Method for preparing sample for analysis and analysis method

Abstract

A method for analyzing an O-binding sugar chain that has been specifically modified in a sialic acid binding manner, the method comprising: a modification step for adding a modifier capable of specifically modifying the O-binding sugar chain in a sialic acid binding manner to a sample containing a glycoprotein that binds to the O-binding sugar chain; a dissociation step for dissociating, from the glycoprotein, the O-binding sugar chain that has been specifically modified by the sialic acid binding method; and an analysis step for analyzing the dissociated O-binding sugar chain that has been specifically modified by the sialic acid binding method.

Description

Technical Field

[0001] The present invention relates to a method for preparing a sample for analysis and an analysis method. Background Art

[0002] Sialic acid is also contained in glycoproteins in the body, and in this case it is mainly present at the end of the sugar chain. Sialic acid is easily arranged on the outside of the glycoprotein molecule, so it is easy to participate in the recognition of other molecules. The binding mode between sialic acid and adjacent sugars is sometimes different. For example, it is known that α2,3- and α2,6- are the main binding modes in human N-bound sugar chains, and in addition to these, α2,8- and α2,9- are known in O-bound sugar chains and glycosphingolipids. Different binding modes can cause sialic acid to be recognized by different molecules and have different effects, so it is important to analyze the binding mode of sialic acid.

[0003] However, sialic acid has a negative charge and is easily decomposed, so it is not easy to analyze sialic acid-containing sialyl sugar chains. In addition, the difference in sialic acid binding does not change the mass of the sugar chain, so it is impossible to distinguish and analyze the binding method using mass spectrometry.

[0004] In order to distinguish and analyze the binding mode of sialic acid, a method for specifically modifying the binding mode of sialic acid is proposed. This method utilizes the property that α2,3-sialic acid is more susceptible to intramolecular dehydration due to a dehydration condensation agent than α2,6-sialic acid, so that α2,3-sialic acid is lactonized by intramolecular dehydration, and α2,6-sialic acid is reacted with a nucleophilic agent such as an alcohol or an amine. Thus, molecules with different masses are generated according to the binding mode of sialic acid, so that the binding mode of sialic acid can be distinguished and analyzed by mass spectrometry. Non-patent document 1 discloses a sample preparation method for mass spectrometry, in which a solution containing isopropylamine and a dehydration condensation agent is added to the free N-bound sugar chain to lactonize α2,3-sialic acid and amidate α2,6-sialic acid.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Nishikaze T., Tsumoto H., Sekiya S., Iwamoto S., Miura Y., Tanaka K., Analytical Chemistry, 2017, 89, 2353-2360. Summary of the invention

[0008] Problem that the invention aims to solve

[0009] The present inventors have found that when analyzing the sialic acid binding mode, there is a decrease in the accuracy of distinguishing α2,6-sialic acid and α2,3-sialic acid. It is believed that this is because when the O-bound sugar chain released from the glycoprotein is subjected to sialic acid binding mode-specific modification (amidation), the α2,6-sialic acid bound to the N-acetylgalactosamine (GalNAc) at the reducing end of the O-bound sugar chain is not correctly modified, and a part of it undergoes the same reaction as α2,3-sialic acid. The present invention is made in view of the above situation, and its purpose is to distinguish and modify the α2,6-sialic acid and α2,3-sialic acid connected to the O-bound sugar chain, and to free the O-bound sugar chain that has been specifically modified in terms of binding mode from the glycoprotein and analyze it with high accuracy.

[0010] Solutions for solving problems

[0011] The present invention relates to:

[0012] A method for analyzing an O-bound sugar chain that has been specifically modified in terms of sialic acid binding, comprising:

[0013] a modification step, adding a modifying agent capable of specifically modifying the sialic acid binding mode of the O-bound sugar chain to the sample containing the glycoprotein bound to the O-bound sugar chain;

[0014] a releasing step of releasing the O-bound sugar chain specifically modified in the sialic acid binding mode from the glycoprotein; and

[0015] In the analysis step, the released O-bound sugar chains specifically modified in the above-mentioned sialic acid binding mode are analyzed.

[0016] Effects of the Invention

[0017] According to the present invention, α2,6-sialic acid and α2,3-sialic acid linked to O-linked sugar chains can be distinguished and modified, and O-linked sugar chains modified in a specific manner can be released from glycoproteins and analyzed with high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a flowchart showing an analysis method according to one embodiment of the present invention.

[0019] Figure 2 This is a mass spectrum of sugar chains contained in glycoproteins derived from serum and cerebrospinal fluid in Experiment 1. IS stands for internal standard.

[0020] Figure 3The upper row is a mass spectrum of sugar chains contained in the serum-derived glycoprotein in Experiment 2. The lower row is a mass spectrum of sugar chains contained in the serum-derived glycoprotein in Experiment 3.

[0021] Figure 4 The upper row is a mass spectrum of sugar chains when sugar chains are modified by the MTT method using a glycoprotein having an O-linked sugar chain in Experiment 4. The lower row is a mass spectrum of sugar chains when sugar chains are released from a glycoprotein having an O-linked sugar chain in Experiment 4 and then modified specifically in a sialic acid-linked manner.

[0022] Figure 5 This is the mass spectrum of the sugar chains released by β-elimination reaction after sialic acid-specific modification of the glycoprotein with O-linked sugar chains in Experiment 5.

[0023] Figure 6 This is a graph showing the ratio of sialic acid groups T that were not methylamidated when the methylamine concentration was changed in Experiment 5.

[0024] Figure 7 The upper row is a graph showing the amount of O-bound sugar chains recovered when the pH in the β-elimination reaction was changed (vertical axis: relative value to the internal standard) in Experiment 6. The lower row is a graph showing the types of sugar chains recovered and their existence ratios when the pH in the β-elimination reaction was changed in Experiment 6.

[0025] Figure 8 This is a graph showing the ratio of the decomposition product of the sialyl group T that is not methylamidated when the pH in the β-elimination reaction is changed in Experiment 6. The large figure and the small figure differ only in the scale of the vertical axis, and show the same experimental data. In the small figure, items 3 to 5 show the upper limit of the vertical axis, and the actual ratio is shown in the large figure.

[0026] Fig. 9 This is a graph showing the ratio of peeling reaction when the pH in the β-elimination reaction is changed in Experiment 6. The large and small figures differ only in the scale of the vertical axis, and show the same experimental data. In the small figure, items 3 to 5 show the upper limit of the vertical axis, and the actual ratio is shown in the large figure. DETAILED DESCRIPTION

[0027] A method for analyzing an O-bound sugar chain specifically modified in a sialic acid binding mode according to one embodiment of the present invention comprises:

[0028] a modification step, adding a modifying agent capable of specifically modifying the sialic acid binding mode of the O-bound sugar chain to the sample containing the glycoprotein bound to the O-bound sugar chain;

[0029] a releasing step of releasing the O-bound sugar chain specifically modified in the sialic acid binding mode from the glycoprotein; and

[0030] In the analysis step, the released O-bound sugar chains specifically modified in the above-mentioned sialic acid binding mode are analyzed.

[0031] When a dehydration condensation agent is added to a sugar chain for sialic acid binding mode-specific modification, not only α2,3-sialic acid is lactonized in the released O-bound sugar chain, but also α2,6-sialic acid that should be amidated is lactonized, which sometimes makes it difficult to distinguish α2,3-sialic acid from α2,6-sialic acid. The present inventors have found that by lactonizing sugar chains that have not yet been released from proteins, the unexpected lactonization of α2,6-sialic acid can be suppressed, and α2,6-sialic acid can be modified differently from α2,3-sialic acid. The ability to perform binding mode-specific sialic acid modification on O-bound sugar chains can improve the accuracy of distinguishing the sialic acid binding mode. In the case where the sugar chain is an N-bound sugar chain, different sialic acid modifications can be performed on α2,3-sialic acid and α2,6-sialic acid by lactonizing sugar chains that have not yet been released from proteins. According to the present invention, even if a glycoprotein contains an O-bound sugar chain, sialic acid can be modified in a binding mode-specific manner, and the sialic acid binding mode can be discriminated with high accuracy. Here, "sialic acid binding mode-specific modification" and "binding mode-specific sialic acid modification" refer to a modification reaction that acts on sialic acid, and when the sialic acid is α2,3-sialic acid and when it is α2,6-sialic acid, the chemical structure generated by the modification reaction is different.

[0032] Sialic acid is located at the end of the sugar chain that is easily recognized by other molecules. Therefore, by distinguishing the sialic acid binding mode, it is possible to clarify viral infection or protein-protein interactions on the cell surface. In addition, it is also known that the sialic acid binding mode in glycoproteins changes with carcinogenesis, and sialic acid is expected to be used as a biomarker for cancer. The effects of biopharmaceuticals vary depending on the sugar chain modification, so by distinguishing the sialic acid binding mode with high precision, it is also possible to assist in the quality management of biopharmaceuticals.

[0033] A flowchart showing the flow of an analysis method according to one embodiment of the present invention is shown in Figure 1 In step S110, a sample is prepared.

[0034] (sample)

[0035] The sample to be analyzed is not particularly limited and may be derived from an organism or a cell. The sample contains glycoproteins and may also contain free sugar chains or glycolipids. The analysis method of the present invention is suitable for analyzing the sialic acid binding mode in sugar chains. Therefore, it is preferred that the sample contains sugar chains that may have sialic acid at the end, and more preferably contains glycoproteins bound to the sugar chains. The analysis method of the present invention can solve the problem that α2,6-sialic acid in O-bound sugar chains is sometimes lactonized. Therefore, it is preferred that the sugar chains in the sample contain O-bound sugar chains, and more preferably contain O-bound sugar chains with α2,6-sialic acid. That is, it is preferred that the sample contains glycoproteins bound to these sugar chains. The analysis method of the present invention is preferably used in the case where the sample contains glycoproteins bound to O-bound sugar chains, but the sample may also contain glycoproteins bound to N-bound sugar chains. From the viewpoint of improving the accuracy of sialic acid modification specific to the binding mode, it is preferred that the sample does not contain O-bound sugar chains in a free state.

[0036] In this specification, protein refers to a general term for molecules in which two or more amino acids form a peptide bond. In this specification, protein may include peptides (including oligopeptides and polypeptides) with a small number of amino acid residues, such as less than 50 amino acid residues, and proteins with a large number of amino acid residues, such as more than 50 amino acid residues. In this specification, glycoprotein may include glycopeptides.

[0037] For glycoproteins with a large number of amino acid residues in the peptide chain, the peptide chain can be cleaved using an enzyme, etc. For example, when preparing a sample for mass spectrometry analysis, the number of amino acid residues in the peptide chain is preferably 30 or less, more preferably 20 or less, and further preferably 15 or less. In the case where it is necessary to clarify the origin of a peptide bound to a sugar chain, the number of amino acid residues in the peptide chain is preferably 2 or more, and more preferably 3 or more.

[0038] When the peptide chain of the glycoprotein is cut, trypsin, Lys-C, arginine endopeptidase, chymotrypsin, pepsin, thermolysin, proteinase K, pronase E, etc. can be used as digestive enzymes. Two or more of these digestive enzymes can be used in combination. The conditions when cutting the peptide chain are not particularly limited, and an appropriate scheme can be adopted according to the digestive enzyme used. Before cutting the peptide chain, the protein in the sample can be denatured or alkylated. The conditions for denaturation or alkylation are not particularly limited. The cutting process of the peptide chain can be carried out before or after the first reaction described later or after the second reaction. The peptide chain can be cut not only by enzyme cutting, but also by chemical cutting.

[0039] A treatment for blocking the amino groups in the glycoprotein in the sample can be appropriately performed. Such treatments include dimethyl amidation and guanidation of the glycoprotein. Thus, when performing the first reaction or the second reaction, side reactions such as intramolecular dehydration condensation that may occur between the amino groups or carboxyl groups at the ends of the protein main chain can be suppressed.

[0040] <Modification steps>

[0041] In the modification step, a modifier capable of performing sialic acid binding mode-specific modification on the O-bound sugar chain is added. The sialic acid binding mode-specific modification preferably includes a first reaction of lactonization of sialic acid. The sialic acid binding mode-specific modification preferably also includes a second reaction of amidation of the lactone structure obtained by the first reaction. In one aspect of the present embodiment, the above-mentioned modifier preferably contains a lactonization reaction solution and an amidation reaction solution. The lactonization reaction solution and the amidation reaction solution are described below.

[0042] (1st reaction)

[0043] In step S120, a first reaction for lactonization of sialic acid is performed on the glycoprotein bound to the sugar chain. In the first reaction, when sialic acid is bound to the glycoprotein, lactonization of sialic acid is selectively performed according to the binding mode. In the first reaction, in addition to lactonization of α2,3-sialic acid in the sugar chain, α2,8-sialic acid and α2,9-sialic acid are preferably lactonized.

[0044] The first reaction can be carried out by contacting the sample with a solution for sialic acid binding mode-specific lactonization (hereinafter also referred to as lactonization reaction solution). An example of a contact method may be to add a lactonization solution to the sample. The lactonization reaction solution preferably contains a dehydration condensation agent. Preferably, at the same time as the first reaction, α2,6-sialic acid is modified differently from lactonization, preferably amidated. In this case, it is preferred that the lactonization reaction solution contains a nucleophile in addition to the dehydration condensation agent, and the nucleophile contains at least one selected from the group consisting of alcohols, amines and their salts.

[0045] In order to selectively cause a dehydration reaction or a nucleophilic reaction according to the sialic acid binding mode, the type and concentration of the dehydration condensation agent and the nucleophilic agent can be adjusted. The lactone produced by the intramolecular dehydration of the carboxyl group of α2,3-sialic acid is a six-membered ring, and the lactone that may be produced by the intramolecular dehydration of the carboxyl group of α2,6-sialic acid is a seven-membered ring. α2,3-sialic acid, which produces a six-membered ring that is more stable than the seven-membered ring, is more easily lactonized than α2,6-sialic acid. In addition, the carboxyl group of α2,3-sialic acid is in a more hindered position than the carboxyl group of α2,6-sialic acid, so it is more difficult for a large molecule to react with α2,3-sialic acid than α2,6-sialic acid. The type and concentration of the dehydration condensation agent and the nucleophilic agent are adjusted so that different modifications can be made according to the sialic acid binding mode based on the difference in molecular structure caused by the sialic acid binding mode.

[0046] The dehydration condensation agent preferably contains carbodiimide. This is because when carbodiimide is used, the carboxyl group present at a site with large steric hindrance is less likely to be amidated than when a phosphonium dehydration condensation agent (so-called BOP reagent) or a uronium dehydration condensation agent is used as the dehydration condensation agent. Examples of carbodiimides include N,N'-dicyclohexylcarbodiimide (DCC), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N,N'-diisopropylcarbodiimide (DIC), 1-tert-butyl-3-ethylcarbodiimide (BEC), N,N'-di-tert-butylcarbodiimide, 1,3-di-p-toluoylcarbodiimide, bis(2,6-diisopropylphenyl)carbodiimide, bis(trimethylsilyl)carbodiimide, 1,3-bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)carbodiimide (BDDC), and salts thereof (hydrochloride, etc.).

[0047] In order to promote the dehydration condensation by the dehydration condensation agent and suppress the side reaction, a high nucleophilic additive is used in addition to the carbodiimide. As a high nucleophilic additive, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt), 4-(dimethylamino)pyridine (DMAP), ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), N-hydroxy-succinimide (HOSu), 6-chloro-1-hydroxy-benzotriazole (Cl-HoBt), N-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt) and the like are preferably used.

[0048] The amine used as the nucleophile preferably comprises a primary alkylamine and / or a secondary alkylamine containing two or more carbon atoms. The primary alkylamine is preferably ethylamine, propylamine, isopropylamine, butylamine, secondary butylamine, tert-butylamine, etc. The secondary alkylamine is preferably dimethylamine, methylethylamine, diethylamine, propylmethylamine, isopropylmethylamine, etc. From the viewpoint of making the carboxyl group of α2,3-sialic acid, which is present in a sterically hindered position, less likely to be amidated, it is preferred to use an amine having a branched alkyl group such as isopropylamine. When an amine is used as the nucleophile of the lactonization reaction solution, the carboxyl group of a portion of the sialic acid such as α2,6-sialic acid is amidated based on the sialic acid binding mode.

[0049] The alcohol used as the nucleophilic agent is not particularly limited, and for example, methanol and ethanol can be used. When an alcohol is used as the nucleophilic agent in the lactonization reaction solution, the carboxyl groups of some sialic acids such as α2,6-sialic acid can be esterified based on the sialic acid bonding mode. The nucleophilic agent may include a salt of the above nucleophilic agent.

[0050] The concentration of the dehydration condensation agent in the lactonization reaction solution is, for example, 1 mM or more and 5 M or less, preferably 10 mM or more and 3 M or less. When carbodiimide is used in combination with a highly nucleophilic additive such as Oxyma, HOAt or HOBt, it is preferred that the concentration of each is in the above range. The concentration of the nucleophilic agent in the lactonization reaction solution is, for example, 0.01 M or more and 20 M or less, preferably 0.1 M or more and 10 M or less. The reaction temperature of the first reaction can be between -20°C and 100°C or less, preferably between -10°C and 50°C or less.

[0051] The first reaction can be implemented in liquid phase or solid phase. When reacting in liquid phase, it is preferably reacted in a non-aqueous solvent such as dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). By reacting in a non-aqueous solvent, there is a tendency that side reactions are suppressed. The concentration of each component in the liquid phase reaction is not particularly limited and can be appropriately determined according to the types of dehydration condensation agent and amine.

[0052] When the first reaction is carried out in the solid phase, the solid phase carrier is not particularly limited as long as it can fix the glycoprotein. In order to fix the glycoprotein, for example, a solid phase carrier having an epoxy group, a tosyl group, a carboxyl group, an amino group, etc. as a ligand can be used. A solid phase carrier having a hydrazide group, an aminooxy group, etc. as a ligand can be used to fix the glycoprotein containing a sugar chain. By reacting in a state where the sample is fixed to the solid phase carrier, the reaction solution after the reaction is easily removed, and the modification of sialic acid can be efficiently carried out. When magnetic beads are used as solid phase carriers, the magnetic beads bound to the glycoprotein can be collected with a magnet and excess reagents can be removed or the beads can be cleaned with a solvent. When a resin is used as a solid phase carrier, the resin can be recovered after passing it through a filter to remove excess reagents, or the resin can be precipitated by centrifugation and excess reagents in the supernatant can be removed. When the first reaction is carried out in the liquid phase, excess reagents can be removed by ultrafiltration.

[0053] The sample after the first reaction can be purified, desalted, solubilized, concentrated, dried, or the like by a known method as necessary to remove the lactonization reaction solution or reduce the concentration of the lactonization reaction solution.

[0054] (Reaction 2)

[0055] In step S130, it is preferred to perform a second reaction to amidate the lactone structure produced by the first reaction. The second reaction can be performed by contacting the sample after the first reaction with a solution for amidation (hereinafter also referred to as an amidation reaction solution). Through the first reaction, the α2,3-binding and α2,6-binding of sialic acid can be distinguished and modified, but the lactone structure produced by α2,3-sialic acid is sometimes unstable, and there is a situation where it returns to the original carboxylic acid structure by hydrolysis. The lactone structure is specifically amidated by the second reaction, thereby stabilizing the lactone structure. At the same time, through the second reaction, the binding mode specifically modifies sialic acid with different masses, so that α2,3-sialic acid and α2,6-sialic acid can be distinguished. By performing the second reaction, sialic acid can be more specifically and rapidly modified in a binding mode-specific manner. An example of the second reaction can be aminolysis. Aminolysis is a reaction based on the interaction between an amino group and a lactone, and is also suitably performed under anhydrous conditions, so it is a reaction different from hydrolysis. In this specification, the ring opening and amidation of lactones using ammonia, amines or their salts that can be carried out under anhydrous conditions are referred to as aminolysis. As another example of the second reaction, a strong dehydration condensation agent can be used to amidate the lactone structure. As the dehydration condensation agent used for the second reaction, phosphonium condensation agents and uronium condensation agents can be listed, specifically, benzotriazole-1-yloxy tris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazole-1-yloxy tris (pyrrolidinyl) phosphonium hexafluorophosphate (PyBOP), 2- (1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2- (7-aza-1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HA TU), (1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU), tetramethylfluoroformamidinium hexafluorophosphate (TFFH), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), etc. Before the amidation using these powerful dehydrating condensing agents, the ring-opening operation of the lactone can be inserted.

[0056] The amidation reaction solution preferably contains at least one selected from the group consisting of ammonia, amines and their salts. When using amines, it is preferred to use an amine different from the amine used for the lactonization reaction solution or to change the mass by modifying with a stable isotope, etc. By using amines of different masses in the first reaction and the second reaction, sialic acid can be amidated so that the mass is different depending on the binding mode. In one aspect of the present embodiment, the second reaction is preferably an aminolysis reaction. A dehydration condensation agent is not necessary in the aminolysis, and the amidation reaction solution may not contain a dehydration condensation agent. The amidation reaction solution may also contain a dehydration condensation agent, for example, the amidation reaction solution can be prepared by not removing the lactonization reaction solution added to the sample in the first reaction and adding ammonia, amines or their salts. In the second reaction, the lactone structure can be stabilized by such a simple operation.

[0057] The amine contained in the amidation reaction solution is preferably a primary amine, more preferably a primary amine having a straight-chain hydrocarbon group, and further preferably a primary amine having a straight-chain alkyl group. As for the amine contained in the amidation reaction solution, as a primary amine having a straight-chain alkyl group, a primary amine having a carbon number of 10 or less is preferred, a primary amine having a carbon number of 7 or less is more preferred, methylamine, ethylamine, propylamine, butylamine or pentylamine is further preferred, and methylamine is most preferred. From the viewpoint of more efficiently amidating the lactone structure, the amine contained in the amidation reaction solution preferably has an unbranched (hereinafter, "branched" means a branched hydrocarbon chain) straight-chain structure or a small carbon number.

[0058] When the amine contained in the amidation reaction solution is a primary amine having an unsaturated chain hydrocarbon group, the unsaturated chain hydrocarbon group preferably contains a double bond, and the unsaturated chain hydrocarbon group more preferably contains an allyl group, and the amine is preferably an allylamine. The amine contained in the amidation reaction solution can be a primary amine containing a hydroxyl group, and can be ethanolamine. The amine contained in the amidation reaction solution can contain various functional groups other than the alkyl group. The amidation reaction solution can contain salts of the above amines. As a result of the amidation reaction, the sugar chain is modified in a manner containing such a functional group, so that the sugar chain subjected to the modification is not only easy to separate by mass spectrometry, but also easy to separate by chromatography, etc.

[0059] The concentration of ammonia, amines and their salts in the amidation reaction solution is preferably 0.1M or more, more preferably 0.3M or more, further preferably 0.5M or more, further preferably 1.0M or more, and most preferably 3.0M or more. As a preferred example, the amidation reaction solution contains ammonia or a primary amine, particularly methylamine, and the concentration of the ammonia or primary amine such as methylamine is preferably 0.1M or more, more preferably 0.3M or more, further preferably 0.5M or more, further preferably 1.0M or more, and most preferably 3.0M or more. The higher the concentration of ammonia, amines and their salts in the amidation reaction solution, the more efficiently the lactone structure can be amidated. The upper limit of the concentration of ammonia, amines and their salts in the amidation reaction solution is not particularly limited, and for example, it may be 16M or less.

[0060] The solvent of the amidation reaction solution may be an aqueous solvent or an organic solvent. From the viewpoint of preventing lactone hydrolysis and reliably causing rapid amidation, it is preferred that the water content is low. The solvent of the amidation reaction solution is preferably a dehydrated solvent to which a dehydration operation to suppress the water content is applied, and is more preferably an anhydrous solvent. The solvent of the amidation reaction solution preferably comprises at least one of methanol and acetonitrile. The amidation reaction solution may contain water, and the solvent of the amidation reaction solution may be water.

[0061] When the amidation reaction is an aminolysis, the solution is preferably at a pH of 7.7 or more, more preferably at a pH of 8.0 or more, further preferably at a pH of 8.8 or more, and most preferably at a pH of 10.3 or more. The higher the pH of the amidation reaction solution, the more efficiently the lactone structure can be amidated. The upper limit of the pH of the amidation reaction solution is not particularly limited, and for example, may be 14 or less. When a strong dehydration condensation agent is used to amidate the lactone structure, the pH is not particularly limited, and the pH can be adjusted according to the dehydration condensation agent used.

[0062] When the second reaction is aminolysis, the reaction can be completed within a few seconds to a few minutes. The time for contacting the sample with the amidation reaction solution for amidation of the lactone structure is preferably less than 1 hour, more preferably less than 30 minutes, further preferably less than 15 minutes, further preferably less than 5 minutes, and most preferably less than 1 minute. Preferably, the sample can be simply washed with the amidation reaction solution or the sample held on a carrier or the like can be temporarily passed through the amidation reaction solution. There is no particular limit to the lower limit of the time for contacting the sample with the amidation reaction solution for amidation of the lactone structure, and for example, it can be more than 1 second. When a strong dehydration condensation agent is used to amidate the lactone structure, the reaction time is not particularly limited, and the reaction time can be adjusted according to the dehydration condensation agent used. For example, it is preferably more than 30 minutes, more preferably more than 1 hour, and most preferably more than 2 hours. The time from the completion of the contact of the sample with the lactonization reaction solution to the completion of the contact of the sample with the amidation reaction solution is preferably less than 1 and a half hours, more preferably less than 1 hour, and more preferably less than 30 minutes. Since the second reaction is completed in a short time, it is possible to prevent the unstable lactone structure from being decomposed and the quantitative analysis of the sugar chain from being impaired. In addition, by setting the reaction time of the second reaction to be shorter, the sample analysis can be performed more efficiently. The lower limit of the time from the completion of the contact between the sample and the lactonization reaction solution to the completion of the contact between the sample and the amidation reaction solution is not particularly limited, and for example, may be 1 second or more.

[0063] The second reaction can be carried out in a liquid phase or a solid phase. As long as the sample can be brought into contact with the amidation reaction solution, the state of the sample when the amidation reaction occurs is not particularly limited, and it is preferred that the amidation reaction solution is contacted in a state where the glycoprotein is bound or adsorbed to the solid phase carrier. The solid phase carrier used in the second reaction is not particularly limited as long as it can fix the glycoprotein, and the solid phase carrier that can be used in the first reaction can be cited.

[0064] The sample after the second reaction can be purified, desalted, solubilized, concentrated, dried, etc. by a known method as needed to remove the amination reaction solution or reduce the concentration of the amination reaction solution. The removal of excess reagents after the second reaction can be carried out by the method described in the first reaction section according to a solid phase or liquid phase reaction method.

[0065] (Purification of glycoproteins)

[0066] In step S140, the glycoprotein is preferably purified. Through this process, the solution used in the first reaction and / or the second reaction can be removed, thereby improving the efficiency of the downstream process. The purification method and conditions of the glycoprotein can be appropriately selected according to the type, property, molecular weight, etc. of the glycoprotein. The purification of the glycoprotein can be carried out by protein precipitation. As a precipitant, salts such as ammonium sulfate (ammonium sulfate); organic solvents such as acetone, acetonitrile and chloroform; alcohols such as methanol, propanol and ethanol; acids such as trichloroacetic acid (TCA), hydrochloric acid and metaphosphoric acid; water-soluble polymers such as polyethylene glycol and dextran, and combinations of these, etc. can be used. In the protein precipitation method, for example, a precipitant can be added to a sample containing glycoproteins and allowed to stand for an appropriate time at a temperature above -20°C and below 30°C. Afterwards, the sample that has been allowed to stand can be centrifuged to recover the resulting precipitate. The purification of glycoproteins can be carried out by fixing the glycoproteins to a solid phase carrier, or by using gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, etc.

[0067] <Freeing step>

[0068] In step S150, sugar chains are freed from glycoproteins. As a method for freeing sugar chains from glycoproteins, enzyme treatment using O-glycosidase, N-glycosidase, endoglycosidic ceramidase, and chemical freeing methods such as hydrazinolysis and β-elimination can be used. Before the treatment to free sugar chains, peptide chain cleavage of glycoproteins can be performed. Together with the freeing of sugar chains, the reducing end of the sugar chain can be modified by labeling using 3-aminoquinoline (3AQ), anthranilic acid (2AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), etc.

[0069] When N-bound sugar chains are freed from the peptide chains of glycoproteins, it is preferred to use enzyme treatments using peptide-N-glycosidase F (PNGaseF), peptide-N-glycosidase A (PNGase A), endo-β-N-acetylglucosaminidase (Endo M), etc. When O-bound sugar chains are freed from the peptide chains of glycoproteins, it is preferred to use a chemical freeing method. The hydrazine hydrolysis method can be carried out according to a known method, for example, anhydrous hydrazine or aqueous hydrazine is added to a sample containing glycoproteins and heated. The β elimination method can be carried out according to a known method, for example, ammonium carbamate, saturated ammonia, ammonium carbonate, anhydrous trifluoromethanesulfonic acid (anhydrous TFMS), sodium hydroxide, dimethylamine, etc. are added to a sample containing glycoproteins and heated under alkaline conditions. From the viewpoint of suppressing side reactions based on peeling, it is preferred to use ammonium salt powders such as ammonium carbamate to free O-bound sugar chains. When reacting under alkaline conditions, the coexistence of pyrazolone reagents such as PMP in advance can be used to label and inhibit side reactions based on peeling while freeing O-bound sugar chains. From the perspective of preventing the conversion of methylated sialic acid to carboxylic acid in the second reaction and from the perspective of the free efficiency of O-bound sugar chains, the pH of the β-elimination reaction is preferably below pH 11.0, and more preferably below pH 10.0. From the perspective of inhibiting the decomposition and peeling reaction of sialylated sugar chains, the pH of the β-elimination reaction is preferably below pH 9.5. The pH when performing the β-elimination reaction is usually above pH 7.5.

[0070] The release of sugar chains can be carried out in a liquid phase or in a solid phase. For example, the sugar chain cleavage treatment can be performed on a glycoprotein fixed to a solid phase carrier, and the sugar chains can be recovered. After the sugar chain cleavage treatment is performed on a sugar chain protein bound to a solid phase carrier having a hydrazide group, a weakly acidic solution can be used to release the sugar chains and recover the sugar chains. By reacting the sample while it is fixed to a solid phase carrier, the removal or desalting purification of the reaction solution becomes easier, and the preparation of the sample can be simplified.

[0071] Free sugar chains can be purified according to known methods, for example, proteins and sugar chains can also be separated by liquid-liquid extraction using organic solvents such as chloroform. Free sugar chains can also be easily purified using solid phase carriers (columns). Free sugar chains can also be bound to solid phase carriers having hydrazide groups and aminooxy groups and recovered. As solid phase carriers having hydrazide groups, "BlotGlyco" manufactured by Sumitomo Bakelite Co., Ltd. can be cited. Sugar chains can also be adsorbed on a carrier for hydrophilic interaction chromatography (hereinafter also referred to as HILIC) for purification. The carrier for HILIC preferably contains an amide group. In addition to being purified using a carbon column, free sugar chains can also be purified using reverse phase carriers such as C18. Labeled sugar chains can be adsorbed on carbon or C18 for purification.

[0072] <Analysis steps>

[0073] In step S160, the freed sugar chains are analyzed. The sugar chains to be analyzed preferably include freed O-bound sugar chains that have been specifically modified in a sialic acid binding manner. As an analysis method, mass spectrometry, chromatography, electrophoresis, or a combination of these can be performed. Through the above-mentioned lactonization reaction, sialic acid is specifically modified in a binding manner, and the mass of sugar chains that are not easily lactonized, such as α2,6-sialic acid, and sugar chains that are easily lactonized, such as α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid, becomes different. Therefore, these sugar chains can be separated based on the sialic acid binding manner by mass spectrometry, chromatography, electrophoresis, etc.

[0074] The ionization method in mass spectrometry is not particularly limited, and matrix-assisted laser desorption ionization (MALDI) method, electrospray ionization (ESI) method, nano-electrospray ionization (nano-LSI) method, etc. can be used, preferably MALDI method. Ionization in mass spectrometry can use any one of positive ion mode and negative ion mode. Mass spectrometry can be performed in multiple stages. Thus, the sugar chain structure or the structure of the peptide chain other than the sialic acid binding mode can be appropriately analyzed.

[0075] The characteristic of the modified body produced by the result of the first reaction and the second reaction can be analyzed by analytical methods other than mass spectrometry such as chromatography, electrophoresis, etc. The sialic acid binding mode is analyzed. The column used in the liquid chromatography is not particularly limited, and the hydrophobic reverse phase column, carbon column and HILIC normal phase column such as C30, C18, C8 and C4 can be appropriately used. The mode of measuring by mass spectrometry after the liquid chromatography can more accurately analyze the composition in the sample, so it is preferred. In this case, it is more preferred to ionize the eluent from the liquid chromatography directly by ESI etc. in a mass spectrometer by online control. Electrophoresis can be SDS-PAGE method, capillary electrophoresis, microchip electrophoresis, two-dimensional electrophoresis, etc., preferably capillary electrophoresis.

[0076] [Example]

[0077] The present invention will be described in more detail below with reference to Examples, but the present invention is not limited thereto. Unless otherwise specified, % indicates mass %.

[0078] <Experiment 1>

[0079] First, the first reaction and the second reaction are carried out without releasing the sugar chains from the glycoprotein. Then, protein precipitation is performed to remove excess reagents. Then, O-bound sugar chains are released from the glycoprotein and sugar chain analysis is performed by mass spectrometry. The specific method is shown below.

[0080] 1) Freeze-dried products of serum and cerebrospinal fluid were prepared as samples containing glycoproteins.

[0081] 2) 100 μL of a lactonization reaction solution containing 2 M isopropylamine hydrochloride (nucleophilic agent), 500 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (dehydration condensation agent), 500 mM 1-hydroxybenzotriazole monohydrate (highly nucleophilic additive) and dimethyl sulfoxide as a solvent was added to the sample and reacted at room temperature for 1 hour (first reaction).

[0082] 3) 100 μL of 10% methylamine was added and stirred (second reaction).

[0083] 4) 800 μL of a solution containing 2.5% trifluoroacetic acid (TFA) and acetonitrile (ACN) as a solvent was added to precipitate the protein.

[0084] 5) Remove the supernatant and recover the precipitated protein.

[0085] 6) Add dithiothreitol (DTT) to reductively alkylate the glycoprotein.

[0086] 7) Purify the protein by ethanol precipitation.

[0087] 8) To release sugar chains from glycoproteins and label them, 50 μL of a solution containing 0.14 M sodium hydroxide, 0.2 M 1-phenyl-3-methyl-5-pyrazolone (PMP) and 40% methanol was added and reacted at 85° C. for 16 hours.

[0088] 9) Add chloroform, stir vigorously to perform liquid-liquid extraction, and discard the chloroform layer. Repeat this operation three times to remove PMP from the sample.

[0089] 10) The aqueous layer containing sugar chains was recovered and chromatographed on a carbon column (manufactured by SPELCO, "Supelclean TM ENVI-Carb TM The sugar chains were purified by "Bulk Packing" (hereinafter referred to as "Purification"). The column was washed with water, 10% ACN, 0.05% TFA, and 20% ACN, 0.05% TFA, and then eluted with 50% ACN, 0.05% TFA.

[0090] 11) The eluate was concentrated under reduced pressure and purified using iatro beads (normal phase carrier, silica system). Specifically, a sample prepared to 95% ACN was adsorbed on the carrier, washed with 95% ACN, 1% acetic acid and 98% ACN, 2% acetic acid, and then eluted with 50% ACN.

[0091] 12) The eluate was dried and then redissolved in water to prepare a sample for analysis. Mass spectrometry analysis was performed in positive ion mode using a 2,5-dihydroxybenzoic acid (DHB) matrix using a MALDI-TOF-MS (manufactured by Bruker, product name "Ultraflex II").

[0092] It should be noted that the above 2) and 3) correspond to the modification step, the above 8) to 11) correspond to the release step, and the above 12) corresponds to the analysis step.

[0093] The mass spectra of sugar chains contained in glycoproteins derived from serum and cerebrospinal fluid are shown in Figure 2 . A difference of about 28 Da was observed between the sugar chain with α2,3-sialic acid (m / z 1040.49) and the sugar chain with α2,6-sialic acid (m / z 1068.60), indicating that sialic acid was modified in a binding mode-specific manner and that the two could be distinguished by mass spectrometry. For the diallyl sugar chain with both α2,3-sialic acid and α2,6-sialic acid, a peak of m / z 1372.86 was confirmed for α2,3-sialic acid methylamidated and α2,6-sialic acid isopropylamidated, but no peak was observed at a position 28 Da less than this peak. Therefore, it was confirmed that α2,6-sialic acid was not lactonized and that α2,3-sialic acid and α2,6-sialic acid were modified differently.

[0094] Experiment 2

[0095] As a comparative experiment, N-bound sugar chains were released from glycoproteins, and the free sugar chains were recovered using BlotGlyco beads. The sugar chains bound to the beads were subjected to the first reaction and the second reaction to modify sialic acid, and then the free sugar chains recovered from the beads were subjected to mass spectrometry analysis. The obtained mass spectrum is shown in Figure 3 The upper row.

[0096] Experiment 3

[0097] First, the first reaction and the second reaction are carried out in a liquid phase without releasing sugar chains from the glycoprotein. Then, protein precipitation is performed to remove excess reagents. Then, N-bound sugar chains are released from the glycoprotein and sugar chain analysis is performed by mass spectrometry. The mass spectrum obtained is shown in Figure 3 The specific method is as follows.

[0098] 1) A freeze-dried product of serum was prepared as a sample.

[0099] 2) 100 μL of a lactonization reaction solution containing 2 M isopropylamine hydrochloride (nucleophile), 500 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (dehydration condensation agent), 500 mM 1-hydroxybenzotriazole monohydrate (highly nucleophilic additive) and dimethyl sulfoxide as a solvent was added to the sample and stirred for 1 hour (first reaction).

[0100] 3) 100 μL of 10% methylamine was added and stirred (second reaction).

[0101] 4) 800 μL of a solution containing 2.5% trifluoroacetic acid (TFA) and acetonitrile (ACN) as a solvent was added to precipitate the protein.

[0102] 5) Remove the supernatant and recover the precipitated protein.

[0103] 6) Add dithiothreitol (DTT) to reductively alkylate the glycoprotein.

[0104] 7) Purify the protein by ethanol precipitation.

[0105] 8) Digest the protein with trypsin (10 mg / mL).

[0106] 9) PNGaseF (Roche, 2U) was used to release the N-linked sugar chain.

[0107] 10) N-linked sugar chains were purified using a solid phase carrier composed of beads having hydrazide groups as ligands (manufactured by Sumitomo Bakelite Co., Ltd., “BlotGlyco”) according to the manufacturer's instructions.

[0108] 11) While being released from the carrier, the reducing end of the sugar chain is labeled using aoWR, a high sensitivity reagent, to prepare a sample for analysis.

[0109] 12) Mass spectrometry analysis was performed using a DHB matrix using MALDI-TOF-MS (manufactured by Bruker, product name: Ultraflex II) in a positive ion mode.

[0110] like Figure 3 As shown, almost the same mass spectra were obtained in Experiment 2 and Experiment 3. It can be seen that in the case of N-bound sugar chains, when the free sugar chains are subjected to the first reaction and the second reaction, and when the sugar chains are released after the first reaction and the second reaction of the glycoprotein, the sialic acid is similarly modified by the binding mode specificity, and can be distinguished by mass spectrometry analysis. It shows that according to the present invention, when the sugar chain of the glycoprotein contains N-bound sialic acid and when it contains O-bound sialic acid, the sialic acid binding specificity can be quickly and accurately determined.

[0111] <Experiment 4>

[0112] As a comparative experiment, a glycoprotein with an O-bound sugar chain was used to show an experiment without sialic acid binding mode-specific modification and an experiment in which the sugar chain was freed and then subjected to sialic acid binding mode-specific modification. 20 μL of 50% hydroxylamine and 20 μL of 0.5M sodium hydroxide were added to fetuin (100 μg) as a glycoprotein, and the sugar chain was freed by reacting at 50°C for 1 hour. After the reaction was completed, 10 μL of 1M hydrochloric acid was added to quench, and 200 μL of acetone was added and concentrated with a centrifugal evaporator. After concentration, it was redissolved with 50 μL of water, and the free sugar chain was captured with BlotGlyco beads. The methyl esterification method (MTT method) or the first reaction and the second reaction (sialic acid binding mode-specific alkyl amidation method; also referred to as "SALSA method" in this specification) were performed on the solid phase to modify sialic acid. According to the MTT method, non-specific modification of the binding mode was performed, so that all sialic acids were esterified. The first reaction and the second reaction were performed by the same method as Experiment 1. The reducing end of the sugar chain was labeled with aoWR, a highly sensitive reagent, and then purified using a HILIC column. The purified aoWR-labeled sugar chain was mixed with 2,5-dihydroxybenzoic acid (10 mg / mL) and analyzed using MALDI-TOF-MS (Bruker, product name "Ultraflex II").

[0113] The obtained mass spectrum is shown in Figure 4 In the case of the MTT method, sialic acid is methyl esterified, and the sialic acid group T is at m / z 1118.6 [M+H] + The disialyl group T was detected at m / z 1424.0 [M+H] + In the case of the SALSA method, the sialic acid group T of sialic acid with an α2,3 bond is detected at m / z 1117.6 [M+H] + On the other hand, the disialyl group T of sialic acid with α2,3 and α2,6 bonds was detected at m / z 1449.8 [M+H] + Signals modified by methylamide and isopropylamide were observed at α2,6-linked sialic acid, and m / z 1421.9 [M+H] modified by methylamide was also detected. + According to any of the methods, sialic acid-binding-specific modification of O-linked sugar chains is not achieved.

[0114] <Experiment 5>

[0115] The concentration of methylamine was changed to investigate the efficiency of sialic acid-binding-specific modification of O-bound sugar chains. First, fetuin (100 μg) was subjected to the first and second reactions. Specifically, 100 μL of lactonization reaction solution (2M iPA-HCl, 500mM EDC-HCl, 500mM HOBt) was added to fetuin and stirred at room temperature for 1 hour (first reaction). Then 100 μL of 10%, 16.6% or 40% methylamine aqueous solution was added to the reaction solution and stirred (second reaction). Then 1 mL of acetonitrile containing 2.5% trifluoroacetic acid was added and allowed to stand at a temperature of -30°C for 1 hour. Centrifuge at 14000g for 20 minutes, remove the supernatant and remove the SALSA reaction solution. Further, 1 mL of acetonitrile was added to the residue, centrifuged at 14000g for 20 minutes, and the supernatant was removed. The obtained protein-containing residue was re-dissolved with 10 mL of water. Prepare a β-elimination reaction solution for freeing sugar chains from glycoproteins. The β-elimination reaction solution is a solution in which sodium hydroxide (NaOH) (final concentration 0.7 M) and 3-methyl-1-phenyl-5-pyrazolone (PMP) (final concentration 1 M) are dissolved in a 60% methanol aqueous solution. Add 40 μL of the β-elimination reaction solution to fetuin with sialic acid specifically modified in the binding mode, maintain the temperature at 85°C in a thermal cycler, and react for 16 hours. After the reaction is completed, add 50 pmol of bisPMP-labeled chitotetraose and neutralize with 10 μL of 4M hydrochloric acid. Add chloroform and stir vigorously to perform liquid-liquid extraction. Remove the chloroform layer and repeat this 3 times. Recover the water layer containing the sugar chains and purify it using a chromatographic carbon column and iatro beads. The purification method is the same as that of Experiment 1. The purified bisPMP-labeled sugar chain was mixed with 2,5-dihydroxybenzoic acid (10 mg / mL) containing 10% sodium 2,5-dihydroxybenzoate, and analyzed by MALDI-TOF.

[0116] The mass spectrum results are shown in Figure 5 In all reactions, the sialic acid group T of sialic acid with α2,3 bond was almost always at m / z 1040.7 [M+Na] + The disialyl group T of both sialic acid with α2,3 bond and α2,6 bond was observed at m / z 1373.1 [M+Na] + Observed at . After the sugar chains on the glycoprotein were modified with sialic acid by the SALSA method, the sugar chains were released by the β-elimination reaction and the binding mode was specifically derivatized. The sugar chains were cleaved from the glycoprotein in the derivatized state by the β-elimination reaction. The effect of methylamine concentration was further studied. The m / z 1040.7 [M+Na] + The α2,3 bond of the sialic acid group T detected at m / z 1027.6 [M+Na] when the carboxylic acid is not modified+ The ratio of unmodified sialic acid T to methylamidation-modified sialic acid T was 4.6% when methylamine was 10% and 2.6% when methylamine was 40%. Similarly, the ratio of unmodified disialyl T to methylamidation-modified disialyl T was 1.5% when methylamine was 10% and 0.8% when methylamine was 40%. Figure 6 ). In the aminolysis reaction, the higher the amine concentration, the higher the efficiency of methylamidation of sialic acid.

[0117] <Experiment 6>

[0118] The pH during the β-elimination reaction was changed and the free O-bound sugar chains were analyzed. Human plasma (10 μL) was modified specifically in the sialic acid binding mode by the same method as in Experiment 5. A 40% methylamine aqueous solution was used in the second reaction. After washing the glycoprotein by the same method as in Experiment 5, 0.1 mL of water was added to the precipitate and suspended in a water bath type ultrasonic processor. 0.4 mL of the previously prepared β-elimination reaction solution was added to the suspension and mixed. The β-elimination reaction solution was prepared by dissolving PMP (final concentration 1 M) and NaOH (final concentration 0.7-0.975 M) in a 60% acetonitrile solution. Five pH values ​​were set by changing the concentration of NaOH. The composition and pH are shown in Table 1. The reaction was carried out at a temperature of 85°C for 16 hours, and PMP labeling was carried out simultaneously with the β-elimination of the O-bound sugar chains. After the reaction was completed, 0.4 mL of 4M HCl was added for neutralization. The operation of adding 0.4 mL of chloroform and vigorously stirring and discarding the chloroform layer by liquid-liquid extraction was repeated 4 times. The water layer containing the labeled sugar chain was recovered and purified using a chromatographic carbon column and iatro beads. The PMP labeled sugar chain was mixed with 2,5-dihydroxybenzoic acid and analyzed by MALDI-TOF. The purification and analysis methods were the same as those in Experiment 5.

[0119] The analysis results are shown in Figure 7 to Figure 9 The recovery of O-bound sugar chains in project 3 was the highest ( Figure 7 On the other hand, in items 3 to 5, the ratio of the decomposition product of the sialyl group T that is not methylamidated increases compared to items 1 and 2 ( Figure 8 ). In items 3 to 5, the ratio of the decomposition product (peeling reaction) of the β-elimination reaction increased compared with items 1 and 2 ( Fig. 9 ).

[0120]

Table 1

[0121] Table 1 Composition and pH of β-elimination reaction solution

[0122] project 1 2 3 4 5 PMP(M) 1 1 1 1 1 NaOH(M) 0.7 0.75 0.93 0.95 0.97 pH 8.7 9.1 9.9 10.1 11.9

[0123] [Way]

[0124] Those skilled in the art will appreciate that the above-described multiple exemplary implementations and embodiments are specific examples of the following aspects.

[0125] (Item 1)

[0126] A method for analyzing an O-bound sugar chain specifically modified in a sialic acid binding mode contained in a sample in one embodiment comprises: a modification step of adding a modifying agent capable of specifically modifying the O-bound sugar chain in a sialic acid binding mode to a sample containing a glycoprotein bound to the O-bound sugar chain; a releasing step of releasing the O-bound sugar chain specifically modified in a sialic acid binding mode from the glycoprotein; and an analyzing step of analyzing the released O-bound sugar chain specifically modified in a sialic acid binding mode. According to the method described in item 1, even if the sugar chain contains O-bound sialic acid, the sialic acid binding mode can be determined with high accuracy.

[0127] (Item 2)

[0128] In the method described in item 1, the sialic acid binding mode-specific modification includes a first reaction, wherein the first reaction lactonizes sialic acid. The first reaction selectively lactonizes sialic acid according to the binding mode. In the first reaction, α2,3-sialic acid in the sugar chain is preferably lactonized.

[0129] (Item 3)

[0130] In the method described in item 2, the sialic acid binding form-specific modification further comprises a second reaction for amidating the lactone structure generated by the first reaction. According to the method described in item 3, binding form-specific sialic acid modification can be performed with higher accuracy.

[0131] (Item 4)

[0132] In the method described in Item 3, the second reaction is carried out using a solution of at least one selected from the group consisting of ammonia, amines, and salts thereof. According to the method described in Item 4, binding mode-specific sialic acid modification can be efficiently performed.

[0133] (Item 5)

[0134] In the method described in Item 3 or Item 4, the second reaction is an aminolysis reaction.

[0135] (Item 6)

[0136] In the method according to any one of items 3 to 5, the sialic acid after the second reaction is modified in a manner that differs in mass depending on the binding mode. According to the method according to item 6, sialic acid modified in a manner that is specific to the binding mode can be distinguished by an analytical method such as mass spectrometry.

[0137] (Item 7)

[0138] In the method according to item 6, the sialic acid binding forms include α-2,3 sialic acid and α-2,6 sialic acid. According to the method according to item 7, α-2,3 sialic acid and α-2,6 sialic acid can be distinguished.

[0139] (Item 8)

[0140] The method according to any one of items 2 to 7, wherein a solution containing a dehydrating condensation agent is added to the sample to carry out the first reaction. According to the method according to item 8, different modifications can be carried out according to the sialic acid binding mode based on the difference in molecular structure caused by the sialic acid binding mode.

[0141] (Item 9)

[0142] In the method according to any one of items 2 to 8, α-2,6 sialic acid is amidated after the first reaction.

[0143] (Item 10)

[0144] The method according to any one of items 2 to 9 further comprises the step of purifying the glycoprotein by protein precipitation after the first reaction. According to the method according to item 10, the solution used in the upstream step can be removed, thereby improving the reaction efficiency of the downstream step.

[0145] (Item 11)

[0146] In the method according to any one of items 1 to 10, the sugar chains are released from the glycoprotein by a β-elimination reaction or a hydrazinolysis reaction. According to the method according to item 11, the sugar chains can be released from the glycoprotein chemically.

[0147] (Item 12)

[0148] In the method according to item 11, the β-elimination reaction is carried out at a pH of 11.0 or less. According to the method according to item 12, the O-bound sugar chain can be efficiently released.

[0149] (Item 13)

[0150] The method according to any one of items 1 to 12 may further comprise the step of purifying the freed sugar chains using a solid phase carrier. According to the method according to item 13, the free sugar chains can be easily separated from the protein.

[0151] (Item 14)

[0152] In the method according to any one of items 1 to 13, the analysis comprises at least one selected from the group consisting of mass spectrometry, chromatography and electrophoresis. According to the method according to item 14, sugar chain analysis can be performed by distinguishing the sialic acid binding mode based on the mass difference generated specifically by the binding mode and the influence of chromatography or electrophoresis on separation.

Claims

1. A method for analyzing an O-bound sugar chain that has been specifically modified in terms of sialic acid binding, comprising: a modification step, adding a modifying agent capable of specifically modifying the sialic acid binding mode of the O-bound sugar chain to the sample containing the glycoprotein bound to the O-bound sugar chain; a releasing step, wherein the O-bound sugar chain that has been specifically modified in the sialic acid binding mode is released from the glycoprotein; and In the analysis step, the released O-bound sugar chains that have been specifically modified in the sialic acid binding mode are analyzed.

2. The method according to claim 1, wherein: The sialic acid binding mode specific modification includes a first reaction, and the first reaction is to lactonize sialic acid.

3. The method according to claim 2, wherein: The sialic acid binding mode-specific modification further includes a second reaction, and the second reaction is used to amidate the lactone structure obtained by the first reaction.

4. The method according to claim 3, wherein: The second reaction is performed using a solution containing at least one selected from the group consisting of ammonia, amines, and salts thereof.

5. The method according to claim 3 or 4, wherein: The second reaction is an aminolysis reaction.

6. The method according to claim 3 or 4, wherein: The sialic acid after the second reaction is modified in a manner different from that of the binding.

7. The method according to claim 6, wherein: The sialic acid binding modes include α-2,3 sialic acid and α-2,6 sialic acid.

8. The method according to claim 2 or 3, wherein: A solution containing a dehydration condensation agent is added to the sample to perform the first reaction.

9. The method according to claim 2 or 3, wherein: α-2,6 sialic acid is amidated after the first reaction.

10. The method according to claim 2 or 3, wherein: The method further comprises the step of purifying the glycoprotein by protein precipitation after the first reaction.

11. The method according to claim 1 or 2, wherein: The sugar chain is released from the glycoprotein by a β-elimination reaction or a hydrazinolysis reaction.

12. The method according to claim 11, wherein: The beta elimination reaction is carried out below pH 11.

0.

13. The method according to claim 1 or 2, wherein: The method further comprises the step of purifying the released sugar chains using a solid phase carrier.

14. The method according to claim 1 or 2, wherein: The analysis includes at least one selected from the group consisting of mass spectrometry, chromatography, and electrophoresis.

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