Method for preparing analytical samples and analytical methods

Abstract

Provided is a method for analyzing an O-linked sugar chain that has been undergone sialic acid linkage-specific modification, the method comprising: a modification step for adding a modifier, which performs sialic acid linkage-specific modification on the O-linked sugar chain, to a sample containing a glycoprotein to which the O-linked sugar chain is linked; a release step for releasing, from the glycoprotein, the O-linked sugar chain that has been undergone the sialic acid linkage-specific modification; and an analysis step for analyzing the released O-linked sugar chain that has been undergone the sialic acid linkage-specific modification.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing and analyzing analytical samples. [Background technology]

[0002] Sialic acid is also found in glycoproteins in living organisms, primarily at the ends of sugar chains. Because sialic acid is easily positioned on the outside of glycoprotein molecules, it is readily recognized by other molecules. Sialic acid can exhibit different bonding patterns with adjacent sugars. For example, in human N-linked sugar chains, α2,3- and α2,6- are the main bonding patterns, while in O-linked sugar chains and sphingoglycolipids, α2,8- and α2,9- are also known. Since differences in bonding patterns can lead to sialic acid being recognized by different molecules and potentially playing different roles, analyzing the bonding patterns of sialic acid is important.

[0003] However, because sialic acid has a negative charge and is easily decomposed, analyzing sialyl sugar chains containing sialic acid is not easy. Furthermore, since the mass of the sugar chain does not change regardless of the sialic acid bonding mode, it is not possible to distinguish and analyze the bonding mode using mass spectrometry.

[0004] To distinguish and analyze the bonding modes of sialic acid, a method has been proposed to perform a bonding mode-specific modification on sialic acid. This method utilizes the property that α2,3-sialic acid is more readily subjected to intramolecular dehydration by a dehydrating condensation agent than α2,6-sialic acid. By lactonizing α2,3-sialic acid through intramolecular dehydration, α2,6-sialic acid is reacted with a nucleophile such as an alcohol or amine. As a result, molecules of different masses are produced depending on the bonding mode of sialic acid, allowing for the distinguishing and analysis of the bonding mode of sialic acid by mass spectrometry. Non-patent document 1 discloses a method for preparing a sample used in mass spectrometry, in which a solution containing isopropylamine and a dehydrating condensation agent is added to a free N-linked glycan to lactonize α2,3-sialic acid and amidate α2,6-sialic acid. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nishikaze T., Tsumoto H., Sekiya S., Iwamoto S., Miura Y., Tanaka K., Analytical Chemistry, 2017, 89, 2353-2360. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The inventors have found that when analyzing the binding mode of sialic acid, the accuracy of distinguishing between α2,6-sialic acid and α2,3-sialic acid may decrease. This is thought to be because when sialic acid is subjected to binding mode-specific modification (amidation) to O-linked glycans released from glycoproteins, the α2,6-sialic acid bound to N-acetylgalactosamine (GalNAc) at the reducing end of the O-linked glycan is not properly modified, and some of the reaction proceeds similarly to that of α2,3-sialic acid. The present invention has been made in view of the above circumstances, and aims to enable the distinguishing modification of α2,6-sialic acid and α2,3-sialic acid attached to O-linked glycans, and to enable accurate analysis of O-linked glycans that have undergone binding mode-specific modification after being released from glycoproteins. [Means for solving the problem]

[0007] The present invention A method for analyzing O-linked glycans that have undergone specific modification of the sialic acid linkage mode, A modification step involves adding a modifying agent to a sample containing a glycoprotein to which the O-linked glycan is bound, which modifies the O-linked glycan in a manner specific to the mode of sialic acid binding. A release step in which the O-linked glycan, which has undergone the sialic acid linkage-specific modification, is released from the glycoprotein, The present invention relates to a method comprising an analytical step of analyzing an O-linked glycan on which the bond mode of the released sialic acid has been specifically modified. [Effects of the Invention]

[0008] According to the present invention, it is possible to distinguish between α2,6-sialic acid and α2,3-sialic acid attached to O-linked glycans and perform modifications accordingly, and it becomes possible to release O-linked glycans that have undergone bond-mode-specific modification from glycoproteins and analyze them with high accuracy. [Brief explanation of the drawing]

[0009] [Figure 1] This is a flowchart showing an analytical method relating to one embodiment of the present invention. [Figure 2] In Experiment 1, the images show mass spectrometry spectra of glycans contained in glycoproteins derived from serum and cerebrospinal fluid. IS indicates the internal standard. [Figure 3] The top panel shows the mass spectrometry spectrum of glycans contained in serum-derived glycoproteins in Experiment 2. The bottom panel shows the mass spectrometry spectrum of glycans contained in serum-derived glycoproteins in Experiment 3. [Figure 4] The upper panel shows the mass spectrometry spectrum of the glycans when glycosylation was performed using the MTT method on a glycoprotein containing O-linked glycans in Experiment 4. The lower panel shows the mass spectrometry spectrum of the glycans when sialic acid linkage-specific modification was performed after the glycans were released from the glycoprotein containing O-linked glycans in Experiment 4. [Figure 5] This is the mass spectrometry spectrum of the glycans released by a β-elimination reaction after sialic acid-specific modification of a glycoprotein containing O-linked glycans in Experiment 5. [Figure 6] This graph shows the percentage of sialyl T that was not methylamidated when the methylamine concentration was changed in Experiment 5. [Figure 7]The upper graph shows the recovery amount of O-linked glycans (vertical axis: relative value to internal standard) when the pH in the β-elimination reaction was changed in Experiment 6. The lower graph shows the types and their abundance ratios of the recovered glycans when the pH in the β-elimination reaction was changed in Experiment 6. [Figure 8] This is a graph showing the ratio of the decomposed product of methylamidated sialyl T when the pH in the β-elimination reaction was changed in Experiment 6. The large graph and the small graph only have different scales on the vertical axis and show the same experimental data. In the small graph, Entry3 to Entry5 indicate the upper limit value of the vertical axis, but the actual ratio is shown in the large graph. [Figure 9] This is a graph showing the ratio of the peeling reaction when the pH in the β-elimination reaction was changed in Experiment 6. The large graph and the small graph only have different scales on the vertical axis and show the same experimental data. In the small graph, Entry3 to Entry5 indicate the upper limit value of the vertical axis, but the actual ratio is shown in the large graph.

Mode for Carrying Out the Invention

[0010] A method for analyzing an O-linked glycan modified with a sialic acid binding mode-specific modification according to an embodiment of the present invention includes: a modification step of adding a modifier for performing a sialic acid binding mode-specific modification to the O-linked glycan to a sample containing a glycoprotein to which the O-linked glycan binds; a release step of releasing the O-linked glycan modified with the sialic acid binding mode-specific modification from the glycoprotein; an analysis step of analyzing the released O-linked glycan modified with the sialic acid binding mode-specific modification; and includes.

[0011] When a dehydrating condensing agent is added to a sugar chain to perform a modification specific to the binding mode of sialic acid, the released O-linked sugar chain not only lactonizes α2,3-sialic acid but also α2,6-sialic acid to be amidated, making it sometimes difficult to distinguish between α2,3-sialic acid and α2,6-sialic acid. The inventors of the present invention have found that by performing a lactonization reaction on a sugar chain that has not been released from a protein, unexpected lactonization of α2,6-sialic acid can be suppressed, and a modification different from that of α2,3-sialic acid can be performed on α2,6-sialic acid. When the O-linked sugar chain can be modified with sialic acid in a binding mode-specific manner, the discrimination accuracy of the binding mode of sialic acid can be improved. Even when the sugar chain is an N-linked sugar chain, different sialic acid modifications can be performed on α2,3-sialic acid and α2,6-sialic acid by performing a lactonization reaction on a sugar chain that has not been released from a protein. According to the present invention, even if a glycoprotein contains an O-linked sugar chain, sialic acid can be modified in a binding mode-specific manner, and the binding mode of sialic acid can be accurately discriminated. Here, "modification specific to the binding mode of sialic acid" and "modification of sialic acid in a binding mode-specific manner" mean a modification reaction acting on sialic acid, in which the chemical structure generated by the modification reaction is different when the sialic acid is α2,3-sialic acid and when it is α2,6-sialic acid.

[0012] Since sialic acid is located at the terminal of a sugar chain that is easily recognized by other molecules, elucidating the binding mode of sialic acid can clarify virus infection or protein-protein interactions on the cell surface. It is also known that the binding mode of sialic acid in glycoproteins changes with carcinogenesis, and the use of sialic acid as a biomarker for cancer is expected. Since the effects of biopharmaceuticals vary depending on sugar chain modification, accurately discriminating the binding mode of sialic acid can also assist in the quality control of biopharmaceuticals.

[0013] A flowchart showing the flow of the analysis method according to an embodiment of the present invention is shown in FIG. 1. In step S110, a sample is prepared.

[0014] (sample) The sample to be analyzed is not particularly limited and may be of biological or cellular origin. The sample may contain glycoproteins and may further contain free glycans or glycolipids. The analytical method according to the present invention is suitably used for analyzing the binding mode of sialic acid in glycans. Therefore, it is preferable that the sample contains glycans that may have sialic acid at their termini, and more preferably that it contains glycoproteins to which such glycans are bound. The analytical method according to the present invention can solve the problem that α2,6-sialic acid may be lactonized in O-linked glycans. Therefore, it is preferable that the glycans in the sample contain O-linked glycans, and more preferably that they contain O-linked glycans equipped with α2,6-sialic acid. That is, it is preferable that the sample contains glycoproteins to which these glycans are bound. The analytical method according to the present invention is suitably used when the sample contains glycoproteins to which O-linked glycans are bound, but the sample may also contain glycoproteins to which N-linked glycans are bound. From the viewpoint of improving the accuracy of binding mode-specific sialic acid modification, it is preferable that the sample does not contain free O-linked glycans.

[0015] In this specification, "protein" refers to a general term for molecules in which two or more amino acids are linked by peptide bonds. In this specification, "protein" 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. In this specification, "glycoprotein" may include glycopeptides.

[0016] If the peptide chain of a glycoprotein has a large number of amino acid residues, the peptide chain may be cleaved by an enzyme or the like. For example, when preparing a sample for mass spectrometry, 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. If it is required to clearly identify the origin of the peptide to which the sugar chain is attached, the number of amino acid residues in the peptide chain is preferably 2 or more, and more preferably 3 or more.

[0017] For cleaving the peptide chains of glycoproteins, digestive enzymes such as trypsin, Lys-C, arginine endopeptidase, chymotrypsin, pepsin, thermolysin, proteinase K, and pronase E can be used. Two or more of these digestive enzymes may be used in combination. The conditions for peptide chain cleavage are not particularly limited, and an appropriate protocol is adopted depending on the digestive enzyme used. Before peptide chain cleavage, the protein in the sample may be denatured or alkylated. The conditions for denaturation or alkylation are not particularly limited. Peptide chain cleavage may be performed before or after the first reaction described later, or after the second reaction. Peptide chains may be cleaved by chemical cleavage or other methods instead of enzymatic cleavage.

[0018] Appropriate treatments may be performed to block the amino groups in the glycoproteins in the sample. Examples of such treatments include dimethylamidation and guanidylation of the glycoprotein. This can suppress side reactions such as intramolecular dehydration condensation that may occur between the amino groups or carboxyl groups at the ends of the protein backbone during the first or second reaction.

[0019] <Modification Step> In the modification step, a modifier is added to the O-linked sugar chain to perform a modification specific to the mode of sialic acid attachment. The mode of sialic acid attachment modification preferably includes a first reaction to lactonize the sialic acid. The mode of sialic acid attachment modification preferably further includes a second reaction to amidize the lactone structure obtained by the first reaction. In one aspect of this embodiment, the modifier preferably includes a lactonization reaction solution and an amidation reaction solution. The lactonization reaction solution and the amidation reaction solution will be described later.

[0020] (First reaction) In step S120, a first reaction is carried out to lactonize sialic acid in glycoproteins to which sugar chains are attached. In the first reaction, if sialic acid is attached to the glycoprotein, the sialic acid is selectively lactonized according to 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.

[0021] The first reaction can be carried out by contacting the sample with a solution for lactonization of sialic acid in a manner specific to its bonding mode (hereinafter also referred to as the lactonization reaction solution). One example of the contact method is to add the lactonization solution to the sample. The lactonization reaction solution preferably contains a dehydrating condensation agent. Simultaneously with the first reaction, it is preferable that α2,6-sialic acid undergoes a modification different from lactonization, preferably amidation. In this case, in addition to the dehydrating condensation agent, the lactonization reaction solution preferably contains a nucleophile, which is at least one selected from the group consisting of alcohols, amines, and salts thereof.

[0022] The type and concentration of the dehydrating condensing agent and nucleophile should be adjusted to selectively induce either a dehydration reaction or a nucleophilic reaction based on the bonding mode of sialic acid. The lactone produced by intramolecular dehydration of the carboxyl group of α2,3-sialic acid is a six-membered ring, while the lactone that can be produced by intramolecular dehydration of the carboxyl group of α2,6-sialic acid is a seven-membered ring. α2,3-sialic acid, which produces a more stable six-membered ring than a seven-membered ring, is more easily lactonized than α2,6-sialic acid. Also, because the carboxyl group of α2,3-sialic acid is in a position with relatively greater steric hindrance compared to the carboxyl group of α2,6-sialic acid, larger 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 bonding mode of sialic acid, the type and concentration of the dehydrating condensing agent and nucleophile should be adjusted so that different modifications occur depending on the bonding mode of sialic acid.

[0023] The dehydration condensation agent preferably contains carbodiimide. This is because, when carbodiimide is used, carboxyl groups located in sterically hindered regions are less likely to be amidated compared to when a phosphonium-based dehydration condensation agent (so-called BOP reagent) or a uronium-based dehydration condensation agent is used. 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 their salts (hydrochloride salts, etc.).

[0024] In order to promote dehydration condensation by a dehydration condensation agent and suppress side reactions, it is preferable to use a highly nucleophilic additive in addition to carbodiimide. Preferred highly nucleophilic additives include 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt), 4-(dimethylamino)pyridine (DMAP), 2-cyano-2-(hydroxyimino)ethyl acetate (Oxyma), N-hydroxysuccinimide (HOSu), 6-chloro-1-hydroxybenzotriazole (Cl-HoBt), and N-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt).

[0025] The amine used as a nucleophile preferably includes primary and / or secondary alkylamines containing two or more carbon atoms. Preferred primary alkylamines include ethylamine, propylamine, isopropylamine, butylamine, sec-butylamine, tert-butylamine, etc. Preferred secondary alkylamines include dimethylamine, ethylmethylamine, diethylamine, propylmethylamine, isopropylmethylamine, etc. From the viewpoint of preventing carboxyl groups located in sterically hindered sites, such as the carboxyl group of α2,3-sialic acid, from being amidated, it is preferable to use an amine having a branched alkyl group, such as isopropylamine. When an amine is used as a nucleophile in the lactonization reaction solution, some carboxyl groups of sialic acids, such as α2,6-sialic acid, are amidated based on the bonding mode of sialic acid.

[0026] The alcohol used as the nucleophile is not particularly limited, and for example, methanol and ethanol can be used. When an alcohol is used as the nucleophile in the lactonization reaction solution, the carboxyl groups of some sialic acids, such as α2,6-sialic acid, are esterified based on the bonding mode of sialic acid. The nucleophile may also include a salt of the nucleophile described above.

[0027] The concentration of the dehydrating condensing agent in the lactonization reaction solution is, for example, 1 mM to 5 M, preferably 10 mM to 3 M. When carbodiimide is used in combination with a highly nucleophilic additive such as Oxyma, HOAt, or HOBt, it is preferable that the concentrations of each are within the above range. The concentration of the nucleophile in the lactonization reaction solution is, for example, 0.01 M to 20 M, preferably 0.1 M to 10 M. The reaction temperature for the first reaction may be around -20°C to 100°C, preferably -10°C to 50°C.

[0028] The first reaction can be carried out in either the liquid or solid phase. When the reaction is carried out in the liquid phase, it is preferable to carry it out in a non-aqueous solvent such as dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). Carrying the reaction in a non-aqueous solvent tends to suppress side reactions. The concentrations of each component in the liquid phase reaction are not particularly limited and can be appropriately determined depending on the type of dehydrating condensation agent and amine, etc.

[0029] When the first reaction is carried out in a solid phase, the solid support is not particularly limited as long as it can immobilize the glycoprotein. To immobilize the glycoprotein, for example, a solid support having an epoxy group, tosyl group, carboxyl group, amino group, etc. as a ligand can be used. A solid support having a hydrazide group, aminooxy group, etc. as a ligand may be used to immobilize a glycoprotein containing sugar chains. By carrying out the reaction with the sample immobilized on the solid support, the removal of the reaction solution after the reaction becomes easier, and sialic acid modification can be carried out efficiently. If magnetic beads are used as the solid support, the magnetic beads to which the glycoprotein is bound can be collected with a magnet, and excess reagent can be removed, or the beads can be washed with a solvent. If resin is used as the solid support, the resin may be recovered after removing the excess reagent by passing it through a filter, or the resin may be precipitated by centrifugation and the excess reagent in the supernatant may be removed. When the first reaction is carried out in a liquid phase, the excess reagent may be removed by ultrafiltration.

[0030] The sample after the first reaction may be purified, desalted, solubilized, concentrated, dried, or otherwise treated by known methods to remove the lactonization reaction solution or reduce its concentration.

[0031] (Second reaction) In step S130, it is preferable to carry out a second reaction to amidate the lactone structure produced by the first reaction. The second reaction can be carried out by contacting the sample after the first reaction with a solution for amidation (hereinafter also referred to as the amidation reaction solution). The first reaction can differentiate and modify the α2,3-bond and α2,6-bond of sialic acid, but the lactone structure produced from α2,3-sialic acid may be unstable and may revert to the original carboxylic acid structure by hydrolysis. The second reaction stabilizes the lactone structure by specifically amidating it. At the same time, the second reaction allows for differentiation between α2,3-sialic acid and α2,6-sialic acid by modifying sialic acid in a way that is specific to the bond mode and has a different mass. By carrying out the second reaction, sialic acid can be modified in a way that is specific to the bond mode with greater specificity and speed. An example of the second reaction may be aminolysis. Aminolysis is a reaction based on the interaction between an amino group and a lactone, and is preferable to hydrolysis because it can be suitably carried out even under anhydrous conditions. In this specification, the ring-opening and amidation of a lactone with ammonia, an amine, or a salt thereof, which can be carried out under anhydrous conditions, is referred to as aminolysis. As another example of the second reaction, the lactone structure may be amidated using a strong dehydrating condensation agent.Examples of dehydrating condensing agents used in the second reaction include phosphonium-based condensing agents and uronium-based condensing agents, specifically benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (H Examples include 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), etc. A lactone cleavage operation may be performed before amidation with these strong dehydration condensing agents.

[0032] The amidation reaction solution preferably contains at least one selected from the group consisting of ammonia, amines, and salts thereof. When using an amine, it is preferable to use a different amine than the one used in the lactonization reaction solution, or to change its mass by modification with a stable isotope, etc. By using amines with different masses in the first and second reactions, sialic acid can be amidated in such a way that the mass differs depending on the bonding mode. In one aspect of this embodiment, the second reaction is preferably an aminolysis reaction. Dehydrating condensation agents are not required for aminolysis, and the amidation reaction solution does not need to contain a dehydrating condensation agent. The amidation reaction solution may contain a dehydrating condensation agent, for example, 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 with such a simple operation.

[0033] 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. Among the primary amines containing a linear alkyl group in the amidation reaction solution, primary amines with 10 or fewer carbon atoms are preferred, primary amines with 7 or fewer carbon atoms are more preferred, methylamine, ethylamine, propylamine, butylamine, or pentylamine are even more preferred, and methylamine is the most preferred. 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 branching (hereinafter, "branching" refers to branching of the hydrocarbon chain) or has a small number of carbon atoms.

[0034] 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, more preferably contains an allyl group, and the amine is preferably an allylamine. The amine contained in the amidation reaction solution may be a primary amine containing a hydroxyl group, or it 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 functional groups, making the modified sugar chain easier to separate not only by mass spectrometry but also by chromatography and other methods.

[0035] The concentrations of ammonia, amines, and their salts in the amidation reaction solution are preferably 0.1 M or higher, more preferably 0.3 M or higher, even more preferably 0.5 M or higher, even more preferably 1.0 M or higher, and most preferably 3.0 M or higher. As a preferred example, the amidation reaction solution contains ammonia or a primary amine, particularly methylamine, and the concentration of the ammonia or primary amine such as methylamine is preferably 0.1 M or higher, more preferably 0.3 M or higher, even more preferably 0.5 M or higher, even more preferably 1.0 M or higher, and most preferably 3.0 M or higher. The higher the concentrations of ammonia, amines, and their salts in the amidation reaction solution, the more efficiently the lactone structure can be amidated. There is no particular upper limit to the concentrations of ammonia, amines, and their salts in the amidation reaction solution, but for example, it may be 16 M or less.

[0036] The solvent in the amidation reaction solution may be an aqueous solvent or an organic solvent, but a low water content is preferable from the viewpoint of preventing the hydrolysis of lactone and ensuring rapid amidation. The solvent in the amidation reaction solution is preferably a dehydrated solvent that has undergone a dehydration process to reduce its water content, and an anhydrous solvent is even more preferable. The solvent in the amidation reaction solution preferably contains at least one of methanol and acetonitrile. The amidation reaction solution may contain water, and the solvent in the amidation reaction solution may be water.

[0037] When the amidation reaction is aminolysis, the solution is preferably pH 7.7 or higher, more preferably pH 8.0 or higher, even more preferably pH 8.8 or higher, and most preferably pH 10.3 or higher. A higher pH in the amidation reaction solution allows for more efficient amidation of the lactone structure. The upper limit of the pH of the amidation reaction solution is not particularly limited, but may be, for example, 14 or lower. When amidating the lactone structure using a strong dehydrating condensation agent, the pH is not particularly limited and may be adjusted according to the dehydrating condensation agent used.

[0038] If the second reaction is aminolysis, the reaction can be completed within a few seconds to a few minutes. The time for which the sample is in contact 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 over the sample held on a support or the like. There is no particular lower limit to the time for which the sample is in contact with the amidation reaction solution to amidate the lactone structure, but it may be, for example, 1 second or more. When amidating the lactone structure using a strong dehydrating condensation agent, the reaction time is not particularly limited and may be adjusted according to the dehydrating condensation agent used. For example, 30 minutes or more is preferred, more preferably 1 hour or more, and most preferably 2 hours 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. Since the second reaction is completed in a short time, it is possible to prevent the unstable lactone structure from decomposing and impairing the quantitative accuracy in the analysis of the sugar chain. Furthermore, by setting a short reaction time for the second reaction, the sample can be analyzed more efficiently. There is no particular limit to the time from when the sample has finished contacting the lactonization reaction solution until when the sample has finished contacting the amidation reaction solution, but it may be, for example, 1 second or more.

[0039] The second reaction can be carried out in either a liquid or 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 sample into contact with the amidation reaction solution in a state where the glycoprotein is bound to or adsorbed onto a solid support. The solid support used in the second reaction is not particularly limited as long as it can immobilize the glycoprotein, and examples include solid supports that can be used in the first reaction.

[0040] The sample after the second reaction is subjected to purification, desalting, solubilization, concentration, drying, etc., by known methods, as necessary. Amidation Remove the reaction solution or Amidation The concentration of the reaction solution may be reduced. Excess reagent after the second reaction can be removed by the method described in the section for the first reaction, depending on whether the reaction is solid-phase or liquid-phase.

[0041] (Purification of glycoproteins) In step S140, it is preferable to purify the glycoprotein. This step removes the solution used in the first and / or second reaction, improving the efficiency of downstream steps. The method and conditions for purifying the glycoprotein can be appropriately selected depending on the type, properties, molecular weight, etc. of the glycoprotein. The purification of the glycoprotein may be carried out by protein precipitation. Suitable precipitants 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 the protein precipitation method, for example, a precipitant is added to a sample containing glycoprotein, and the sample is allowed to stand at -20°C to 30°C for an appropriate time. After that, the precipitate formed by centrifugation of the sample can be collected. The purification of the glycoprotein may also be carried out by immobilizing the glycoprotein on a solid support, and gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, etc. may be used.

[0042] <Free step> In step S150, the glycans are released from the glycoprotein. Methods for releasing the glycans from the glycoprotein include enzymatic treatment using O-glycosidase, N-glycosidase, endoglycoceramidase, etc., and chemical release methods such as hydrazine degradation and β-elimination. The peptide chain of the glycoprotein may be cleaved before the glycan release process. Modifications such as labeling the reducing end of the glycan with 3-aminoquinoline (3AQ), anthranilic acid (2AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), etc., may be performed along with the release of the glycans.

[0043] When releasing N-linked glycans from the peptide chain of glycoproteins, enzymatic treatment with peptide-N-glycosidase F (PNGase F), peptide-N-glycosidase A (PNGase A), endo-β-N-acetylglucosaminidase (Endo M), etc., is preferably used. When releasing O-linked glycans from the peptide chain of glycoproteins, chemical release methods are preferably used. The hydrazine decomposition method can be carried out according to known methods, for example, by adding anhydrous hydrazine or hydrated hydrazine to a sample containing glycoproteins and heating it. The β-elimination method can be carried out according to known methods, for example, by adding ammonium carbamate, saturated ammonia, ammonium carbonate, anhydrous trifluoromethanesulfonic acid (anhydrous TFMS), sodium hydroxide, dimethylamine, etc., to a sample containing glycoproteins and heating it 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 reacting under alkaline conditions, pyrazolone reagents such as PMP may be added to 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 O-linked glycan release, the pH of the β-elimination reaction is preferably 11.0 or lower, and more preferably 10.0 or lower. From the viewpoint of suppressing the decomposition and peeling reaction of sialyzed glycans, 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.

[0044] The release of glycans may be carried out in the liquid phase or the solid phase. For example, the glycans may be recovered by performing the above-described glycan cleavage treatment on a glycoprotein immobilized on a solid support. Alternatively, a glycan protein bound to a solid support having a hydrazide group may be subjected to glycan cleavage treatment, and then the glycans may be released and recovered using a weakly acidic solution. By carrying out the reaction with the sample immobilized on a solid support, the removal of the reaction solution or desalting and purification becomes easier, simplifying the preparation of the sample.

[0045] Free glycans may be purified according to known methods, for example, by liquid-liquid extraction using an organic solvent such as chloroform to separate the protein from the glycans. Free glycans can also be easily purified using a solid support (column). Free glycans can also be recovered by binding them to a solid support having a hydrazide group or an aminooxy group. Examples of solid supports having a hydrazide group include "BlotGlyco" manufactured by Sumitomo Bakelite Co., Ltd. Glycans may also be purified by adsorbing them onto a support for hydrophilic interaction chromatography (hereinafter also called HILIC). Supports for HILIC preferably contain an amide group. Free glycans may also be purified using a reversed-phase support such as C18 in addition to a carbon column. Labeled glycans may also be purified by adsorbing them onto carbon or C18.

[0046] <Analysis Steps> In step S160, the released glycans are analyzed. The glycans to be analyzed preferably include O-linked glycans that have undergone specific modification of the sialic acid bonding mode. The analytical method can be mass spectrometry, chromatography, electrophoresis, or a combination thereof. Due to the lactonization reaction described above, sialic acid is modified specifically for its bonding mode, and the mass differs between glycans that are not easily lactonized, such as α2,6-sialic acid, and glycans that are easily lactonized, such as α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid. Therefore, these glycans can be separated based on the sialic acid bonding mode by mass spectrometry, chromatography, electrophoresis, etc.

[0047] The ionization method in mass spectrometry is not particularly limited, and methods such as matrix-assisted laser desorption / ionization (MALDI), electrospray (ESI), and nano-electrospray ionization (nano-LSI) can be used, but MALDI is preferred. In mass spectrometry, either positive ion mode or negative ion mode may be used for ionization. Mass spectrometry may be performed in multiple steps. This allows for the appropriate analysis of the structure of sugar chains other than the sialic acid bonding mode, or the structure of peptide chains.

[0048] Based on the characteristics of the modified product resulting from the first and second reactions, the binding mode of sialic acid may be analyzed using analytical methods other than mass spectrometry, such as chromatography and electrophoresis. 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. It is preferable to perform mass spectrometry after liquid chromatography to perform a more precise analysis of the components in the sample. In this case, it is even more preferable to directly ionize the eluate from the liquid chromatograph in a mass spectrometer using ESI or the like under online control. Electrophoresis may be SDS-PAGE, capillary electrophoresis, microchip electrophoresis, two-dimensional electrophoresis, etc., but capillary electrophoresis is preferred. [Examples]

[0049] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, percentages indicate mass percentages.

[0050] <Experiment 1> First, the first and second reactions were carried out without releasing the glycans from the glycoprotein. Next, protein precipitation was performed to remove excess reagent. Subsequently, O-linked glycans were released from the glycoprotein, and the glycans were analyzed by mass spectrometry. The specific method is described below.

[0051] 1) Freeze-dried serum and cerebrospinal fluid were prepared as samples containing glycoproteins. 2) 100 μL of a lactonization reaction solution containing 2M isopropylamine hydrochloride (nucleophile), 500 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (dehydrating condensation agent), 500 mM 1-hydroxybenzotriazole monohydrate (highly nucleophilic additive), and dimethyl sulfoxide as a solvent was added to the sample and reacted at room temperature for 1 hour (first reaction). 3) Add 100 μL of 10% methylamine and stir (second reaction). 4) 800 μL of a solution containing 2.5% trifluoroacetic acid (TFA) and acetonitrile (ACN) as a solvent was added to precipitate the protein. 5) Remove the supernatant and collect the precipitated protein. 6) Dithiothreitol (DTT) was added to reduce and alkylate the glycoprotein. 7) Ethanol precipitation was performed to purify the protein. 8) Glycosaccharides were released and labeled from glycoproteins. 50 μL of a solution containing 0.14 M sodium hydroxide, 0.2 M 1-phenyl-3-methyl-5-pyrazolone (PMP), and 40% methanol was added and the mixture was reacted at 85°C for 16 hours. 9) Chloroform was added and the mixture was vigorously stirred to perform liquid-liquid extraction, after which the chloroform layer was discarded. This procedure was repeated three times to remove PMP from the sample. 10) Collect the aqueous layer containing the sugar chains and process it through a graphite carbon column (SPELCO's "Supelclean") TM ENVI-Carb TM The glycans were purified using "Bulk Packing". The column was washed with water, 10% ACN, 0.05% TFA, and 20% ACN, 0.05% TFA, and then eluted with 50% ACN, 0.05% TFA. 11) The eluted material was concentrated under reduced pressure and purified using Iatrobeads (normal phase support, silica-based). Specifically, the sample prepared in 95% ACN was adsorbed onto the support, washed with 95% ACN, 1% acetic acid, and 98% ACN, 2% acetic acid, and then eluted with 50% ACN. 12) After drying the eluate, it was redissolved in water to prepare the sample for analysis. Using a 2,5-dihydroxybenzoic acid (DHB) matrix, time-of-flight mass spectrometry (MALDI-TOF-MS) (Bruker, product name "Ultraflex II") was performed in positive ion mode. Steps 2) and 3) above correspond to modification steps, and steps 8) to 11) above correspond to release steps. Step 12) above corresponds to analysis steps.

[0052] Figure 2 shows the mass spectrometry spectra of glycans contained in glycoproteins derived from serum and cerebrospinal fluid, respectively. A difference of approximately 28 Da was observed between glycans containing α2,3-sialic acid (m / z 1040.49) and glycans containing α2,6-sialic acid (m / z 1068.60), indicating that sialic acid undergoes binding-mode-specific modification, and that this can be distinguished by mass spectrometry. For diallyl glycans containing both α2,3-sialic acid and α2,6-sialic acid, a peak at m / z 1372.86 was observed, indicating methylamidation of α2,3-sialic acid and isopropylamidation of α2,6-sialic acid. However, no peak was observed at a position 28 Da less than this peak. Therefore, it was confirmed that α2,6-sialic acid is not lactonized, and that α2,3-sialic acid and α2,6-sialic acid undergo different modifications.

[0053] <Experiment 2> As a comparative experiment, N-linked glycans were released from glycoproteins, the free glycans were recovered using BlotGlyco beads, and the glycans bound to the beads were modified with sialic acid using the first and second reactions. Subsequently, mass spectrometry was performed on the free glycans recovered from the beads. The obtained mass spectrometry spectra are shown in the upper panel of Figure 3.

[0054] <Experiment 3> First, the first and second reactions were carried out in the liquid phase without releasing the glycans from the glycoprotein. Next, protein precipitation was performed to remove excess reagent. Subsequently, N-linked glycans were released from the glycoprotein, and the glycans were analyzed by mass spectrometry. The obtained mass spectrometry spectra are shown in the lower panel of Figure 3. The specific procedure is described below.

[0055] 1) Lipo-dried serum was prepared as a sample. 2) 100 μL of a lactonization reaction solution containing 2M isopropylamine hydrochloride (nucleophile), 500 mM N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (dehydrating condensation agent), 500 mM 1-hydroxybenzotriazole monohydrate (highly nucleophilic additive), and dimethyl sulfoxide as a solvent was added to the sample and stirred for 1 hour (first reaction). 3) Add 100 μL of 10% methylamine and stir (second reaction). 4) 800 μL of a solution containing 2.5% trifluoroacetic acid (TFA) and acetonitrile (ACN) as a solvent was added to precipitate the protein. 5) Remove the supernatant and collect the precipitated protein. 6) Dithiothreitol (DTT) was added to reduce and alkylate the glycoprotein. 7) Ethanol precipitation was performed to purify the protein. 8) Proteins were digested with trypsin (10 mg / mL). 9) N-linked glycans were released using PNGaseF (Roche, 2U). 10) N-linked glycans were purified according to the protocol using a solid support consisting of beads having a hydrazide group as a ligand (Sumitomo Bakelite Co., Ltd., "BlotGlyco"). 11) Simultaneously with the release from the carrier, the reducing end of the glycan was labeled using the high-sensitivity reagent aoWR, and a sample for analysis was prepared. 12) Using a DHB matrix, mass spectrometry was performed in positive ion mode using MALDI-TOF-MS (Bruker, "product name Ultraflex II").

[0056] As shown in Figure 3, nearly identical mass spectrometry spectra were obtained in Experiment 2 and Experiment 3. In N-linked glycans, it was found that sialic acid underwent similarly mode-specific modifications whether the first and second reactions were performed on a free glycan or on a glycoprotein followed by the release of the glycan, and these modifications could be distinguished by mass spectrometry. The present invention demonstrates that the binding specificity of sialic acid can be rapidly and accurately determined whether the glycoprotein's glycan contains N-linked sialic acid or O-linked sialic acid.

[0057] <Experiment 4> As a comparative experiment, we present experiments using glycoproteins with O-linked glycans, comparing an experiment where sialic acid was not modified in a manner specific to its linkage mode, with an experiment where sialic acid was modified in a manner specific to its linkage mode after the release of the glycans. Fetwin (100 μg), a glycoprotein, was mixed with 20 μL of 50% hydroxylamine and 20 μL of 0.5 M sodium hydroxide, and reacted at 50°C for 1 hour to release the glycans. After the reaction, 10 μL of 1 M hydrochloric acid was added to quench the reaction, and 200 μL of acetone was added and the mixture was concentrated using a centrifugal evaporator. After concentration, the mixture was redissolved in 50 μL of water, and the free glycans were captured with BlotGlyco beads. Sialic acid was modified on a solid phase by methyl esterification (MTT method) or by the first and second reactions (sialic acid linkage mode specific alkylamidation method; also referred to as the "SALSA method" herein). According to the MTT method, linkage mode nonspecific modification occurs, so all sialic acids are esterified. The first and second reactions were carried out in the same manner as in Experiment 1. After labeling the reducing end of the glycans with the high-sensitivity reagent aoWR, the glycans were purified by HILIC column chromatography. The purified aoWR-labeled glycans were then subjected to 2,5- Dihydro The sample was mixed with benzoic acid (10 mg / mL) and analyzed using MALDI-TOF-MS (Bruker, product name "Ultraflex II").

[0058] The obtained mass spectrometry spectrum is shown in Figure 4. In the MTT method, sialic acid is methyl esterified, and sialyl T is m / z 1118.6 [M+H] +Dithialyl T is m / z 1424.0 [M+H] + It was detected. In the SALSA method, sialyl T, which has α2,3 linked sialic acid, has an m / z of 1117.6 [M+H]. + It was detected as a methylamide compound. On the other hand, dicialyl T, which has α2,3 and α2,6 sialic acid bonds, has an m / z of 1449.8 [M+H]. + Signals were observed with methylamide and isopropylamide modifications, but the α2,6 bond sialic acid was modified with methylamide (m / z 1421.9[M+H]). + It was also detected. Neither method achieved modification specific to the sialic acid linkage mode of O-linked glycans.

[0059] <Experiment 5> The concentration of methylamine was varied to examine the efficiency of the linkage mode-specific modification of sialic acid in O-linked glycans. First, the first and second reactions were carried out on fetuin (100 μg). Specifically, 100 μL of a lactonization reaction solution (2 M iPA-HCl, 500 mM EDC-HCl, 500 mM HOBt) was added to fetuin and stirred at room temperature for 1 hour (the first reaction). Subsequently, 100 μL of a 10%, 16.6% or 40% aqueous methylamine solution was added to the reaction solution and stirred (the second reaction). Then, 1 mL of acetonitrile containing 2.5% trifluoroacetic acid was added and allowed to stand at -30°C for 1 hour. Centrifugation was performed at 14,000 g for 20 minutes, and the supernatant was removed to remove the SALSA reaction solution. Further, 1 mL of acetonitrile was added to the residue and centrifuged at 14,000 g for 20 minutes, and the supernatant was removed. The obtained residue containing the protein was redissolved in 10 mL of water. A β-elimination reaction solution for releasing glycans from glycoproteins was prepared. The β-elimination reaction solution is a solution in which sodium hydroxide (NaOH) (final concentration 0.7 M) and 3-methyl-1-phenyl-5-pyrazolone (PMP) (final concentration 1 M) are dissolved in a 60% aqueous methanol solution. 40 μL of the β-elimination reaction solution was added to fetuin in which sialic acid was modified in a linkage mode-specific manner, and the temperature was maintained at 85°C using a thermal cycler and reacted for 16 hours. After completion of the reaction, 50 pmol of bisPMP-labeled chitotetraose was added, and then neutralized with 10 μL of 4 M hydrochloric acid. Chloroform was added and stirred vigorously for liquid-liquid extraction. The chloroform layer was removed, and this was repeated three times. The aqueous layer containing the glycans was recovered and purified using a graphite carbon column and iatrobeads. The purification method was the same as in Experiment 1. The purified bisPMP-labeled glycans were mixed with 2,5-dihydroxybenzoic acid (10 mg / mL) containing 10% sodium 2,5-dihydroxybenzoate and analyzed by MALDI-TOF.

[0060] The results of the mass spectrometry spectra are shown in Fig. 5. In all reactions, most of the sialyl T with α2,3-linked sialic acid was observed at m / z 1040.7 [M+Na] + and the d sialyl T with two α2,3- and α2,6-linked sialic acids was observed at m / z 1373.1 [M+Na] +This was observed. After directly modifying the glycans on the glycoprotein with sialic acid using the SALSA method, the glycans were released by a β-elimination reaction. This confirmed that derivatization specific to the binding mode proceeded, and that the glycans were cleaved from the glycoprotein while remaining derivatized by the β-elimination reaction. Furthermore, the effect of methylamine concentration was investigated. Methylamidation modification resulted in m / z 1040.7[M+Na] + The α2,3-linked sialyl T detected is m / z 1027.6[M+Na] when the carboxylic acid is unmodified. + It was detected. The ratio of unmodified sialyl T to methylamidated sialyl T was 4.6% when methylamine was 10% and 2.6% when methylamine was 40%. Similarly, the ratio of unmodified dicialyl T to methylamidated dicialyl T was 1.5% when methylamine was 10% and 0.8% when methylamine was 40% (Figure 6). In the aminolysis reaction, the efficiency of methylamidation of sialic acid was higher with increasing amine concentration.

[0061] <Experiment 6> The released O-linked glycans were analyzed by varying the pH during the β-elimination reaction. Using the same method as in Experiment 5, sialic acid was used for specific sialic acid linkage modification in human plasma (10 μL). A 40% methylamine aqueous solution was used for the second reaction. After washing the glycoprotein using the same method as in Experiment 5, 0.1 mL of water was added to the precipitate, and the mixture was suspended using a water bath ultrasonic device. 0.4 mL of the pre-prepared β-elimination reaction solution was added to the suspension and mixed. The β-elimination reaction solution was prepared by dissolving PMP (final concentration 1 M) and NaOH (final concentration 0.7-0.975 M) in a 60% acetonitrile solution. Five different pH levels were set by varying the concentration of NaOH. The composition and pH are shown in Table 1. The reaction was carried out at 85°C for 16 hours, and PMP labeling was performed simultaneously with the β-elimination of O-linked glycans. After the reaction was complete, 0.4 mL of 4 M HCl was added to neutralize the mixture. 0.4 mL of chloroform was added and the mixture was vigorously stirred. The chloroform layer was discarded by liquid-liquid extraction, and this procedure was repeated four times. The aqueous layer containing the labeled glycans was collected and purified using a graphite carbon column and Iatro beads. The PMP-labeled glycans were mixed with 2,5-dihydrobenzoic acid and analyzed by MALDI-TOF. The purification and analysis methods were the same as in Experiment 5.

[0062] The analysis results are shown in Figures 7 to 9. The amount of O-linked glycans recovered was highest in Entry 3 (Figure 7). On the other hand, in Entry 3 to 5, the proportion of degradation products of methylamidated sialyl T was increased compared to Entry 1 and 2 (Figure 8). In Entry 3 to 5, the proportion of degradation products from the β-elimination reaction (peeling reaction) was increased compared to Entry 1 and 2 (Figure 9).

[0063] [Table 1]

[0064] [Aspect] Those skilled in the art will understand that the above-described exemplary embodiments and examples are specific examples of the following embodiments.

[0065] (Section 1) A method for analyzing O-linked glycans that have undergone sialic acid linkage-specific modification in a sample according to one embodiment includes a modification step of adding a modifier that performs sialic acid linkage-specific modification to the O-linked glycan to a sample containing a glycoprotein to which the O-linked glycan is bound; a release step of releasing the O-linked glycan that has undergone sialic acid linkage-specific modification from the glycoprotein; and an analysis step of analyzing the released O-linked glycan that has undergone sialic acid linkage-specific modification. According to the method described in paragraph 1, even if the glycan contains O-linked sialic acid, the linkage mode of the sialic acid can be accurately determined.

[0066] (Section 2) In the method described in paragraph 1, the specific modification of the sialic acid bond mode includes a first reaction that lactonizes the sialic acid. The first reaction selectively lactonizes the sialic acid according to the bond mode. In the first reaction, α2,3-sialic acid in the sugar chain is preferably lactonized.

[0067] (Section 3) In the method described in paragraph 2, the mode-specific modification of the sialic acid further comprises a second reaction for amidating the lactone structure produced by the first reaction. The method described in paragraph 3 allows for more precise mode-specific modification of the sialic acid.

[0068] (Section 4) In the method described in paragraph 3, the second reaction is carried out using a solution containing at least one selected from the group consisting of ammonia, amines, and salts thereof. According to the method described in paragraph 4, modification of sialic acid in a manner specific to the bonding mode can be efficiently performed.

[0069] (Section 5) In the method described in paragraph 3 or 4, the second reaction is an aminolysis reaction.

[0070] (Section 6) In any of the methods described in paragraphs 3 to 5, the sialic acid after the second reaction undergoes modifications with different masses depending on the bonding mode. According to the method described in paragraph 6, sialic acid that has undergone bonding mode-specific modifications can be distinguished by analytical methods such as mass spectrometry.

[0071] (Section 7) In the method described in paragraph 6, the bonding modes of the sialic acid include α-2,3-sialic acid and α-2,6-sialic acid. According to the method described in paragraph 7, α-2,3-sialic acid and α-2,6-sialic acid can be distinguished.

[0072] (Section 8) In the method described in any of paragraphs 2 to 7, the first reaction is carried out by adding a solution containing a dehydrating condensation agent to the sample. According to the method described in paragraph 8, different modifications can be made depending on the bonding mode of sialic acid, based on the differences in molecular structure due to the bonding mode of sialic acid.

[0073] (Section 9) In the method described in any of paragraphs 2 to 8, α-2,6-sialic acid is amidated after the first reaction.

[0074] (Section 10) The method according to any of paragraphs 2 to 9 further comprises purifying the glycoprotein by protein precipitation after the first reaction. The method according to paragraph 10 makes it possible to remove the solution used in the upstream step and improve the reaction efficiency of the downstream step.

[0075] (Section 11) In the method described in any of paragraphs 1 to 10, the sugar chain is released from the glycoprotein by a β-elimination reaction or a hydrazine degradation reaction. According to the method described in paragraph 11, the sugar chain can be chemically released from the glycoprotein.

[0076] (Section 12) In the method described in item 11, the β-elimination reaction is carried out at a pH of 11.0 or lower. According to the method described in item 12, O-linked glycans can be efficiently released.

[0077] (Section 13) Paragraphs 1 to 12 either The method described in paragraph 13 further comprises purifying the liberated glycans using a solid support. According to the method described in paragraph 13, liberated glycans can be easily separated from proteins.

[0078] (Section 14) In the method described in any of paragraphs 1 to 13, the analysis includes at least one selected from the group consisting of mass spectrometry, chromatography, and electrophoresis. According to the method described in paragraph 14, the glycans can be analyzed by distinguishing the binding modes of sialic acid based on the mass difference that arises in a manner specific to the binding mode, or on the effect on separation by chromatography or electrophoresis.

Claims

1. A method for analyzing O-linked glycans that have undergone specific modification of the sialic acid linkage mode, A modification step involves adding a modifying agent to a sample containing a glycoprotein to which the O-linked glycan is bound, which modifies the O-linked glycan in a manner specific to the mode of sialic acid binding. A release step in which the O-linked glycan, which has undergone the sialic acid linkage-specific modification, is released from the glycoprotein, The analysis step includes analyzing the O-linked glycan on which the released sialic acid has undergone a specific modification of its bonding mode, The aforementioned sialic acid bonding modes include α2,3-sialic acid and α2,6-sialic acid. The modifier that performs the specific modification of the bonding mode of the sialic acid comprises a lactonization reaction solution and an amidation reaction solution. The lactonization reaction solution comprises a dehydrating condensing agent and a nucleophile. The dehydration condensing agent comprises carbodiimide, The amidation reaction solution contains ammonia or a primary amine, The aforementioned mode-specific modification of sialic acid further comprises a first reaction for lactonizing the α2,3-sialic acid and a second reaction for amidating the lactone structure of the α2,3-sialic acid obtained by the first reaction. The first reaction is carried out by contacting the sample with the lactonization reaction solution. The second reaction is carried out by contacting the sample after the first reaction with the amidation reaction solution.

2. The method according to claim 1, wherein the second reaction is an aminolysis reaction with the primary amine.

3. The method according to claim 1, wherein the sialic acid after the second reaction has undergone modifications with different masses depending on the bonding mode.

4. The method according to claim 1, wherein α-2,6-sialic acid is amidated after the first reaction.

5. The method according to claim 1, further comprising purifying the glycoprotein by a protein precipitation method after the first reaction.

6. The method according to claim 1, wherein the sugar chain is released from the glycoprotein by a β-elimination reaction or a hydrazine degradation reaction.

7. The method according to claim 6, wherein the β-elimination reaction is carried out at a pH of 11.0 or lower.

8. The method according to claim 1, further comprising purifying the liberated sugar chain using a solid support.

9. The method according to claim 1, wherein the analysis comprises at least one selected from the group consisting of mass spectrometry, chromatography, and electrophoresis.

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