記載の方法により糖タンパク質から糖鎖を遊離する工程と、
遊離した糖鎖を標識する工程であって、糖鎖オキシムの標識を含む工程と
を含む、糖鎖の分析方法に関する。
また、本発明は、別の態様において、
〔11〕糖タンパク質から糖鎖を遊離するためのキットであって、
(a)ヒドロキシルアミン類と(b)塩基性試薬とを含むキット。
また、本発明の糖タンパク質から糖鎖を遊離するためのキットは、一実施の形態において、
〔12〕上記〔11〕に記載のキットであって、
(a)アミン類をさらに含むことを特徴とする。
また、本発明の糖タンパク質から糖鎖を遊離するためのキットは、一実施の形態において、
〔13〕糖タンパク質から糖鎖を遊離するためのキットであって、
(a)ヒドロキシルアミン類と併用して使用するための(b)塩基性試薬と(c)アミン類とを含むキットであることを特徴とする。
一実施形態において、本発明は、糖タンパク質から糖鎖を遊離させる方法であって、前記糖タンパク質に、(a)ヒドロキシルアミン類と、(b)塩基性試薬とを含む反応液を接触させて、前記糖タンパク質から遊離した糖鎖と前記反応液との混合液を得る工程を含む方法を提供する。糖タンパク質を、(a)ヒドロキシルアミン類と、(b)塩基性試薬とを含む反応液と接触させることにより、当該糖タンパク質から糖鎖を遊離することができる。
一実施形態において、本発明は、糖タンパク質が有する糖鎖の分析方法であって、上述した方法により糖タンパク質から糖鎖を遊離する工程と、遊離した糖鎖を標識する工程と、を含む、糖鎖の分析方法を提供する。
一実施形態において、本発明は、糖タンパク質から糖鎖を遊離するためのキットであって、(a)ヒドロキシルアミン類と(b)塩基性試薬とを含むキットを提供する。また、本実施形態のキットは、一形態の形態において、(a)ヒドロキシルアミン類と(b)塩基性試薬とに加えて、さらに(c)アミン類を含むキットである。また、本実施形態の糖タンパク質から糖鎖を遊離するためのキットは、別の一実施形態において、(a)ヒドロキシルアミン類と併用して使用するための(b)塩基性試薬と(c)アミン類とを含むキットである。
一実施形態において本発明は、糖タンパク質を含む試料が収容される容器を保持する容器保持部と、前記容器に試薬を導入する試薬導入部と、を備え、前記試薬導入部が、前記容器に(a)ヒドロキシルアミン類と、(b)塩基性試薬とを含む反応液を導入する反応液導入部を含む、糖タンパク質から糖鎖を遊離させる装置を提供する。なお、以下に説明する装置の構成はあくまで一例であり、本実施形態の装置はこの構成に拘束されるものではない。
<モノクローナル抗体(IgG1)の糖鎖分析>
(N結合型糖鎖遊離反応)
モノクローナル抗体(IgG1、40μg)を酢酸カルシウムで飽和させた25%アンモニア水溶液30μLに溶解し、50%ヒドロキシルアミン水溶液を20μLを添加して混合した後、ドラフト中、ヒートブロックを用いて80℃にて1時間加熱した。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。
グラファイトカーボンを充填した固相抽出カートリッジ(HyperSep Hypercarb 25mg、サーモサイエンティフィック社製)を用いて中和した反応液を脱塩し、続いてピコリンボランと2-アミノベンズアミドを用いて糖鎖を蛍光標識した。蛍光標識糖鎖はSephadex G-15(GEヘルスケア社製)で精製した後、HPLCにて分析した。図1にそのクロマトグラムを示した。図1中、横軸は溶出時間を示し、縦軸は蛍光強度(相対値)を示す。
従来の方法と比較するため酵素(PNGaseF)を用いて糖鎖遊離反応を行った。モノクローナル抗体(IgG1、40μg)を100mMジチオスレイトールと0.5%SDSを含む500mMトリス塩酸緩衝液(pH8.6)40μLに溶解し、80℃で10分間加熱した。その後、室温まで冷却した後、5%Nonidet P-40(40μL)と蒸留水15μLを加え、さらにPNGaseFを5μL(16mU、Takara bio Inc.)添加し37℃にて16時間インキュベートした。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。その後、固相抽出カートリッジSepPak C18(50mg、Waters Corp.)に供し、蒸留水で洗浄した。濾液と洗浄液を合わせて、実施例1に記載した方法により固相抽出カートリッジ(HyperSep Hypercarb 25mg)にて脱塩処理、2-アミノベンズアミドによる蛍光標識、Sephadex G-15(GEヘルスケア社製)による精製を経てHPLCに供した。図2にそのクロマトグラムを示した。図2中、横軸は溶出時間を示し、縦軸は蛍光強度(相対値)を示す。図1および図2に示すように、本実施例の方法により、従来法と同様のクロマトグラムが得られることが明らかとなった。
<ヒト血清由来IgGの糖鎖分析>
(N結合型糖鎖遊離反応)
ヒト血清由来IgG(200μg)の水溶液20μLをサンプルチューブに入れ、そのチューブに50%ヒドロキシルアミン水溶液を10μL添加し、次いで25%アンモニア水溶液の25μLと5μLの1.2M水酸化リチウムとを加えて混合した後、ドラフト中、ヒートブロックを用いて50℃、1時間加熱した。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。
実施例1と同様の方法により、反応液を脱塩後、糖鎖を蛍光標識してHPLCにて分析した。図3にそのクロマトグラムを示した。図3中、横軸は溶出時間を示し、縦軸は蛍光強度(相対値)を示す。その結果、これまでに報告されている、ヒト血清由来IgGから遊離させた糖鎖の分析結果と同様の結果が得られた。
<ウシフェツインの糖鎖分析>
(O結合型糖鎖遊離反応)
ウシフェツイン(20μg)を蒸留水38.5μLに溶解し、50%ヒドロキシルアミン10μLとジアザビシクロウンデセン1.5μLを添加し混合した後、ドラフト中、ヒートブロックを用いて60℃にて5分間加熱した。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。
実施例1と同様の方法により、反応液を脱塩後、糖鎖を蛍光標識してHPLCにて分析した。図4にそのクロマトグラムを示した。図4中、横軸は溶出時間を示し、縦軸は蛍光強度(相対値)を示す。その結果、これまでに報告されている、ウシフェツインから遊離させた糖鎖の分析結果と同様の結果が得られた。
<ウシアポトランスフェリンの糖鎖分析>
(N結合型糖鎖遊離反応)
ウシアポトランスフェリン(200μg)の水溶液20μLをサンプルチューブに入れ、そのチューブに50%ヒドロキシルアミン水溶液を20μL添加し、次いで1.0M 水酸化リチウムを10μL加えて混合した後、ドラフト中、ヒートブロックを用いて50℃、1時間加熱した。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。
実施例1と同様の方法により、反応液を脱塩後、糖鎖を蛍光標識してHPLCにて分析した。図5にそのクロマトグラムを示した。図5中、横軸は溶出時間を示し、縦軸は蛍光強度(相対値)を示す。その結果、Nグリコリルノイラミン酸を含有する糖鎖も分析できることが明らかとなった。
<セイヨウワサビペルオキシダーゼの糖鎖分析>
(N結合型糖鎖遊離反応)
セイヨウワサビペルオキシダーゼ(200μg)を含む水溶液20μLをサンプルチューブに入れ、そのチューブに50%ヒドロキシルアミン水溶液を20μL添加し、次いで1.0M 水酸化リチウムを10μL加えて混合した後、ドラフト中、ヒートブロックを用いて50℃、1時間加熱した。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。
実施例1と同様の方法により、反応液を脱塩後、糖鎖を蛍光標識してHPLCにて分析した。図6にそのクロマトグラムを示した。図6中、横軸は溶出時間を示し、縦軸は蛍光強度(相対値)を示す。その結果、還元末端のNアセチルグルコサミンの3位にフコースを有するN結合型糖鎖の分析も可能であることが明らかとなった。
<ウシ顎下腺ムチンの糖鎖分析>
(O結合型糖鎖遊離反応)
ウシ顎下腺ムチン(50μg)の水溶液20μLと50%ヒドロキシルアミン20μLとジアザビシクロウンデセン10μLを混合した後、ドラフト中、ヒートブロックを用いて60℃にて5分間加熱した。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。
実施例1と同様の方法により、反応液を脱塩後、糖鎖を蛍光標識してHPLCにて分析した。図7にそのクロマトグラムを示した。図7中、横軸は溶出時間を示し、縦軸は蛍光強度(相対値)を示す。その結果、これまでに報告されているものと同様の結果が得られた。
<各種条件下におけるN結合型糖鎖遊離反応>
(a)ヒドロキシルアミン類、(b)塩基性試薬、および/または、(c)アミン類を含む反応液について、試薬の種類や濃度、温度条件、反応時間などの各種条件を変更した各種試験を行った。各試験例の条件および収量を表1A及び表1Bに示す。なお、試験例1~6において、糖タンパク質はhuman serum IgG 200μgを用い、試験例7~80においては、モノクローナル抗体M-L001 40μgを用いた。また、各試薬の濃度は終濃度を示し、収量は、HPLCにおける全糖鎖ピークの面積値の合計を表す。また、結果の「A」はPNGaseFを用いた従来法とほぼ同等の結果が得られたことを示し、「B」はPNGaseFを用いた従来法に比べて収量が半分以下の結果が得られたことを示し、「C」は本来の糖鎖に加えて分解した糖鎖のピークが検出された結果を示し、「D」は糖鎖がほとんど分析できなかったことを示す。
(2)試験例4~6では、塩基性試薬としてアルカリ土類金属のカルシウム塩または水酸化カルシウムの使用を比較した。その結果、いずれのカルシウム化合物も良好な結果を得ることができ、特に、水酸化カルシウムが最も収量が高かった。なお、水酸化リチウムと比較すると、収量自体は下がっていたが、分解反応の一種である異性化(エピメリゼーション)を抑制することができていた。
(3)試験例8~14では、ヒドロキシルアミン類の濃度条件を変更して比較を行った。その結果、ヒドロキシルアミンは、0.5%、150mMでは効果を得ることができなかった。一方で、2%、600mMから遊離された糖鎖の安定化の効果を得ることができた。
(4)試験例15~20では、反応時間の条件を変更して比較を行った。その結果、アンモニアと酢酸カルシウムを用いた80℃の条件下においては、反応時間が2時間程度において、最も好ましい結果が得られたが、2時間を超えると遊離した糖鎖の分解が進行することがわかった。
(5)試験例21~26では、塩基性試薬(アルカリ触媒)を変更して比較を行った。その結果、弱アルカリでも糖鎖の遊離を行うことができ、各塩基性試薬において好ましい収量および結果を得ることができた。
(6)試験例27~32では、塩基性試薬のうち各種のアンモニア水に溶解したアルカリ土類金属の塩または水酸化物を用い、比較を行った。その結果、酢酸マグネシウム以外は良好な結果を得ることができた。
(7)試験例33~38では、アミンを変更して比較を行った。その結果、メチルアミン、ジメチルアミンは、収量が増加する一方で、異性化やアミドの分解(デアミデーション)が促進した。エチレンジアミン、および、エタノールアミンの使用は、異性化を抑制しつつ、収量を増加させることができた。
(8)試験例39~42では、低pKaを有する各種アミンを用い、比較を行った。その結果、DABCOおよびモルホリンにおいては、良好な収量および結果をえることができた。特に、DABCOは、デアミデーションが全く見られなかった。
(9)試験例43~48では、KOHの濃度を変更(2mM~2M)して比較を行った。その結果、KOHの濃度が高い方が好ましい結果を得ることができた。
(10)試験例49、50では、塩基性試薬としてのDBUを用い、水酸化リチウムと比較した。その結果、DBUを用いた方が、水酸化リチウムよりも好ましい収量を得ることができた。
(11)試験例51~54では、DBU存在下におけるヒドロキシルアミン類の濃度を変更して比較を行った。その結果、ヒドロキシルアミンの濃度が少なくとも10%となるまでは濃度依存的に好ましい収量および結果を得ることができた。
(12)試験例55~57では、DBU存在下、50℃における反応時間(60分~240分)の変更について比較した。その結果、1時間が最も好ましく、1時間以上の反応時間では、反応時間が伸びるほど、収量および結果が悪くなった。
(13)試験例58~61では、室温における反応時間について比較した。その結果、室温では反応速度が低く、酵素を用いた反応の半量程度の収量をえるのに16時間を要した。
(14)試験例62、63では、ヒドロキシルアミン類として、ヒドロキシルアミン塩酸塩の使用についてDMSO中で比較、検討した(表中には、DMSOの表示なし)。その結果、DMSOを溶媒とした場合にはほとんど反応が進行しないことが判明した。
(15)試験例64~67では、DBU以外の各種有機塩基の使用について比較した。その結果、DBUとアンモニアとの組み合わせ以外は、いずれも非常に良好な収量および結果を得られた。
(16)試験例68~71では、DBUの濃度の変更について比較した。その結果、DBUは、濃度が高い方が好ましい収量および結果を得ることができた。具体的には、100mM以上のDBUを用いることで優れた効果を得ることができた。
(17)試験例72~75では、DBU以外の各種有機塩基の使用について比較した。その結果、Proton spongeでは、好ましい収量を得ることができなかった。Proton sponge以外の有機塩基では、好ましい収量および結果を得ることができた。
(18)試験例76~79では、アミン類非存在下におけるDBUの濃度の変更について比較した。その結果、100mM以上のDBUを用いることで優れた効果を得ることができた。
(19)試験例80では、4%セチルトリメチルアンモニウム水酸化物を検討した。その結果、良好な収量および結果を得ることができた。
<各種条件下におけるO結合型糖鎖遊離反応>
(a)ヒドロキシルアミン類、(b)塩基性試薬、および/または、(c)アミン類を含む反応液について、試薬の種類や濃度、温度条件、反応時間などの各種条件を変更した各種試験を行った。各試験例の条件および収量を表2A及び表2Bに示す。なお、試験例1~52においては、糖タンパク質としてウシフェツイン20μgを用いた。また、各試薬の濃度は終濃度を示し、収量は、HPLCにおける全糖鎖ピークの面積値の合計を表す。また、結果の「A」は糖鎖収量が2000以上で、かつピーリング産物が20%以下であったことを示し、「B」はピーリング産物が20%以上または糖鎖収量が1000以上2000以下であったことを示し、「C」は糖鎖収量が200以上1000以下であったことを示し、「D」は糖鎖収量が200以下であったことを示す。
(2)試験例5、6は、塩基なしまたはアンモニアよりも弱い塩基を用いて比較を行った。その結果、アンモニアよりも強い塩基性が糖鎖の遊離には必要不可欠であることがわかった。
(3)試験例7~10は、アルカリの強度の違いによる比較を行った。その結果、いずれの試験例でも好ましい結果を得られた。また、強アルカリ条件下とすることで、収量が増加した。
(4)試験例11~14では、強アルカリ条件下での温度の変化について比較した。その結果、60~75℃までは、温度の上昇に伴い、収量が増加し、非常に好ましい結果が得られた。一方、90℃でも好ましい結果は得られたが、収量が若干低下し、ピーリングが増加し始めた。
(5)試験例15~18では、強アルカリ条件下でのアンモニア濃度の変化について比較した。その結果、強アルカリ条件下においては、O結合型糖鎖の遊離にアンモニアの存在は不要であった。
(6)試験例19では、ヒドロキシルアミンの有無の影響について比較した。その結果、ヒドロキシルアミン不存在の条件では、糖鎖のほとんどがピーリングを引き起こしていた。
(7)試験例20~26では、アントラニル酸存在下で反応時間を変更した際の影響について比較した。また、21~26では反応液にDMSOを添加している。その結果、アントラニル酸を添加する場合にはDMSOが必要で、試験例の条件において反応時間30分までは、ピーリングを抑えたO結合型糖鎖の遊離を行うことができた。一方、30分以上ではピーリングが生じ、1時間以上では、ピーリングとは異なる分解物が生じていた。
(8)試験例27、28では、LiOHの代わりに、高濃度KOHの使用を検討した。その結果は、高濃度のKOHでは全くO結合型糖鎖を遊離させることができなかった。
(9)試験例29、30では、アルカリ(KOH、NaOH)の検討を行った。その結果、いずれのアルカリも、遊離したO結合型糖鎖としてジシアリル糖鎖が少なく、LiOHの方が好ましい結果を示した。
(10)試験例31、32では、DABCOの効果の検討を行った。その結果、DABCOの添加により収量は減少するものの、ピーリングを抑制することができた。
(11)試験例33、34では、塩基性試薬としてのDBUとLiOHとの比較を行った。その結果、DBUを使用した試験の方が、収量が良く、また、ジシアリル糖鎖の量も相対的に多い結果となった。
(12)試験例35~38は、200mMDBU、10%ヒドロキシルアミンを用いた際の反応時間の長さについて比較した。その結果、室温でもO結合型糖鎖の遊離反応は時間の経過ごとに進行した。また、4時間の反応後であっても、ピーリングした糖鎖はほとんどなかった。
(13)試験例40~44では、他の有機塩基の使用を検討した。その結果、TMG、t-ブチルTMG、TBD、MTBDのいずれもO結合型糖鎖の遊離を行うことができ、特に、TMD、TBD、MTBDの方がより好ましい収量および結果を得ることができた。
(14)試験例45~47では、DBUの濃度について検討した。その結果、DBUの濃度が高い方が、収量が増加した。
(15)試験例48~51では、ヒドロキシルアミンの濃度の検討を行った。その結果、ヒドロキシルアミンの量はDBUよりも相対的に多くすることでピーリングを抑えることが可能であった。
(16)試験例52は、塩基性試薬として、4%セチルトリメチルアンモニウム水酸化物を用いた試験結果を示す。良好な収量および結果を得ることができた。
<モノリスシリカによる試薬除去を組みあわせたウシフェツインのO結合型糖鎖分析>
(50%ヒドロキシルアミンを用いたO型糖鎖遊離反応)
ウシフェツイン(20μg)を50%ヒドロキシルアミン50μLに溶解し、1,8-ジアザビシクロ[5.4.0]ウンデカ-7-エン(DBU)10μLを添加して混合した後、ドラフト中、ヒートブロックを用いて60℃で20分間加熱した。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。
中和した反応液にアセトン200μL又はサリチルアルデヒド200μLをそれぞれ添加し攪拌した。さらに、上記の反応液にアセトニトリル16mLを添加し、あらかじめアセトニトリルで平衡化したモノリスシリカ(シリカモノリススピンカラム、Cleanup column、住友ベークライト社製)に全量アプライし、続いてアセトニトリル1.2mLで洗浄した。続いて、10%酢酸100μLを用いてモノリスシリカから糖鎖を溶出し、900μLの純水と混合した後、実施例1と同様にしてグラファイトカーボンカートリッジによる脱塩処理を行い、0.1%トリフルオロ酢酸(TFA)を含む50%アセトニトリル1mLで糖鎖を溶出し、減圧乾固した。
乾固した糖鎖を10%酢酸25μLに溶解し、ピコリンボランと2-アミノ安息香酸を用いて糖鎖を蛍光標識した。蛍光標識糖鎖は、モノリスシリカ(シリカモノリススピンカラム、Cleanup column、住友ベークライト社製)で過剰の試薬を除去した後、HPLCで分析した。
<10%ヒドロキシルアミンを用いたO結合型糖鎖遊離反応>
ウシフェツイン(20μg)を10%ヒドロキシルアミン50μLに溶解し、DBU 10μLを添加して混合した後、ドラフト中、ヒートブロックを用いて60℃で20分間加熱した。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。
実施例1と同様にして、グラファイトカーボンを充填した固相抽出カートリッジ(HyperSep Hypercarb 25mg、サーモサイエンティフィック社製)を用いて中和した反応液を脱塩処理し、0.1%TFAを含む50%アセトニトリル1mLで糖鎖を溶出し、減圧乾固した。
実施例9と同様にして糖鎖を蛍光標識し、HPLCで分析した。
<50%ヒドロキシルアミンを用いたO結合型糖鎖遊離反応>
ウシフェツイン(20μg)を50%ヒドロキシルアミン50μLに溶解し、DBU 10μLを添加して混合した後、ドラフト中、ヒートブロックを用いて60℃で20分間加熱した。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。
実施例1と同様にして、グラファイトカーボンを充填した固相抽出カートリッジ(HyperSep Hypercarb 25mg、サーモサイエンティフィック社製)を用いて中和した反応液を脱塩処理し、0.1%TFAを含む50%アセトニトリル1mLで糖鎖を溶出し、減圧乾固した。
実施例9と同様にして糖鎖を蛍光標識し、HPLCで分析した。
<モノリスシリカからの糖鎖回収法の検討>
(50%ヒドロキシルアミンを用いたO結合型糖鎖遊離反応)
ウシフェツイン(20μg)を50%ヒドロキシルアミン50 μLに溶解し、DBU 10μLを添加して混合した後、ドラフト中、ヒートブロックを用いて60℃で20分間加熱した。その後、直ちに氷浴中で冷却し1N塩酸で反応液を中和した。
中和した反応液にアセトン200μLを添加し攪拌した。さらに、上記の反応液にアセトニトリル20mLを添加し、あらかじめアセトニトリルで平衡化したモノリスシリカ(シリカモノリススピンカラム、Cleanup column、住友ベークライト社製)に全量アプライし、続いてアセトニトリル1.2mLで洗浄した。
続いて、10%酢酸25μLをモノリスシリカに添加し、溶液を回収した。さらに、ピコリンボランと2-アミノ安息香酸の混合液25μLをモノリスシリカに添加後、溶液を回収し、先の溶液と混合して50℃で3時間反応させ、糖鎖を蛍光標識した。蛍光標識糖鎖は、モノリスシリカ(シリカモノリススピンカラム、Cleanup column、住友ベークライト社製)で過剰の試薬を除去した後、HPLCで分析した。
Claims (21)
- 糖タンパク質から糖鎖を遊離させる方法であって、前記糖タンパク質に、(a)ヒドロキシルアミン類と、(b)塩基性試薬とを含む反応液を接触させて、前記糖タンパク質から遊離した糖鎖と前記反応液との混合液を得る工程を含む方法。
- 前記反応液が前記(a)ヒドロキシルアミン類を2~70%(w/w)含む、請求項1に記載の方法。
- 前記反応液が(c)アミン類を更に含む、請求項1又は2に記載の方法。
- 前記(c)アミン類が、アンモニア水、メチルアミン水溶液、ジメチルアミン水溶液、エチルアミン、ジエチルアミン、エタノールアミン、エチレンジアミン、ブチルアミン、モルホリン、DABCO、アントラニル酸からなる群より選択される少なくとも一つの化合物である、請求項3に記載の方法。
- 前記糖タンパク質から遊離した糖鎖が糖鎖オキシムを含む、請求項1~4のいずれか一項に記載の方法。
- 前記糖タンパク質に前記反応液を接触させる工程において、前記反応液のpHが、8~14の範囲内である、請求項1~5のいずれか一項に記載の方法。
- 前記(a)ヒドロキシルアミン類が、ヒドロキシルアミン、ヒドロキシルアミンの塩、O置換ヒドロキシルアミン及びO置換ヒドロキシルアミンの塩からなる群より選択される少なくとも一つの化合物である、請求項1~6のいずれか一項に記載の方法。
- 前記(b)塩基性試薬が、アルカリ金属の水酸化物、アルカリ金属の弱酸塩、アルカリ土類金属の水酸化物、アンモニア水溶液に溶解したアルカリ土類金属の塩及び有機塩基からなる群より選択される少なくとも一つである、請求項1~7のいずれか一項に記載の方法。
- 前記アルカリ金属の水酸化物が、水酸化リチウム、水酸化ナトリウム又は水酸化カリウムであり、
前記アルカリ金属の弱酸塩が、重炭酸ナトリウム又は炭酸ナトリウムであり、
前記アルカリ土類金属の水酸化物が、水酸化カルシウム、水酸化バリウム又は水酸化ストロンチウムであり、
前記アルカリ土類金属の塩が、酢酸カルシウム、塩化カルシウム、酢酸バリウム又は酢酸マグネシウムであり、
前記有機塩基が、1,8-ジアザビシクロ[5.4.0]ウンデカ-7-エン、1,5,7-トリアザビシクロ[4.4.0]デカ-5-エン、7-メチル-1,5,7-トリアザビシクロ[4.4.0]デカ-5-エン、1,1,3,3-テトラメチルグアニジン、2-tert-ブチル-1,1,3,3-テトラメチルグアニジン又はセチルトリメチルアンモニウムヒドロキシドである、請求項8に記載の方法。 - 前記混合液にケトン、アルデヒド又は酸無水物を添加し、前記混合液中に残存する(a)ヒドロキシルアミン類をケトキシム、アルドキシム又はアミドに変換する工程と、
前記混合液を、糖鎖に親和性を有する固相と接触させて、前記固相に前記糖タンパク質から遊離した糖鎖を吸着させる工程と、
前記固相から前記糖鎖を溶出させる工程と、
を更に含む、請求項1~9のいずれか一項に記載の方法。 - 糖タンパク質が有する糖鎖の分析方法であって、
請求項1~10のいずれか一項に記載の方法により糖タンパク質から糖鎖を遊離する工程と、
遊離した糖鎖を標識する工程であって、糖鎖オキシムの標識を含む工程と、
を含む、糖鎖の分析方法。 - 糖タンパク質から糖鎖を遊離するためのキットであって、(a)ヒドロキシルアミン類と(b)塩基性試薬とを含むキット。
- (c)アミン類を更に含む、請求項12に記載のキット。
- ケトン、アルデヒド又は酸無水物と、糖鎖に親和性を有する固相とを更に含む、請求項12又は13に記載のキット。
- 請求項1~10のいずれか一項に記載の方法を行うための説明書を更に含む、請求項12~14のいずれか一項に記載のキット。
- 糖タンパク質から糖鎖を遊離するためのキットであって、(a)ヒドロキシルアミン類と併用して使用するための(b)塩基性試薬と(c)アミン類とを含むキット。
- ケトン、アルデヒド又は酸無水物と、糖鎖に親和性を有する固相とを更に含む、請求項16に記載のキット。
- 請求項1~10のいずれか一項に記載の方法を行うための説明書を更に含む、請求項16又は17に記載のキット。
- 糖タンパク質を含む試料が収容される容器を保持する容器保持部と、前記容器に試薬を導入する試薬導入部と、を備え、
前記試薬導入部が、前記容器に(a)ヒドロキシルアミン類と、(b)塩基性試薬とを含む反応液を導入する反応液導入部を含む、糖タンパク質から糖鎖を遊離させる装置。 - 前記試薬導入部が、前記容器にケトンを導入するケトン導入部、アルデヒドを導入するアルデヒド導入部又は酸無水物を導入する酸無水物導入部を更に含む、請求項19に記載の装置。
- 糖鎖に親和性を有する固相を含む容器を保持する固相保持部を更に備える、請求項19又は20に記載の装置。
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JP6667856B2 (ja) | 2020-03-18 |
EP3521319B1 (en) | 2024-01-10 |
KR102100181B1 (ko) | 2020-04-13 |
US20190309003A1 (en) | 2019-10-10 |
KR20190038665A (ko) | 2019-04-08 |
JPWO2018062167A1 (ja) | 2019-06-24 |
JP2020073670A (ja) | 2020-05-14 |
EP3521319C0 (en) | 2024-01-10 |
CN113004430B (zh) | 2022-12-30 |
EP3521319A4 (en) | 2020-06-03 |
US11325935B2 (en) | 2022-05-10 |
CN113004430A (zh) | 2021-06-22 |
EP3521319A1 (en) | 2019-08-07 |
CN109983034A (zh) | 2019-07-05 |
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