Method for analyzing sialyl sugar chain

Abstract

Provided is a method for analyzing a sialyl sugar chain having sialic acid bound thereto, the method comprising: a step for preparing a first sample containing a first modified material derived from the sialyl sugar chain and a second sample containing a second modified material derived from the sialyl sugar chain; a step for analyzing each of the first sample and the second sample by a chromatographic method or an electrophoresis method to obtain first analysis data associated with the first sample and second analysis data associated with the second sample; a step for comparing the first analysis data with the second analysis data; and a step for determining that the sialic acid bound to the sialyl sugar chain comprises at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid and α2,9-sialic acid when the mobility attributed to the first modified material, which is shown in the first analysis data, and the mobility attributed to the second modified material, which is shown in the second analysis data, are different from each other. In the method, the first modified material is a compound produced by the esterification or amidation or both thereof of the sialic acid bound to the sialyl sugar chain, and the second modified material is a compound produced by the esterification or amidation or both thereof of the sialic acid bound to the sialyl sugar chain, which is different from the α2,3-sialic acid, the α2,8-sialic acid and the α2,9-sialic acid.

Description

Analytical methods for sialylglycans

[0001] The present invention relates to a method for analyzing sialyl sugar chains.

[0002] Sialic acid is also found in glycoproteins in vivo, where it is primarily present at the terminal end of the glycan. Because sialic acid is likely to be located on the outside of glycoprotein molecules, it is easily involved in recognition by other molecules. Sialic acid may have different linkage modes between adjacent sugars. For example, in human N-linked glycans, the main linkage modes are known to be α2,3- and α2,6-. In addition, α2,8- and α2,9-linkage modes are known to exist in O-linked glycans and glycosphingolipids. Analysis of the linkage modes of sialic acid is important because different linkage modes can lead to recognition by different molecules and play different roles.

[0003] JP 2016-194500 A JP 2019-152475 A

[0004] Nishikaze T., Tsumoto H., Sekiya S., Iwamoto S., Miura Y., Tanaka K., Analytical Chemistry, 2017, 89, 2353-2360.

[0005] Because sialic acid has a negative charge and is easily degraded, it is not easy to analyze sialic acid-containing sialylglycans. Furthermore, because the mass (molecular weight) of the glycan does not change depending on the type of sialic acid bond, it is not possible to distinguish between different bond types using mass spectrometry.

[0006] In order to distinguish and analyze the binding modes of sialic acid, methods for performing binding mode-specific modifications on sialic acid have been proposed. For example, Patent Document 1 discloses a method for preparing an analytical sample for analyzing glycans contained in a sample, which comprises carrying out a first reaction to generate different modified forms depending on the binding mode of sialic acid when sialic acid is bound to the glycan of the analyte, and reacting the analyte containing the glycan with an amine containing two or more carbon atoms and a dehydration condensing agent in the first reaction.

[0007] Furthermore, Patent Document 2 discloses a method for preparing a sample containing a sugar chain, the method comprising: performing a lactonization reaction to lactonize at least a portion of the sialic acid contained in the sugar chain; and adding to the sample an amidation reaction solution containing at least one selected from the group consisting of ammonia, an amine, and a salt thereof, which is reacted with the lactonized sialic acid, to perform an amidation reaction to amidate the lactone of the lactonized sialic acid. Non-Patent Document 1 discloses a method for preparing a sample to be used in mass spectrometry, in which a solution containing isopropylamine and a dehydration condensing agent is added to free N-linked sugar chains to lactonize α2,3-sialic acid and amidate α2,6-sialic acid.

[0008] These methods utilize the fact that α2,3-sialic acid is more susceptible to intramolecular dehydration by dehydration condensation agents than α2,6-sialic acid, and generate molecules with different masses depending on the bond type of sialic acid. Therefore, the bond types of sialic acids can be distinguished and analyzed by mass spectrometry.

[0009] It is known that differences in the binding mode of sialic acid are involved in various biological phenomena in living organisms, for example, changes in the binding mode of sialic acid are known to occur with canceration. Therefore, distinguishing the binding mode of sialic acid as described above has attracted attention for its use as a biomarker for sialic acid and for the quality control of biopharmaceuticals, and further improvements (e.g., speeding up and simplifying) in analytical methods for sialylglycans are expected.

[0010] The present invention has been made in view of the above circumstances, and aims to provide a method for analyzing sialylglycan that can distinguish the binding mode of sialic acid bound to a sialylglycan by chromatography or electrophoresis.

[0011] As a result of extensive research, the present inventors have discovered that the binding mode of sialic acid bound to sialylglycan can be distinguished by analyzing, by chromatography or electrophoresis, two types of samples that are modified differently depending on the binding mode of sialic acid, and have thus completed the present invention.

[0012] The present invention provides a method for analyzing a sialic acid-bound sialylglycan, comprising the steps of: preparing a first sample containing a first modification derived from the sialylglycan; and a second sample containing a second modification derived from the sialylglycan; analyzing each of the first and second samples by chromatography or electrophoresis to obtain first analytical data derived from the first sample and second analytical data derived from the second sample; comparing the first analytical data with the second analytical data; and determining that the sialic acid bound to the sialylglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid when the mobility attributed to the first modification shown in the first analytical data is different from the mobility attributed to the second modification shown in the second analytical data; and the first modification is a compound produced by esterification, amidation, or both of the sialic acid bound to the sialylglycan, The second modified form relates to a method for analyzing a sialylglycan, wherein the sialic acid bonded to the sialylglycan other than the α2,3-sialic acid, the α2,8-sialic acid, and the α2,9-sialic acid is a compound produced by esterification, amidation, or both.

[0013] According to the present invention, it is possible to provide a method for analyzing sialyl-glycan that can distinguish the binding mode of sialic acid bound to the sialyl-glycan.

[0014] Fig. 1 is a flowchart showing an analytical method according to one embodiment of the present invention. Fig. 2 is a fluorescence chromatogram showing the results of HPLC analysis performed in the examples. Fig. 3 is a graph showing the results of microchip electrophoresis analysis performed in the examples.

[0015] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0016] <<Method for Analyzing Sialylglycan>> A method for analyzing sialylglycan according to one embodiment of the present invention is a method for analyzing sialic acid-bound sialylglycan, comprising the steps of: preparing a first sample containing a first modified form derived from the sialylglycan, and a second sample containing a second modified form derived from the sialylglycan; analyzing each of the first and second samples by chromatography or electrophoresis to obtain first analytical data derived from the first sample and second analytical data derived from the second sample; comparing the first analytical data with the second analytical data; and determining that the sialic acid bound to the sialylglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid, when the mobility attributed to the first modified form shown in the first analytical data differs from the mobility attributed to the second modified form shown in the second analytical data. The first modified form is a compound in which the sialic acid bound to the sialyl sugar chain is produced by esterification, amidation, or both, and the second modified form is a compound in which the sialic acid bound to the sialyl sugar chain other than the α2,3-sialic acid, the α2,8-sialic acid, and the α2,9-sialic acid is produced by esterification, amidation, or both.

[0017] 1 is a flowchart showing the flow of an analysis method according to one embodiment of the present invention. Each step will be described below.

[0018] <Step of preparing a first sample and a second sample> In this step, a first sample containing a first modified product derived from a sialylglycan and a second sample containing a second modified product derived from a sialylglycan are prepared.

[0019] (Sialylglycan) In this embodiment, the term "sialylglycan" refers to a glycan containing at least one sialic acid as a sugar constituting the glycan. The sialylglycan may be a sialylglycan constituting a part of a glycoprotein, a sialylglycan constituting a part of a glycolipid, or a sialylglycan released from these. When the sialylglycan is a part of a glycoprotein, the sialylglycan may be an O-linked glycan or an N-linked glycan. In one aspect of this embodiment, the sialylglycan may be a labeled glycan in which the reducing end of a released sialylglycan is modified with a labeling compound to enable fluorescence or UV detection. Examples of the labeling compound include 2-aminobenzoic acid (2-AA) and 8-aminopyrene-1,3,6-trisulfonic acid (APTS), which are used in the examples.

[0020] (First Sample) In this embodiment, the first sample may be a liquid or a solid. From the viewpoint of facilitating subsequent steps, the first sample is preferably a liquid. In one aspect of this embodiment, the first sample may be derived from a living organism or a cell.

[0021] (First Modified Product) In this embodiment, the first modified product is derived from the sialylated glycan to be analyzed. The first modified product is a compound produced by esterifying, amidating, or both, the sialic acid bound to the sialyl glycan. That is, the esterified or amidated sialic acid in the first modified product may be α2,3-sialic acid, α2,6-sialic acid, α2,8-sialic acid, or α2,9-sialic acid. In one aspect of this embodiment, the first modified product can also be considered as a type of glycan. The first modified product may constitute a part of a glycoprotein or a part of a glycolipid, or may be a compound released from these.

[0022] As used herein, "protein" refers collectively to molecules in which two or more amino acids are peptide-bonded. Proteins herein may include peptides (including oligopeptides and polypeptides) with a small number of amino acid residues, for example, fewer than 50 amino acid residues, and proteins with a large number of amino acid residues, for example, 50 or more amino acid residues. As used herein, glycoproteins may include glycopeptides.

[0023] When the peptide chain constituting the glycoprotein has a large number of amino acid residues, the peptide chain may be cleaved with a digestive enzyme, etc. For example, when preparing a sample for liquid chromatography or electrophoresis, the number of amino acid residues in the peptide chain is preferably 30 or less, more preferably 20 or less, and even more preferably 15 or less. There is no particular lower limit on the number of amino acid residues in the peptide chain, but 2 or more is preferred, and 3 or more is more preferred.

[0024] Examples of digestive enzymes used to cleave the peptide chains constituting glycoproteins include trypsin, Lys-C, arginine endopeptidase, chymotrypsin, pepsin, thermolysin, proteinase K, and pronase E. These digestive enzymes may be used singly or in combination of two or more. The conditions for cleaving the peptide chains are not particularly limited as long as they do not affect the sialylglycan to be measured, and an appropriate protocol may be adopted depending on the digestive enzyme used. Prior to cleaving the peptide chains, the proteins in the sample may be denatured or alkylated. The conditions for the denaturation or alkylation are not particularly limited. The peptide chain cleavage may be performed before or after the first reaction or after the second reaction, which will be described later. In one aspect of this embodiment, the peptide chains may be cleaved by chemical cleavage or the like, rather than enzymatic cleavage.

[0025] Treatment to block amino groups in glycoproteins in the sample may be carried out as appropriate. Examples of such treatments include dimethylamidation and guanidylation of glycoproteins. This can suppress side reactions such as intramolecular dehydration condensation that may occur between the glycoprotein and the amino or carboxyl group at the end of the protein main chain when the first or second reaction described below is carried out.

[0026] The first modified product can be produced by esterifying, amidating, or both, the sialic acid bound to the sialylglycan. In one aspect of this embodiment, the first modified product may be produced by subjecting the sialylglycan to a linkage-specific modification of sialic acid. Here, "linkage-specific modification of sialic acid" and "linkage-specific modification of sialic acid" refer to a modification reaction that acts on sialic acid, in which the chemical structure produced by the modification reaction differs between α2,3-sialic acid (or α2,8-sialic acid or α2,9-sialic acid) and α2,6-sialic acid. Specifically, the linkage-specific modification of sialic acid preferably includes a first reaction in which sialic acid is lactonized and a second reaction in which the lactone structure produced by the first reaction is amidated. The first and second reactions are described below.

[0027] (First Reaction) In the first reaction, when sialic acid is bound to a glycoprotein, the sialic acid is selectively lactonized depending on the binding mode. In the first reaction, preferably, in addition to α2,3-sialic acid in the sugar chain, α2,8-sialic acid and α2,9-sialic acid are lactonized.

[0028] The first reaction can be carried out by contacting a sample containing the above-mentioned sialylglycan with a solution for linkage-specific lactonization of sialic acid (hereinafter also referred to as a "lactonization reaction solution"). One example of a contacting method is to add the lactonization solution to the above-mentioned sample. The lactonization reaction solution preferably contains a dehydration condensation agent. Simultaneously with the first reaction, α2,6-sialic acid is preferably subjected to a modification other than lactonization, preferably amidation or esterification. In this case, the lactonization reaction solution preferably contains, in addition to the dehydration condensation agent, a nucleophile containing at least one selected from the group consisting of alcohols, amines, and salts thereof.

[0029] The types and concentrations of the dehydration condensation agent and nucleophile may be adjusted to selectively induce dehydration or nucleophilic reactions based on the bond mode of sialic acid. The lactone produced by intramolecular dehydration of the carboxy group of α2,3-sialic acid is a six-membered ring, while the lactone produced by intramolecular dehydration of the carboxy 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 a seven-membered ring, is more easily lactonized than α2,6-sialic acid. Furthermore, since the carboxy group of α2,3-sialic acid is located in a position that is relatively more sterically hindered than the carboxy group of α2,6-sialic acid, large molecules are less likely to react with α2,3-sialic acid than with α2,6-sialic acid. Based on the differences in molecular structure due to the bond mode of sialic acid, the types and concentrations of the dehydration condensation agent and nucleophile are adjusted to achieve different modifications depending on the bond mode of sialic acid.

[0030] The dehydration condensation agent preferably contains a carbodiimide, because when a carbodiimide is used, a carboxy group present at a site with large steric hindrance is less likely to be amidated than when a phosphonium-based dehydration condensation agent (so-called BOP reagent) or a uronium-based 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-toluylcarbodiimide, bis(2,6-diisopropylphenyl)carbodiimide, bis(trimethylsilyl)carbodiimide, 1,3-bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)carbodiimide (BDDC), and salts thereof (such as hydrochlorides).

[0031] In order to promote the dehydration condensation by the dehydration condensation agent and to suppress side reactions, it is preferable to use a highly nucleophilic additive in addition to the carbodiimide. In one aspect of this embodiment, the "highly nucleophilic additive" can also be understood as a type of dehydration condensation agent. As the highly nucleophilic additive, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt), 4-(dimethylamino)pyridine (DMAP), 2-cyano-2-(hydroxyimino)ethyl 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), etc. are preferably used.

[0032] The amine used as the nucleophile preferably contains a primary and / or secondary alkylamine containing two or more carbon atoms. Preferred primary alkylamines include ethylamine, propylamine, isopropylamine, butylamine, sec-butylamine, and tert-butylamine. Preferred secondary alkylamines include dimethylamine, ethylmethylamine, diethylamine, propylmethylamine, and isopropylmethylamine. From the viewpoint of preventing amidation of carboxy groups present at sites with significant steric hindrance, such as the carboxy group of α2,3-sialic acid, it is preferred to use an amine having a branched alkyl group, such as isopropylamine. That is, the nucleophile preferably contains an amine compound having a branched alkyl group or a salt thereof. When an amine is used as the nucleophile in the lactonization reaction solution, the carboxy groups of some sialic acids, such as α2,6-sialic acid, are amidated based on the bonding mode of the sialic acid.

[0033] The alcohol used as the nucleophile is not particularly limited, and examples thereof include methanol and ethanol. When an alcohol is used as the nucleophile in the lactonization reaction solution, the carboxy groups of some sialic acids, such as α2,6-sialic acid, are esterified based on the bonding mode of the sialic acid. The nucleophile may include a salt of the above-mentioned nucleophile.

[0034] The concentration of the dehydration condensation agent in the lactonization reaction solution is preferably, for example, 1 mM to 5 M, and more preferably 10 mM to 3 M. When a carbodiimide is used in combination with a highly nucleophilic additive (e.g., Oxyma, HOAt, HOBt, etc.), the concentrations of each are preferably within the above ranges. The concentration of the nucleophile in the lactonization reaction solution is, for example, preferably 0.01 M to 20 M, and more preferably 0.1 M to 10 M. The reaction temperature for the first reaction may be approximately -20°C to 100°C, and preferably -10°C to 50°C.

[0035] The first reaction can be carried out in either a liquid phase or a solid phase. When the reaction is carried out in a liquid phase, it is preferable to carry out the reaction in a non-aqueous solvent such as dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). By carrying out the reaction in a non-aqueous solvent, side reactions tend to be suppressed. The concentration of each component in the liquid phase reaction is not particularly limited and can be determined appropriately depending on the type of dehydration condensation agent and amine, etc.

[0036] When the first reaction is carried out in a solid phase, the solid-phase support is not particularly limited as long as it can immobilize sialylglycan (including sialylglycan bound to proteins or lipids). To immobilize sialylglycan, for example, a solid-phase support having an epoxy group, a tosyl group, a carboxy group, an amino group, or the like as a ligand can be used. A glycoprotein containing a glycan can also be immobilized using a solid-phase support having a hydrazide group, an aminooxy group, or the like as a ligand. By carrying out the reaction with the sialylglycan immobilized on the solid-phase support, the reaction solution can be easily removed after the reaction, allowing for efficient modification of sialic acid. When magnetic beads are used as the solid-phase support, the magnetic beads to which the sialylglycan is bound can be collected with a magnet, and excess reagent can be removed, or the beads can be washed with a solvent. When a resin is used as the solid-phase support, the excess reagent can be removed by passing the mixture through a filter, and then the resin can be recovered. Alternatively, the resin can be precipitated by centrifugation, and the excess reagent in the supernatant can be removed. When the first reaction is carried out in a liquid phase, the excess reagent can be removed by ultrafiltration.

[0037] The product after the first reaction (hereinafter, sometimes referred to as "intermediate") may be subjected to treatment such as purification, desalting, solubilization, concentration, drying, etc. by a known method, as necessary, to remove the lactonization reaction solution or reduce the concentration of the lactonization reaction solution.

[0038] (Second Reaction) The second reaction may be carried out by contacting the product (intermediate) from the first reaction with a solution for amidation (hereinafter also referred to as the "amidation reaction solution"). The first reaction can differentiate between α2,3- and α2,6-linkages of sialic acid, but the lactone structure generated from α2,3-sialic acid may be unstable and tends to revert to the original carboxylic acid structure upon hydrolysis. The second reaction specifically amidates the lactone structure, stabilizing the lactone structure. At the same time, the second reaction results in linkage-specific modification of sialic acid with different masses, making it possible to differentiate between α2,3-sialic acid and α2,6-sialic acid. By carrying out the second reaction, sialic acid can be more quickly and specifically modified in a linkage-specific manner. An example of the second reaction may be aminolysis. Aminolysis is a reaction based on the interaction between an amino group and a lactone structure. The aminolysis is different from hydrolysis because it is favorable even under anhydrous conditions. In this specification, the ring-opening and amidation of a lactone structure with ammonia, an amine, or a salt thereof, which is possible even under anhydrous conditions, is referred to as "aminolysis." In one aspect of this embodiment, "stabilization of a lactone structure" and "stabilizing a lactone structure" can also be understood as replacing an unstable lactone structure with another stable structure (such as a methylamide group) by performing aminolysis.

[0039] As another example of the second reaction, the lactone structure may be amidated using a strong dehydration condensation agent. Examples of the dehydration condensation agent used in the second reaction include phosphonium-based condensation agents and uronium-based condensation agents, and specific examples thereof include benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (H ATU), (1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU), tetramethylfluoroformamidinium hexafluorophosphate (TFFH), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), and the like. Amidation with these strong dehydrating condensing agents may be preceded by a lactone cleavage procedure.

[0040] The amidation reaction solution preferably contains at least one selected from the group consisting of ammonia, amines, and salts thereof. When an amine is used, it is preferable to use an amine different from the amine used in the lactonization reaction solution, or to change the mass by modification with a stable isotope, etc. By using amines with different masses in the first and second reactions, sialic acids can be amidated so that the mass differs depending on the bond type. A dehydrating condensing agent is not required for aminolysis, and the amidation reaction solution does not need to contain a dehydrating condensing agent. The amidation reaction solution may contain a dehydrating condensing agent. For example, the amidation reaction solution may be prepared by adding ammonia, an amine, or a salt thereof without removing the lactonization reaction solution added to the sample in the first reaction. In the second reaction, the lactone structure can be stabilized by such a simple operation.

[0041] The amine contained in the amidation reaction solution is preferably a primary amine, more preferably a primary amine having a linear hydrocarbon group, and even more preferably a primary amine having a linear alkyl group. The amine contained in the amidation reaction solution is preferably a primary amine having 10 or fewer carbon atoms, more preferably a primary amine having 7 or fewer carbon atoms, still more preferably methylamine, ethylamine, propylamine, butylamine, or pentylamine, and most preferably methylamine. From the viewpoint of more efficiently amidating the lactone structure, it is preferable that the amine contained in the amidation reaction solution has a linear structure without branches (hereinafter, "branch" refers to a branch in a hydrocarbon chain) or has a small number of carbon atoms.

[0042] 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 more preferably contains an allyl group. The amine is preferably allylamine. The amine contained in the amidation reaction solution may be a primary amine containing a hydroxy group or may be ethanolamine. The amine contained in the amidation reaction solution may contain various functional groups other than alkyl groups. The amidation reaction solution may contain a salt of the above-mentioned amine. As a result of the amidation reaction, the sugar chain is modified to contain such a functional group, making the modified sugar chain easier to separate not only by mass spectrometry but also by chromatography, etc.

[0043] The concentrations of ammonia, amine, and salts thereof in the amidation reaction solution are preferably 0.1 M or more, more preferably 0.3 M or more, even more preferably 0.5 M or more, even more preferably 1.0 M or more, and most preferably 3.0 M or more. In a suitable 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 0.1 M or more, more preferably 0.3 M or more, even more preferably 0.5 M or more, even more preferably 1.0 M or more, and most preferably 3.0 M or more. The higher the concentrations of ammonia, amine, and salts thereof in the amidation reaction solution, the more efficiently the lactone structure can be amidated. The upper limit of the concentrations of ammonia, amine, and salts thereof in the amidation reaction solution is not particularly limited, but may be, for example, 16 M or less.

[0044] The solvent for the amidation reaction solution may be an aqueous solvent or an organic solvent, but is preferably a solvent with a low water content from the viewpoint of preventing hydrolysis of the lactone structure and ensuring rapid amidation. The solvent for the amidation reaction solution is preferably a dehydrated solvent that has been subjected to a dehydration operation to reduce the water content, and is more preferably an anhydrous solvent. The solvent for the amidation reaction solution preferably contains at least one of methanol and acetonitrile. In another aspect of this embodiment, the amidation reaction solution may contain water, and the solvent for the amidation reaction solution may be water.

[0045] The amidation reaction solution preferably has a pH of 7.7 or higher, more preferably a pH of 8.0 or higher, even more preferably a pH of 8.8 or higher, and most preferably a pH of 10.3 or higher. As the pH of the amidation reaction solution increases, the lactone structure can be amidated more efficiently. The upper limit of the pH of the amidation reaction solution is not particularly limited, but may be, for example, 14 or lower. When a lactone structure is amidated using a strong dehydrating condensing agent, the pH is not particularly limited, and the pH may be adjusted depending on the dehydrating condensing agent used.

[0046] The second reaction can be completed within a few seconds to a few minutes. The time for contacting the sample with the amidation reaction solution to amidate the lactone structure is preferably less than 1 hour, more preferably less than 30 minutes, even more preferably less than 15 minutes, even more preferably less than 5 minutes, and most preferably less than 1 minute. Preferably, the sample may be washed with the amidation reaction solution, or the amidation reaction solution may be temporarily passed through a sample held on a carrier or the like. The lower limit of the time for contacting the sample with the amidation reaction solution to amidate the lactone structure is not particularly limited, but may be, for example, 1 second or more. The time from the end of contact between the sample and the lactonization reaction solution to the end of contact between the sample and the amidation reaction solution is preferably less than 1.5 hours, more preferably less than 1 hour, and even more preferably less than 30 minutes. Because the second reaction is completed in a short time, it is possible to prevent decomposition of unstable lactone structures and loss of quantitative accuracy in glycan analysis. Furthermore, setting the reaction time of the second reaction to a short time enables more efficient sample analysis. The lower limit of the time from the end of contact between the sample and the lactonization reaction solution to the end of contact between the sample and the amidation reaction solution is not particularly limited, but may be, for example, 1 second or more.

[0047] The second reaction can be carried out in either a liquid phase or a solid phase. The state of the sample during the amidation reaction is not particularly limited as long as it can be brought into contact with the amidation reaction solution, but it is preferable to bring the glycoprotein into contact with the amidation reaction solution in a state where the glycoprotein is bound or adsorbed to a solid-phase carrier. The solid-phase carrier used in the second reaction is not particularly limited as long as it is capable of immobilizing the glycoprotein, and examples of the solid-phase carriers that can be used in the first reaction include those that can be used in the first reaction.

[0048] The sample after the second reaction may be subjected to treatment such as purification, desalting, solubilization, concentration, drying, etc. by known methods, as necessary, to remove the amination reaction solution or reduce the concentration of the amination reaction solution. Excess reagents after the second reaction can be removed by the methods described in the section on the first reaction, depending on whether the reaction is solid phase or liquid phase.

[0049] Taking into account the details of the first and second reactions described above, the first modified product can also be understood as a compound produced by contacting an intermediate produced by contacting the sialyl sugar chain with a dehydration condensation agent and a nucleophilic agent with at least one selected from the group consisting of ammonia, amines, and salts thereof.

[0050] (Second Sample) In this embodiment, the second sample may be a liquid or a solid. From the viewpoint of facilitating subsequent steps, the second sample is preferably a liquid. In one aspect of this embodiment, the second sample may be derived from a living organism or a cell.

[0051] (Second Modified Product) In this embodiment, the second modified product is derived from the sialic acid chain to be analyzed. The second modified product is a compound produced by esterification, amidation, or both of the sialic acid bound to the sialic acid chain other than the α2,3-sialic acid, the α2,8-sialic acid, and the α2,9-sialic acid. In one aspect of this embodiment, the second modified product can also be understood as a type of glycan. The second modified product may constitute a part of a glycoprotein or a part of a glycolipid, or may be a compound released therefrom. The sialic acid chain from which the second modified product is derived and the sialic acid chain from which the first modified product is derived are usually the same. In one aspect of this embodiment, when the sialic acid chain to be analyzed does not contain α2,3-sialic acid, α2,8-sialic acid, or α2,9-sialic acid, the second modified product may be the same compound as the first modified product.

[0052] The second modified product can be produced by esterifying, amidating, or both, a "sialic acid bound to a sialylglycan" other than α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid. Here, examples of "sialic acids other than α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid" include α2,6-sialic acid. In one aspect of this embodiment, the second modified product is preferably a compound produced by a method comprising the first reaction and a third reaction in which the lactone structure produced by the first reaction is ring-opened. In another aspect of this embodiment, the second modified product can also be understood as a compound produced by placing an intermediate produced by contacting the sialylglycan with a dehydration condensation agent and a nucleophile in a basic environment. The reagents and reaction conditions described above are used for the first reaction. The third reaction will be described below.

[0053] (Third Reaction) The third reaction can be carried out by hydrolyzing the lactone structure generated from α2,3-sialic acid to open the ring and return it to the original carboxylic acid structure. The third reaction is preferably carried out by placing the intermediate generated after the first reaction in a basic environment.

[0054] The basic environment is preferably an environment in which at least one selected from the group consisting of quaternary ammonium cations, tertiary amines, secondary amines, branched primary amines having two or more carbon atoms directly bonded to the carbon atom bonded to the amino group, alkali metal hydroxides, alkaline earth metal hydroxides, tetraalkylammonium hydroxides, guanidine, guanidine derivatives, and salts thereof, and alkaline buffer solutions is present. The alkaline buffer solution is preferably at least one selected from the group consisting of Tris buffer, Good's buffer, borate buffer, and carbonate buffer solution. In one aspect of this embodiment, the basic environment is more preferably an environment in which t-butylamine, trimethylamine, or both are present. The use of such a compound with high steric hindrance suppresses aminolysis, allowing the lactone structure to be efficiently converted back to the original carboxylic acid structure.

[0055] The basic environment preferably has a pH of 8 or higher, more preferably a pH of 9 or higher, even more preferably a pH of 9.5 or higher, and most preferably a pH of 10 or higher. As the pH of the basic environment increases, the lactone structure can be opened more efficiently. The upper limit of the pH of the basic environment is not particularly limited, but may be, for example, 14 or lower.

[0056] The third reaction can be completed within a few seconds to a few minutes. The reaction time of the third reaction is preferably 1 hour or less, more preferably 30 minutes or less, even more preferably 10 minutes or less, even more preferably 5 minutes or less, and most preferably 3 minutes or less. The lower limit of the reaction time of the third reaction is not particularly limited, but may be, for example, 1 second or more.

[0057] The third reaction can be carried out in either a liquid phase or a solid phase. The state of the sample is not particularly limited as long as it can be placed in a basic environment, but it is preferable to place the sialylglycan in a basic environment while it is bound or adsorbed to a solid-phase carrier. The solid-phase carrier used in the third reaction is not particularly limited as long as it is capable of immobilizing the sialylglycan, and examples thereof include solid-phase carriers that can be used in the first reaction.

[0058] The sample after the third reaction may be purified, desalted, solubilized, concentrated, dried, or the like by known methods, as needed, to remove or reduce the concentration of the reagents used in the reaction. Excess reagents after the third reaction can be removed by the methods described in the section on the first reaction, depending on whether the reaction is solid phase or liquid phase.

[0059] The methods for preparing the first and second samples have been described above. When the glycoprotein is in the form of a sialylglycan bound to a protein, the glycoprotein may be purified and the sialylglycan may be released from the glycoprotein, as described below.

[0060] (Purification of Glycoproteins) Purifying glycoproteins allows removal of solutions used in the first, second, and / or third reactions, improving the efficiency of downstream processes. The method and conditions for purifying glycoproteins can be appropriately selected depending on the type, properties, molecular weight, etc. of the glycoprotein. Glycoprotein purification may be performed by protein precipitation. Precipitants that can be used include salts such as 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 thereof. In protein precipitation, for example, a precipitant is added to a sample containing glycoproteins and allowed to stand at temperatures between -20°C and 30°C for an appropriate period of time. The resulting precipitate can then be collected by centrifuging the sample. Glycoprotein purification may be performed by immobilizing the glycoprotein on a solid support, or by using gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, etc.

[0061] (Release of sialylglycan from glycoprotein) Methods for releasing sialylglycan from glycoprotein include enzymatic treatment using O-glycosidase, N-glycosidase, endoglycoceramidase, etc., and chemical release methods such as hydrazinolysis and β-elimination. Before the treatment for releasing the glycochain, the peptide chain of the glycoprotein may be cleaved. Modification such as labeling of the reducing end of the glycochain with 3-aminoquinoline (3AQ), anthranilic acid (2AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), 8-aminopyrene-1,3,6-trisulfonic acid (APTS), etc. may be performed in conjunction with the release of the glycochain.

[0062] To release N-linked glycans from peptide chains of glycoproteins, enzymatic treatment with peptide-N-glycosidase F (PNGase F), peptide-N-glycosidase A (PNGase A), endo-β-N-acetylglucosaminidase (Endo M), or the like is preferably used. To release O-linked glycans from peptide chains of glycoproteins, chemical release methods are preferably used. The hydrazinolysis method may be performed according to known methods, for example, by adding anhydrous hydrazine or hydrated hydrazine to a sample containing glycoproteins and heating. The β-elimination method may be performed according to known methods, for example, by adding ammonium carbamate, saturated ammonia, ammonium carbonate, anhydrous trifluoromethanesulfonic acid (anhydrous TFMS), sodium hydroxide, dimethylamine, or the like to a sample containing glycoproteins and heating under alkaline conditions. From the viewpoint of suppressing side reactions due to peeling, it is preferable to release O-linked glycans using ammonium salt powder such as ammonium carbamate. When carrying out the reaction under alkaline conditions, a pyrazolone reagent such as PMP may be coexistent to simultaneously release and label the O-linked glycans, thereby suppressing side reactions due to peeling. From the viewpoint of preventing the conversion of methylated sialic acid to carboxylic acid in the second reaction and from the viewpoint of the efficiency of release of O-linked glycans, the pH of the β-elimination reaction is preferably 11.0 or lower, more preferably 10.0 or lower. From the viewpoint of suppressing decomposition of sialylated glycans and peeling reactions, the pH of the β-elimination reaction is preferably 9.5 or lower. The pH when carrying out the β-elimination reaction is usually 7.5 or higher.

[0063] The release of the sugar chains may be carried out in a liquid phase or in a solid phase. For example, the above-mentioned sugar chain cleavage treatment may be performed on a glycoprotein immobilized on a solid support, and the sugar chains may be recovered. After the sugar chain cleavage treatment is performed on a glycoprotein bound to a solid support having a hydrazide group, the sugar chains may be released and recovered using a weakly acidic solution. By carrying out the reaction while the sample is immobilized on a solid support, removal of the reaction solution or desalting and purification becomes easier, simplifying sample preparation.

[0064] Free glycans may be purified according to known methods, for example, by separating proteins and glycans by liquid-liquid extraction using an organic solvent such as chloroform. Free glycans can also be conveniently purified using a solid-phase support (column). Free glycans can also be recovered by binding to a solid-phase support having a hydrazide group or an aminooxy group. An example of a solid-phase support having a hydrazide group is "BlotGlyco" manufactured by Sumitomo Bakelite Co., Ltd. The glycans may also be purified by adsorbing them to a support for hydrophilic interaction chromatography (hereinafter also referred to as HILIC). The support for HILIC preferably contains an amide group. Free glycans may also be purified using a reverse-phase support such as C18, in addition to a carbon column. Labeled glycans may also be purified by adsorbing them to carbon or C18.

[0065] <Step of Obtaining First Analytical Data and Second Analytical Data> In this step, the first sample and the second sample are each analyzed by chromatography or electrophoresis to obtain first analytical data derived from the first sample and second analytical data derived from the second sample. Here, "first analytical data derived from the first sample" refers to analytical data obtained by analyzing the first sample by chromatography or electrophoresis. "Second analytical data derived from the second sample" refers to analytical data obtained by analyzing the second sample by chromatography or electrophoresis. It goes without saying that the analytical method used to obtain the first analytical data is the same as the analytical method used to obtain the second analytical data.

[0066] The chromatography method used in this step is not particularly limited as long as it is a known method, and any method can be used. In one aspect of this embodiment, the chromatography method is preferably at least one selected from the group consisting of liquid chromatography and supercritical fluid chromatography, and more preferably liquid chromatography. The analytical equipment and analytical conditions used for the chromatography method are not particularly limited, and examples include the analytical equipment and analytical conditions described in the Examples below. The column used for liquid chromatography is not particularly limited, and hydrophobic reversed-phase columns such as C30, C18, C8, and C4, carbon columns, and normal-phase columns for HILIC can be used as appropriate.

[0067] The analytical data obtained by the chromatography method is not particularly limited, and may be a chromatogram chart or a retention time determined from the chromatogram.

[0068] The electrophoresis method used in this step is not particularly limited as long as it is a known method, and any method can be used. In one aspect of this embodiment, the electrophoresis method is preferably at least one selected from the group consisting of capillary electrophoresis, SDS-PAGE, microchip electrophoresis, two-dimensional electrophoresis, and isoelectric focusing, and more preferably at least one selected from the group consisting of capillary electrophoresis, SDS-PAGE, and microchip electrophoresis. The analytical equipment and analytical conditions used for the electrophoresis method are not particularly limited, and known analytical equipment and analytical conditions can be used.

[0069] The analytical data obtained by electrophoresis is not particularly limited, and may be a chart showing the results of electrophoresis, or the mobility determined from the chart.

[0070] <Step of Comparing First Analysis Data and Second Analysis Data> In this step, the first analysis data and the second analysis data are compared. The method of comparison is not particularly limited, and examples include a method of overlaying the two chromatograms and examining the presence or absence of peaks at different positions, and a method of calculating and examining the difference in retention time between the two.

[0071] <Step of determining whether sialic acid bound to sialylglycan contains sialic acid of a predetermined bond type> In this step, when the mobility attributed to the first modification shown in the first analytical data differs from the mobility attributed to the second modification shown in the second analytical data, it is determined that the sialic acid bound to the sialylglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid. Here, the above-mentioned "mobility" is a concept that includes the position and retention time of a peak in a chromatogram in a chromatography method, and the mobility and migration time in an electrophoresis method.

[0072] The method for assigning mobilities in the first analytical data and the second analytical data is not particularly limited. For example, if the chemical structure of a first modified form or a second modified form contained in a sample to be analyzed has been predicted and the mobility derived from the predicted chemical structure is a known mobility, the mobility corresponding to the known mobility in the first analytical data or the second analytical data, respectively, can be determined to be the mobility attributed to the first modified form or the second modified form. If the chemical structure of the first modified form or the second modified form contained in a sample to be analyzed cannot be predicted, analytical data (corresponding to the "third analytical data" described below) derived from a sample containing sialylglycan from which the first modified form and the second modified form are derived (corresponding to the "third sample" described below) can be compared with the first analytical data or the second analytical data to determine that the changed mobility is the mobility attributed to the first modified form or the second modified form. Note that if no changed mobility is observed in the comparison of the first analytical data, second analytical data, and third analytical data (if the mobilities of all three analytical data are the same), it can be determined that the sample to be analyzed does not contain sialylglycan.

[0073] In this embodiment, examples of "when the mobility attributable to the first modified form shown in the first analytical data differs from the mobility attributable to the second modified form shown in the second analytical data" include the following cases: (1) when analytical data is obtained by chromatography, the position of the peak attributable to the first modified form shown in the first analytical data is different from the position of the peak attributable to the second modified form shown in the second analytical data; (2) when analytical data is obtained by chromatography, the absolute value of the difference between the retention time attributable to the first modified form shown in the first analytical data and the retention time of the peak attributable to the second modified form shown in the second analytical data is greater than 0; or (3) when analytical data is obtained by electrophoresis, the absolute value of the difference between the mobility attributable to the first modified form shown in the first analytical data and the mobility attributable to the second modified form shown in the second analytical data is greater than 0.

[0074] When the sialylglycan to be analyzed contains α2,3-sialic acid, α2,8-sialic acid, or α2,9-sialic acid, the first modified form and the second modified form differ in chemical structure, molecular weight, and charge state, and when the two are analyzed by chromatography or electrophoresis, this difference manifests itself as a difference in mobility. That is, when the mobility attributed to the first modified form shown in the first analytical data differs from the mobility attributed to the second modified form shown in the second analytical data, it can be determined that the sialic acid bound to the sialylglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid. On the other hand, when the sialic acid to be analyzed contains only sialic acid other than α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid (e.g., α2,6-sialic acid), the first modified product and the second modified product have the same chemical structure, molecular weight, and charge state, and when they are analyzed by chromatography or electrophoresis, no difference in mobility is observed. In other words, when the mobility assigned to the first modified product shown in the first analytical data and the mobility assigned to the second modified product shown in the second analytical data are the same, it can be determined that the sialic acid bound to the sialylglycan is α2,6-sialic acid.

[0075] <Analytical Method Including Third Sample> The analytical method for sialylglycan according to this embodiment may further comprise the steps of: preparing a third sample containing the sialylglycan; analyzing the third sample by chromatography or electrophoresis to obtain third analytical data derived from the third sample; comparing the third analytical data with the first analytical data and the second analytical data; and determining that the sialic acid bound to the sialylglycan comprises at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid, and a sialic acid other than the α2,3-sialic acid, the α2,8-sialic acid, and the α2,9-sialic acid, when the mobility assigned to the first modified form shown in the first analytical data, the mobility assigned to the second modified form shown in the second analytical data, and the mobility assigned to the sialylglycan shown in the third analytical data are different.

[0076] (Step of preparing a third sample) The third sample according to this embodiment contains a sialylglycan. The sialylglycan is a sialylglycan from which the first modified product and the second modified product are derived. The third sample may be a liquid or a solid. The third sample is preferably a liquid. In one aspect of this embodiment, the third sample may be derived from a living organism or a cell. The third sample may be prepared at the same time as the above-mentioned "step of preparing a first sample and a second sample," or may be prepared at a different time.

[0077] In the step of analyzing the third sample by chromatography or electrophoresis to obtain third analytical data derived from the third sample, the analysis is performed using the same analytical method as used to analyze the first sample and the second sample, and the third analytical data is obtained. The third analytical data may be obtained at the same time as the above-mentioned "step of obtaining first analytical data and second analytical data," or may be obtained at a different time. In the step of comparing the third analytical data with the first analytical data and the second analytical data, the respective analytical data can be compared using the same method as described above.

[0078] When the mobility assigned to the first modified form shown in the first analytical data, the mobility assigned to the second modified form shown in the second analytical data, and the mobility assigned to the sialylglycan shown in the third analytical data are different, it can be determined that the sialic acid bound to the sialicglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid, and a sialic acid other than α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid (e.g., α2,6-sialic acid).

[0079] In one aspect of this embodiment, when the mobility assigned to the first modified form shown in the first analytical data is different from the mobility assigned to the second modified form shown in the second analytical data, and the mobility assigned to the second modified form shown in the second analytical data is the same as the mobility assigned to the sialylglycan shown in the third analytical data (e.g., A1, A2, A3 in Figures 2 and 3), it can be determined that the sialic acid bound to the sialylglycan includes at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid.

[0080] In another aspect of this embodiment, when the mobility assigned to the first modified form shown in the first analytical data is the same as the mobility assigned to the second modified form shown in the second analytical data, and the mobility assigned to the second modified form shown in the second analytical data is different from the mobility assigned to the sialylglycan shown in the third analytical data (e.g., B1, B2, B3 in Figures 2 and 3), it can be determined that the sialic acid bound to the sialylglycan contains a sialic acid other than α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid (e.g., α2,6-sialic acid).

[0081] The analytical method for sialylglycan according to this embodiment has been described above. Conventionally, when analyzing the molecular weight and molecular structure of a target molecule using mass spectrometry, chromatography, or electrophoresis, if the target molecule is charged, the expected measurement results may not be obtained. For example, when a sialic acid-containing sialic acid is analyzed directly by capillary electrophoresis (CE), the negatively charged sialic acid is detected earlier than expected based on the molecular weight of the sialic acid, making it impossible to accurately evaluate the molecular weight and molecular structure. Therefore, attempts have been made to neutralize the sialic acid contained in the sialic acid (e.g., by esterification or amidation) before subjecting it to measurement by capillary electrophoresis or the like. Thus, while conventional analytical methods may neutralize the charge of a target molecule before analysis, there has been no idea of ​​deliberately returning a neutralized target molecule to its original "charged state" before subjecting it to analysis. The present inventors have reexamined this point. There are two main types of sialic acid, α2,3-sialic acid and α2,6-sialic acid. To distinguish between these two types using CE or microchip electrophoresis, the inventors discovered that a simple, rapid, and effective method for neutralizing both types and then converting α2,3-sialic acid back into charged sialic acid is possible, leading to the completion of the present invention.

[0082] Because sialic acid is located at the end of glycans, where it is easily recognized by other molecules, determining the binding mode of sialic acid can elucidate viral infections or protein-protein interactions on cell surfaces. It is also known that the binding mode of sialic acid in glycoproteins changes with cancer, raising hopes that sialic acid could be used as a cancer biomarker. Because the effectiveness of biopharmaceuticals varies depending on glycosylation, accurately and quickly determining the binding mode of sialic acid can also aid in the quality control of biopharmaceuticals.

[0083] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these. Percentages are by mass unless otherwise specified.

[0084] [Experiment 1: Method for Analyzing Sialyl Sugar Chains Using HPLC] <Reagents> The following reagents were used. (Sugar chains) α2,3-Sialyl sugar chain: α2,3-A2 (manufactured by ACROSCALE Inc.) α2,6-Sialyl sugar chain: α2,6-A2GN1 (manufactured by Tokyo Chemical Industry Co., Ltd.) Neutral sugar chain: NA2 (manufactured by Sigma-Aldrich) (Reaction reagents) Reagent A: dimethyl sulfoxide solution (DMSO solution) of isopropylamine hydrochloride (nucleophile: 15-23%) and 1-hydroxybenzotriazole (highly nucleophilic additive: 5.0-9.0%) Reagent B: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (>98.0%) (EDC, dehydration condensation agent) Reagent C: aqueous solution of methylamine (16%) The above-mentioned Reagents A to C are SialoCapper ＴＭ The one included in the -ID Kit (Shimadzu Corporation) was used.

[0085] <Step of Preparing First Sample and Second Sample> (Preparation of First Sample Containing First Modified Form) Each of the three types of sugar chains (1 nmol) described above was added to SialoCapper. ＴＭ The reaction was carried out using a β-ID Kit (Shimadzu Corporation) according to the attached instructions. Specifically, a mixture of Reagent A and Reagent B was added to each glycan and allowed to react for one hour with stirring (first reaction). At this time, the concentrations of the dehydration condensation agent and highly nucleophilic additive in each reaction solution were 500 mM, and the concentration of the nucleophile was 2 M. Next, Reagent C was added to each reaction solution and mixed for several seconds with a vortex mixer (second reaction). Subsequently, the reaction solution was purified using a HILIC microtip according to the instructions, and the solvent was removed using a SpeedVac. As a result of this procedure, α2,3-sialic acid (α2,8-sialic acid or α2,9-sialic acid) was methylamidated, and α2,6-sialic acid was isopropylamidated. In other words, this procedure yields the first modified form derived from each glycan.

[0086] 15 mg of 2-aminobenzoic acid (2-AA) was dissolved in 300 μL of 30% acetic acid (AcOH) / DMSO solvent. 200 μL of the resulting solution was used to dissolve 12 mg of sodium cyanoborohydride to obtain a reaction solution. 10 μL of the above reaction solution was added to each glycan and stirred thoroughly, followed by reaction at 37°C for 18 hours to convert the reducing end of each glycan to 2AA. 190 μL of acetonitrile was added to the glycan sample containing the 2AA-conjugated glycan, and the mixture was purified using a HILIC microtip to remove excess reagent. Specifically, the HILIC microtip was washed with water and then equilibrated with 90% acetonitrile and 0.1% trifluoroacetic acid. The above glycan sample diluted with acetonitrile was added to the equilibrated HILIC microtip and allowed to pass through by centrifugation. Thereafter, the glycans were washed with 90% acetonitrile and 0.1% trifluoroacetic acid, and finally eluted from the HILIC microtip with water. A first sample containing the first modified product was prepared by the above procedure.

[0087] (Preparation of Second Sample Containing Second Modified Form) First, a mixture of Reagent A and Reagent B was added to each of the three types of glycans (1 nmol) described above, and the mixture was allowed to react for one hour with stirring (first reaction). At this time, the concentrations of the dehydration condensation agent and highly nucleophilic additive in each reaction solution were 500 mM, and the concentration of the nucleophile was 2 M. Next, the same amount of water was added instead of Reagent C, and the mixture was mixed for several seconds using a vortex mixer. Purification was then performed using a HILIC microtip according to the manufacturer's instructions. A trimethylamine aqueous solution was added to the eluate from the HILIC microtip so that the final trimethylamine concentration was 14%, and the mixture was mixed for several seconds using a vortex mixer. The lactone structure was cleaved in a basic environment (pH 12-13) (third reaction). The solvent was then removed from the reaction solution using a SpeedVac. As a result of this procedure, the α2,3-sialic acid remains as the original carboxy group, but the α2,6-sialic acid is isopropylamidated, i.e., secondary modified products derived from each sugar chain are obtained by this procedure.

[0088] 15 mg of 2-aminobenzoic acid (2-AA) was dissolved in 300 μL of 30% AcOH / DMSO solvent. 200 μL of the resulting solution was used to dissolve 12 mg of sodium cyanoborohydride to obtain a reaction solution. 10 μL of the above reaction solution was added to each glycan and stirred thoroughly, followed by reaction at 37°C for 18 hours to convert the reducing end of each glycan to 2AA. 190 μL of acetonitrile was added to the glycan sample containing the 2AA-conjugated glycan, and the mixture was purified using a HILIC microtip to remove excess reagent. Specifically, the HILIC microtip was washed with water and then equilibrated with 90% acetonitrile and 0.1% trifluoroacetic acid. The above glycan sample diluted with acetonitrile was added to the equilibrated HILIC microtip and allowed to pass through by centrifugation. Thereafter, the glycans were washed with 90% acetonitrile and 0.1% trifluoroacetic acid, and finally eluted from the HILIC microtip with water. By the above procedure, a second sample containing the second modified product was prepared.

[0089] Furthermore, a sugar chain that was subjected to only 2AA conversion without carrying out the first, second and third reactions was prepared as a third sample.

[0090] <Step of Obtaining First Analytical Data and Second Analytical Data> The obtained first sample and second sample were each analyzed by high performance liquid chromatography (HPLC) to obtain first analytical data derived from the first sample and second analytical data derived from the second sample ( FIG. 2 ). Furthermore, the third sample was analyzed by HPLC to obtain third analytical data derived from the third sample ( FIG. 2 ). The HPLC analysis conditions are shown in Table 1 below.

[0091]

[0092] <Step of Comparing First Analytical Data and Second Analytical Data> Figure 2 is a fluorescence chromatogram showing the results of the above-mentioned HPLC analysis. In Figure 2, the horizontal axis represents retention time (minutes), and the vertical axis represents detected fluorescence intensity (mV). A schematic diagram of the molecules to be measured is shown at the top of Figure 2. In this schematic diagram, diamonds represent sialic acid (here, N-acetylneuraminic acid), white circles represent galactose, black squares represent N-acetylglucosamine, and gray circles represent mannose. In Figure 2, the upper row shows the analytical results of the unmodified glycans (third analytical data derived from the third sample). In Figure 2, the lower row shows the analytical results of the first-modified glycans (first analytical data), and the middle row shows the analytical results of the second-modified glycans (second analytical data). Comparing the first analytical data and the second analytical data, no change in retention time (mobility) was observed for the α2,6-sialyl glycans (B2 and B3 in Figure 2) and the neutral glycans (C2 and C3 in Figure 2). On the other hand, when the first analytical data and the second analytical data were compared, a change in retention time (mobility) was observed for the α2,3-sialyl sugar chains (A2 and A3 in FIG. 2).

[0093] <Step of determining whether sialic acid bound to a sialylglycan contains sialic acid of a specific bond type> The chromatograms in Figure 2 will be considered below. In the case of the A2-type glycan with α2,3-linked sialic acid shown in the left column, a comparison of the chromatograms A2 and A3 in Figure 2 reveals a shift in retention time, and therefore it can be determined that this glycan contains α2,3-sialic acid. If it has been confirmed in advance that the target glycan contains sialic acid by fractionation using an anion exchange chromatography column such as DEAE (diethylaminoethylcellulose), it can also be determined that this glycan contains α2,3-sialic acid by comparing A1 and A2 in Figure 2, as there is no shift in retention time. When the presence or absence of sialic acid is unknown, it is preferable to make a comprehensive judgment by comparing all of A1, A2, and A3, since there is a possibility that sialic acid is not present when only A1 and A2 are compared. This is because in the case of sugar chains that do not contain sialic acid, the retention time does not shift depending on whether or not there is sialic acid modification, as in the case of C1, C2, and C3 in the right column in FIG.

[0094] In the case of the A2-type glycan with α2,6-sialic acid in the middle column of Figure 2, a comparison of the chromatograms B1 and B2 in Figure 2 reveals a shift in retention time, which allows us to determine that this glycan contains α2,6-sialic acid. If it has been confirmed in advance that the glycan of interest contains sialic acid by fractionation using an anion exchange chromatography column such as DEAE, the glycan can also be determined to contain α2,6-sialic acid by comparing B2 and B3, as there is no shift in retention time. If the presence or absence of sialic acid is unknown, it is not possible to determine the presence or absence of sialic acid by comparing B2 and B3 alone; in such cases, it is preferable to make a comprehensive judgment by comparing all of B1, B2, and B3. If a glycan is detected at the same retention time in all of B1, B2, and B3, it can be determined that the glycan does not contain sialic acid.

[0095] When a single glycan molecule contains both α2,3-sialic acid and α2,6-sialic acid, the retention times will be shifted for all three glycans: a glycan with no sialic acid modification (third sample), a glycan with only α2,6-sialic acid amidated (second modification, second sample), and a glycan with all sialic acids amidated (first modification, first sample). Therefore, it is preferable to make a judgment by comparing all three chromatograms.

[0096] In addition to α2,3-sialic acid and α2,6-sialic acid, other sialic acid linkages such as α2,8-sialic acid and α2,9-sialic acid also exist depending on the sample. However, α2,8-sialic acid and α2,9-sialic acid can also be modified with sialic acid, and their reactivity under sialic acid modification conditions is equivalent to that of α2,3-sialic acid. Therefore, by using this technology, it is possible to determine the presence or absence of sialic acid and whether it is α2,6-sialic acid or other sialic acids (α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid).

[0097] That is, in the above-mentioned analytical method, when the mobility assigned to the first modified form shown in the first analytical data is different from the mobility assigned to the second modified form shown in the second analytical data, it can be determined that the sialic acid bound to the sialylglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid.

[0098] Furthermore, in the above-mentioned analytical method, when the mobility assigned to the first modified form shown in the first analytical data, the mobility assigned to the second modified form shown in the second analytical data, and the mobility assigned to the sialylglycan shown in the third analytical data are different, it can be determined that the sialic acid bound to the sialicglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid, and a sialic acid other than α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid (α2,6-sialic acid).

[0099] [Experiment 2: Analysis of sialyl sugar chains using microchip electrophoresis] <Reagents> The same sugar chains and reaction reagents as in Experiment 1 were used.

[0100] <Step of Preparing First Sample and Second Sample> (Preparation of First Sample Containing First Modified Form) Each of the three types of sugar chains (1 nmol) described above was added to SialoCapper. ＴＭThe reaction was carried out using a β-ID Kit (Shimadzu Corporation) according to the attached instructions. Specifically, a mixture of Reagent A and Reagent B was added to each glycan and allowed to react for one hour with stirring (first reaction). At this time, the concentrations of the dehydration condensation agent and highly nucleophilic additive in each reaction solution were 500 mM, and the concentration of the nucleophile was 2 M. Next, Reagent C was added to each reaction solution and mixed for several seconds with a vortex mixer (second reaction). Subsequently, the reaction solution was purified using a HILIC microtip according to the instructions, and the solvent was removed using a SpeedVac. As a result of this procedure, α2,3-sialic acid (α2,8-sialic acid or α2,9-sialic acid) was methylamidated, and α2,6-sialic acid was isopropylamidated. In other words, this procedure yields the first modified form derived from each glycan.

[0101] Each sugar chain was transferred to a PCR thin-well tube (volume: 0.2 mL), and an aqueous solution (5 μL) of 50 mM APTS (8-aminopyrene-1,3,6-trisulfonic acid) and 500 mM citric acid was further added to dissolve each sugar chain. Next, a DMSO solution (5 μL) of 1.0 M sodium cyanoborohydride was added to the tube, and the reaction was carried out at 55°C for 50 minutes to label the reducing end of each sugar chain with APTS. After the reaction, the reaction was stopped by adding 140 μL of water to the sugar chain sample containing the labeled sugar chain.

[0102] The glycan sample diluted with water was transferred to a 1.5 mL tube, and 200 μL of 20% tetrabutylammonium bromide / 0.5% acetic acid-chloroform solution was added and vigorously stirred. This procedure dissolved excess labeling reagent from the aqueous layer into the chloroform layer (liquid-liquid extraction). Next, the tube was centrifuged at 5000 × g for 30 seconds, and the chloroform layer was removed. This liquid-liquid extraction was performed again, and the recovered aqueous layer was diluted with 300 μL of water and added to a HILIC-SPE (HILIC-SPE: Monospin NH2, GL-Science). After centrifugation at 5000 × g for 60 seconds, the carrier was washed with 400 μL of water, and then the labeled glycans were eluted from the carrier with 50 μL of 0.28% ammonium hydroxide solution. The labeled glycans were dried using a centrifugal concentrator and stored at −20°C until analysis began. A first sample containing a first modified product was prepared according to the above procedure.

[0103] (Preparation of Second Sample Containing Second Modified Form) First, a mixture of Reagent A and Reagent B was added to each of the three types of sugar chains (1 nmol) described above, and the mixture was allowed to react with stirring for one hour (first reaction). At this time, the concentrations of the dehydration condensation agent and highly nucleophilic additive in each reaction solution were 500 mM, and the concentration of the nucleophile was 2 M. Next, the same amount of water was added instead of Reagent C, and the mixture was mixed for several seconds using a vortex mixer. Subsequently, the mixture was purified using a HILIC microtip according to the manufacturer's instructions. A trimethylamine aqueous solution was added to the eluate from the HILIC microtip so that the final trimethylamine concentration was 14%, and the mixture was mixed for several seconds using a vortex mixer. The lactone structure was cleaved in a basic environment (pH 12-13) (third reaction). The solvent was then removed from the reaction solution using a SpeedVac. As a result of this procedure, the α2,3-sialic acid remains as the original carboxy group, but the α2,6-sialic acid is isopropylamidated, i.e., secondary modified products derived from each sugar chain are obtained by this procedure.

[0104] Each sugar chain was transferred to a PCR thin-well tube (volume: 0.2 mL), and an aqueous solution (5 μL) of 50 mM APTS (8-aminopyrene-1,3,6-trisulfonic acid) and 500 mM citric acid was further added to dissolve each sugar chain. Next, a DMSO solution (5 μL) of 1.0 M sodium cyanoborohydride was added to the tube, and the reaction was carried out at 55°C for 50 minutes to label the reducing end of each sugar chain with APTS. After the reaction, the reaction was stopped by adding 140 μL of water to the sugar chain sample containing the labeled sugar chain.

[0105] The glycan sample diluted with water was transferred to a 1.5 mL tube, and 200 μL of 20% tetrabutylammonium bromide / 0.5% acetic acid-chloroform solution was added and vigorously stirred. This procedure dissolved excess labeling reagent from the aqueous layer into the chloroform layer (liquid-liquid extraction). Next, the tube was centrifuged at 5000 × g for 30 seconds, and the chloroform layer was removed. This liquid-liquid extraction was performed again, and the recovered aqueous layer was diluted with 300 μL of water and added to a HILIC-SPE (HILIC-SPE: Monospin NH2, GL-Science). After centrifugation at 5000 × g for 60 seconds, the carrier was washed with 400 μL of water, and then the labeled glycans were eluted from the carrier with 50 μL of 0.28% ammonium hydroxide solution. The labeled glycans were dried using a centrifugal concentrator and stored at −20°C until analysis began. A second sample containing the second modified product was prepared by the above procedure.

[0106] Furthermore, a sugar chain that was only labeled with APTS without undergoing the first, second and third reactions was prepared as a third sample.

[0107] <Step of Obtaining First and Second Analytical Data> First, the four reservoirs (R1, R2, R3, and R4) and the separation channel on the microchip were automatically washed with ultrapure water. Next, the three reservoirs (R2, R3, and R4; sometimes referred to as "buffer reservoirs") and the separation channel were filled with a polymer gel buffer. The resulting first and second samples (3 μL each) were electrically injected from reservoir R1 over 45 seconds, followed by a separation operation for 140 seconds under the conditions of R1 = 330 V, R2 = 330 V, R3 = 0 V, and R4 = 1485 V. The analytical conditions for microchip electrophoresis are shown in Table 2 below. Analysis by microchip electrophoresis was performed using the above method, and first analytical data derived from the first sample and second analytical data derived from the second sample were obtained ( FIG. 3 ). Furthermore, a third sample was analyzed by microchip electrophoresis under the same conditions as above, and third analytical data derived from the third sample were obtained ( FIG. 3 ).

[0108]

[0109] <Step of Comparing First Analytical Data and Second Analytical Data> Figure 3 is a graph showing the results of the analysis performed by the microchip electrophoresis method. In Figure 3, the horizontal axis represents migration time (seconds), and the vertical axis represents detected fluorescence intensity (unit: mV). In Figure 3, the large peak observed around 30 to 40 seconds is thought to be a peak derived from remaining APTS. The top part of Figure 3 shows a schematic diagram of the molecules to be measured. In this schematic diagram, diamonds represent sialic acid (here, N-acetylneuraminic acid), white circles represent galactose, black squares represent N-acetylglucosamine, and gray circles represent mannose. In Figure 3, the upper part shows the analytical results of the glycans before modification (third analytical data derived from the third sample). In Figure 3, the lower part shows the analytical results of the first modified product (first analytical data), and the middle part shows the analytical results of the second modified product (second analytical data). When the first analytical data was compared with the second analytical data, no change in migration time (mobility) was observed for the α2,6-sialyl glycans (B2 and B3 in Figure 3) and neutral glycans (C2 and C3 in Figure 3). On the other hand, when the first analytical data was compared with the second analytical data, a change in migration time (mobility) was observed for the α2,3-sialyl glycans (A2 and A3 in Figure 3).

[0110] <Step of Determining Whether Sialic Acid Binding to a Sialylglycan Contains Sialic Acid of a Predetermined Bonding Mode> The graph in Figure 3 will now be considered. In the case of an A2-type glycan with α2,3-linked sialic acid (shown in the left column), a comparison of graphs A2 and A3 in Figure 3 reveals a shift in migration time, enabling the glycan to be determined to contain α2,3-sialic acid. If the presence of sialic acid in the target glycan has been confirmed in advance by fractionation using an anion exchange chromatography column such as DEAE (diethylaminoethylcellulose), the absence of a shift in migration time when comparing A1 and A2 in Figure 3 also allows the glycan to be determined to contain α2,3-sialic acid. When the presence or absence of sialic acid is unknown, a comparison of A1 and A2 alone may indicate the absence of sialic acid, so it is preferable to make a comprehensive judgment by comparing all three graphs, A1, A2, and A3. This is because, in the case of a glycan that does not contain sialic acid, the migration time does not shift depending on the presence or absence of sialic acid modification, as shown by graphs C1, C2, and C3 in the right column in Figure 3.

[0111] In the case of the A2-type glycan with α2,6-sialic acid in the middle column of Figure 3, a comparison of the graphs B1 and B2 in Figure 3 reveals a shift in migration time, which allows us to determine that this glycan contains α2,6-sialic acid. If it has been confirmed in advance that the glycan of interest contains sialic acid by fractionation using an anion exchange chromatography column such as DEAE, the glycan can also be determined to contain α2,6-sialic acid by comparing B2 and B3, as there is no shift in migration time. If the presence or absence of sialic acid is unknown, it is not possible to determine the presence or absence of sialic acid by comparing B2 and B3 alone; in such cases, it is preferable to make a comprehensive judgment by comparing all of B1, B2, and B3. If a glycan is detected at the same migration time in all of B1, B2, and B3, it can be determined that the glycan does not contain sialic acid.

[0112] When a single glycan molecule contains both α2,3-sialic acid and α2,6-sialic acid, the migration times will be shifted for all three types of glycans: a glycan with no sialic acid modification (third sample), a glycan with only α2,6-sialic acid amidated (second modification, second sample), and a glycan with all sialic acids amidated (first modification, first sample). Therefore, it is preferable to make a judgment by comparing all three types of graphs.

[0113] In addition to α2,3-sialic acid and α2,6-sialic acid, other sialic acid linkages such as α2,8-sialic acid and α2,9-sialic acid also exist depending on the sample. However, α2,8-sialic acid and α2,9-sialic acid can also be modified with sialic acid, and their reactivity under sialic acid modification conditions is equivalent to that of α2,3-sialic acid. Therefore, by using this technology, it is possible to determine the presence or absence of sialic acid and whether it is α2,6-sialic acid or other sialic acids (α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid).

[0114] That is, in the above-mentioned analytical method, when the mobility assigned to the first modified form shown in the first analytical data is different from the mobility assigned to the second modified form shown in the second analytical data, it can be determined that the sialic acid bound to the sialylglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid.

[0115] Furthermore, in the above-mentioned analytical method, when the mobility assigned to the first modified form shown in the first analytical data, the mobility assigned to the second modified form shown in the second analytical data, and the mobility assigned to the sialylglycan shown in the third analytical data are different, it can be determined that the sialic acid bound to the sialicglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid, and a sialic acid other than α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid (α2,6-sialic acid).

[0116] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0117] (Item 1) A method for analyzing a sialic acid-bound sialylglycan according to one embodiment comprises the steps of preparing a first sample containing a first modification derived from the sialylglycan and a second sample containing a second modification derived from the sialylglycan; analyzing the first sample and the second sample by chromatography or electrophoresis to obtain first analytical data derived from the first sample and second analytical data derived from the second sample; comparing the first analytical data with the second analytical data; and comparing the mobility attributed to the first modification shown in the first analytical data with the mobility attributed to the second modification shown in the second analytical data. and determining that the sialic acid bound to the sialylglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid when the mobility attributable to the first modified form is different from that attributable to the second modified form, wherein the first modified form is a compound in which the sialic acid bound to the sialylglycan is produced by esterification, amidation, or both, and the second modified form is a compound in which the sialic acid bound to the sialylglycan is produced by esterification, amidation, or both, other than the α2,3-sialic acid, the α2,8-sialic acid, and the α2,9-sialic acid. According to the method described in item 1, the binding mode of the sialic acid bound to the sialylglycan can be determined by chromatography or electrophoresis.

[0118] (Item 2) The method according to item 1 further comprises a step of determining that the sialic acid bound to the sialylglycan is α2,6-sialic acid when the mobility attributed to the first modified form shown in the first analytical data is the same as the mobility attributed to the second modified form shown in the second analytical data. According to the method according to item 2, the binding mode of the sialic acid bound to the sialylglycan can be determined in more detail by chromatography or electrophoresis.

[0119] (Item 3) The method according to item 1 further comprises the steps of preparing a third sample containing the sialylglycan, analyzing the third sample by chromatography or electrophoresis to obtain third analytical data derived from the third sample, comparing the third analytical data with the first analytical data and the second analytical data, and determining that the sialic acid bound to the sialylglycan comprises at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid, and a sialic acid other than the α2,3-sialic acid, the α2,8-sialic acid, and the α2,9-sialic acid, when the mobility attributed to the first modified form shown in the first analytical data, the mobility attributed to the second modified form shown in the second analytical data, and the mobility attributed to the sialylglycan shown in the third analytical data are different. According to the method described in item 3, the binding mode of sialic acid bound to the sialylglycan can be determined in more detail by chromatography or electrophoresis.

[0120] (Item 4) In the method according to any one of items 1 to 3, the first modified product is a compound produced by contacting an intermediate produced by contacting the sialyl sugar chain with a dehydration condensation agent and a nucleophilic agent with at least one selected from the group consisting of ammonia, amines, and salts thereof. According to the method according to item 4, the first modified product can be produced efficiently.

[0121] (Item 5) In the method according to item 4, the dehydration condensation agent includes a carbodiimide. According to the method according to item 5, it is possible to produce the first modified product more efficiently.

[0122] (Item 6) In the method according to item 4 or 5, the nucleophile includes an amine compound having a branched alkyl group or a salt thereof. According to the method according to item 6, it is possible to efficiently produce the first modified product.

[0123] (Item 7) In the method according to any one of items 1 to 6, the second modified product is a compound produced by placing an intermediate produced by contacting the sialyl sugar chain with a dehydration condensation agent and a nucleophilic agent in a basic environment. According to the method according to item 7, the second modified product can be produced efficiently.

[0124] (Item 8) In the method according to item 7, the basic environment is an environment in which t-butylamine, trimethylamine, or both are present. According to the method according to item 8, it is possible to produce the second modified product more efficiently.

[0125] (Item 9) In the method according to any one of Items 1 to 8, the chromatography method is liquid chromatography. According to the method according to Item 9, α-2,3 sialic acid and the like can be distinguished from α-2,6 sialic acid based on the retention time in a chromatogram.

[0126] (Item 10) In the method according to any one of items 1 to 9, the electrophoresis method is at least one selected from the group consisting of capillary electrophoresis, SDS-PAGE, and microchip electrophoresis. According to the method according to item 10, α-2,3 sialic acid and the like can be distinguished from α-2,6 sialic acid based on their mobility by electrophoresis.

Claims

1. A method for analyzing sialic acid-bound sialylglycan, comprising: Preparing a first sample containing a first modified product derived from the sialyl sugar chain and a second sample containing a second modified product derived from the sialyl sugar chain; analyzing each of the first sample and the second sample by chromatography or electrophoresis to obtain first analytical data derived from the first sample and second analytical data derived from the second sample; comparing the first analytical data with the second analytical data; determining that the sialic acid bound to the sialylglycan contains at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid when the mobility attributed to the first modified form shown in the first analytical data is different from the mobility attributed to the second modified form shown in the second analytical data, the first modified product is a compound in which the sialic acid bound to the sialyl sugar chain is produced by esterification, amidation, or both; The second modified form is a compound in which the sialic acid bound to the sialylglycan is produced by esterification, amidation, or both, and the sialic acid is a sialic acid other than the α2,3-sialic acid, the α2,8-sialic acid, and the α2,9-sialic acid.

2. The method for analyzing sialylglycan according to claim 1, further comprising a step of determining that the sialic acid bound to the sialylglycan is α2,6-sialic acid when the mobility assigned to the first modified form shown in the first analytical data is the same as the mobility assigned to the second modified form shown in the second analytical data.

3. preparing a third sample containing the sialylglycan; analyzing the third sample by a chromatography method or an electrophoresis method to obtain third analytical data derived from the third sample; comparing the third analytical data to the first analytical data and the second analytical data; The method for analyzing sialylglycan according to claim 1, further comprising the step of determining that the sialic acid bound to the sialylglycan comprises at least one selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid, and a sialic acid other than the α2,3-sialic acid, the α2,8-sialic acid, and the α2,9-sialic acid, when the mobility assigned to the first modified form shown in the first analytical data, the mobility assigned to the second modified form shown in the second analytical data, and the mobility assigned to the sialylglycan shown in the third analytical data are different.

4. The method for analyzing sialylglycan according to any one of claims 1 to 3, wherein the first modified product is a compound produced by contacting an intermediate produced by contacting the sialylglycan with a dehydration condensation agent and a nucleophilic agent with at least one selected from the group consisting of ammonia, amines, and salts thereof.

5. The method for analyzing sialylglycan according to claim 4, wherein the dehydration condensation agent comprises a carbodiimide.

6. The method for analyzing sialylglycan according to claim 4, wherein the nucleophilic agent comprises an amine compound having a branched alkyl group or a salt thereof.

7. The method for analyzing sialylglycan according to any one of claims 1 to 3, wherein the second modified product is a compound produced by placing an intermediate produced by contacting the sialylglycan with a dehydration condensation agent and a nucleophilic agent under a basic environment.

8. The method for analyzing sialylglycan according to claim 7, wherein the basic environment is an environment in which t-butylamine, trimethylamine, or both are present.

9. The method for analyzing sialylglycan according to any one of claims 1 to 3, wherein the chromatography method is a liquid chromatography method.

10. The method for analyzing sialylglycan according to any one of claims 1 to 3, wherein the electrophoresis method is at least one selected from the group consisting of capillary electrophoresis, SDS-PAGE, and microchip electrophoresis.