Preparation method of the sample and analysis method

Abstract

The technical problem of the present application is to rapidly stabilize lactone in a sugar chain. A sample preparation method is a sample preparation method for preparing a sample containing a sugar chain, the preparation method including the steps of: performing a lactonization reaction that lactonizes at least a portion of sialic acid contained in the sugar chain; and performing an amidation reaction that amidates the lactone of the lactonized sialic acid by adding an amidation reaction solution to the sample, the amidation reaction solution containing at least one selected from the group consisting of ammonia, amines, and salts thereof that react with the lactonized sialic acid.

Description

[0001] The present application is a divisional application based on the Chinese Invention Application of SHIMADZU CORPORATION, the subject of which is "Sample preparation method and analysis method", the Chinese Invention Application having the application number 201910123558.1 and the filing date of February 18, 2019. TECHNICAL FIELD

[0002] The present application relates to a sample preparation method and an analysis method. BACKGROUND

[0003] Sialic acid is a sugar that exists in large amounts in living organisms. Sialic acid is contained in a sugar chain bound to a protein in living organisms, and is present more in the terminal (non-reducing terminal) of the sugar chain. Therefore, sialic acid is disposed on the outside of the molecule in such a glycoprotein molecule, and can be directly recognized from other molecules, and thus plays an important role.

[0004] There are cases where the linkage type between sialic acid and an adjacent sugar is different. For example, it is known that in human N-type sugar chains, the linkage types are mainly α2,3- and α2,6-, and in O-type sugar chains or glycosphingolipids, in addition to these, there are α2,8- and α2,9- linkage types. Depending on the difference in the linkage type, sialic acid can be recognized from different molecules, and thus can have different roles. In addition, it is also known that the linkage type of sialic acid in expressed glycoproteins changes as cancer progresses, and thus its use as a biomarker for cancer is also expected. Furthermore, it is known that the effect of sugar chain modification on biological drugs, and in the quality management of biological drugs, the analysis of the linkage type of sialic acid is also essential.

[0005] However, since sialic acid has a negative charge, ionization is difficult in positive ion mode or sialic acid is easily decomposed, and thus analysis by mass spectrometry of sialic acid sugar chains containing sialic acid is not easy. In addition, the molecular weight does not change depending on the linkage type of sialic acid, and thus it becomes more difficult to distinguish and analyze the linkage type of sialic acid.

[0006] In order to distinguish and analyze the linkage type of sialic acid, a chemical modification method that performs linkage type-specific modification has been proposed. In such a chemical modification method, using the property that α2,3-sialic acid is more likely to cause intramolecular dehydration by a dehydration condensing agent than α2,6-sialic acid, α2,3-sialic acid is lactonized by intramolecular dehydration, and at the same time, α2,6-sialic acid is reacted with a nucleophilic agent such as an alcohol or an amine. Thereby, molecules of different masses are generated depending on the linkage type of sialic acid, and thus it is possible to distinguish and analyze the linkage type of sialic acid by mass spectrometry.

[0007] However, the lactone of α2,3-sialic acid is unstable and starts to decompose as soon as it is dissolved in water, and a considerable proportion thereof can also decompose in about 48 hours. Therefore, in order to avoid quantitative deterioration due to decomposition of the lactone, modification of the lactone is performed to stabilize it.

[0008] In Patent Literature 1 and Non-Patent Literature 1, a solution containing isopropylamine and a dehydration condensing agent is added to a free sugar chain to lactonize α2,3-sialic acid and to amidate α2,6-sialic acid. Thereafter, in order to hydrolyze and amidate the lactone, a solution containing methylamine hydrochloride and a solution containing a dehydration condensing agent are sequentially added to the sample and allowed to react for 2 hours.

[0009] In Non-Patent Literature 2, a solution containing dimethylamine and a dehydration condensing agent is added to a glycoprotein on a tissue section to lactonize α2,3-sialic acid and to amidate α2,6-sialic acid. Thereafter, as shown in the scheme (B), in order to hydrolyze and amidate the lactone, ammonia is added and allowed to react for 2 hours. Figure 1 (B) shown, in order to hydrolyze and amidate the lactone, ammonia is added and allowed to react for 2 hours.

[0010] In Non-Patent Literature 3, a solution containing ethanol and a dehydration condensing agent is added to a glycoprotein bound to a solid support to lactonize α2,3-sialic acid and to esterify α2,6-sialic acid. Thereafter, as shown in the scheme (Scheme) 1(b) of Non-Patent Literature 3, in order to hydrolyze the lactone, a Tris buffer at pH 10 is added to the sample and allowed to react for 1 hour, and then a solution containing methylamine hydrochloride and a dehydration condensing agent is added to the sample and allowed to react for 30 minutes.

[0011] Prior Art Documents

[0012] Patent Literature

[0013] Patent Literature 1: Japanese Patent No. 6135710

[0014] Non-Patent Literature 1: Tatsuro Nishiwaki, Yuki Tsuyumu, Satoki Sekiya, Shinichi Iwamoto, Yuriko Miura, Koji Tanaka, Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling, Analytical Chemistry (USA), American Chemical Society Publications, February 21, 2017, Vol. 89, No. 4, pp. 2353-2360.

[0015] Non-patent literature 2: Holst S, Heijs B, de Haan N, van Zeijl RJ, Briaire-de Bruijn IH, van Pelt GW, Mehta AS, Angel PM, Mesker WE, Tollenaar RA, Drake RR, Bovee JV, McDonnell LA, Wuhrer M. Linkage-Specific in Situ Sialic Acid Derivatization for N-Glycan Mass Spectrometry Imaging of Formalin-Fixed Paraffin-Embedded Tissues. Analytical Chemistry (United States) Journal of the American Chemical Society, June 7, 2016, Vol. 88, No. 11, pp. 5904-5913.

[0016] Non-patent literature 3: Li H, Gao W, Feng X, Liu P, Liu X. MALDI-MS analysis of sialylated N-glycan linkage isomers using solid-phase two step derivatization method. Analytica Chimica Acta (Netherlands) Elsevier, June 14, 2016, Vol. 924, pp. 77-85. SUMMARY

[0017] PROBLEMS TO BE SOLVED BY THE INVENTION

[0018] In the method of the above-described prior art literature, since an attempt is made to open a lactone ring by hydrolysis and then to react a carboxyl group resulting from the opening with an amine in the presence of a dehydration condensing agent, a long time is required for the reaction and the operation is complicated.

[0019] SOLUTION TO THE PROBLEM

[0020] The preparation method of the sample of the preferred embodiment of the present application is a preparation method of a sample containing a sugar chain, including the steps of: performing a lactonization reaction that lactonizes at least a part of sialic acid contained in the sugar chain; and performing an amidation reaction that amidates a lactone of the sialic acid that has been lactonized by adding an amidation reaction solution to the sample, the amidation reaction solution containing at least one selected from the group consisting of ammonia, amines, and salts thereof that react with the lactonized sialic acid.

[0021] In a further preferred embodiment, the process further comprises the step of, after the lactonization reaction, removing the lactonization reaction solution used for the lactonization reaction from the sample.

[0022] In a further preferred embodiment, the amidation reaction is performed only by contacting the sample with the amidation reaction solution.

[0023] In a further preferred embodiment, the amidation reaction solution does not contain a dehydration condensing agent that reacts with the lactone.

[0024] In a further preferred embodiment, after the amidation reaction solution is added to the sample, no operation of reacting the sample with a dehydration condensing agent is performed.

[0025] In a further preferred embodiment, the time for which the sample is contacted with the amidation reaction solution for performing the amidation reaction is shorter than 30 minutes.

[0026] In a further preferred embodiment, the amine is a primary amine.

[0027] In a further preferred embodiment, the amine contains an alkyl group.

[0028] In a further preferred embodiment, the alkyl group is not branched.

[0029] In a further preferred embodiment, the amine contains at least one of an allyl group and a hydroxyl group.

[0030] In a further preferred embodiment, the pH of the amidation reaction solution is 8.0 or more.

[0031] In a further preferred embodiment, in performing the lactonization reaction, a lactonization reaction solution is added to the sample to lactonize at least a portion of the sialic acid, the lactonization reaction solution containing a dehydration condensing agent that reacts with the sialic acid contained in the sugar chain.

[0032] In a further preferred embodiment, the lactonization reaction solution further contains a nucleophile that reacts with the sialic acid contained in the sugar chain, the nucleophile being different in mass from the ammonia used for the amidation reaction and the amine, the lactonization reaction solution being added to the sample to lactonize a portion of the sialic acid based on the binding mode of the sialic acid, and at least a portion of the nucleophile binds to another portion of the sialic acid.

[0033] In a further preferred embodiment, in the lactonization reaction, at least one of the sialic acids selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid is lactonized.

[0034] In a further preferred embodiment, the lactonization reaction solution is added to the sample to lactonize α2,3-sialic acid and bind a portion of the nucleophile to α2,6-sialic acid.

[0035] In a further preferred embodiment, the contact of the sample with the amidation reaction solution is performed in a state in which the sample is bound or adsorbed to a solid support.

[0036] In a further preferred embodiment, the solvent of the amidation reaction solution comprises an organic solvent.

[0037] The analysis method of the preferred embodiment of the present application includes the steps of preparing a sample by the above-described sample preparation method, and analyzing the prepared sample.

[0038] In a further preferred embodiment, the prepared sample is analyzed by at least one of mass spectrometry and chromatography.

[0039] Effects of the Invention

[0040] According to the present application, it is possible to more rapidly stabilize the molecule after lactonization in the sugar chain. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is a flowchart showing the flow of the analysis method of one embodiment.

[0042] Figure 2 is a mass spectrum obtained by subjecting the reaction product to mass spectrometry in the negative ion mode after subjecting the sugar chain released from the α2,3-sialic acid glycopeptide to a lactonization reaction and an amidation reaction, Figure 2 a is a mass spectrum when the concentration of methylamine at the time of the amidation reaction is 1%, Figure 2 b is a mass spectrum when the concentration of methylamine at the time of the amidation reaction is 10%.

[0043] Figure 3 is a graph showing the relationship between the concentration of an aqueous solution of amine at the time of the amidation reaction and the efficiency of amidation.

[0044] Figure 4 is a graph showing the relationship between the kind of amine at the time of the amidation reaction and the generation ratio of each reaction product.

[0045] Figure 5 is a graph showing the relationship between the kind of amine at the time of the amidation reaction and the generation ratio of each reaction product.

[0046] Figure 6 is a graph showing the ratio of the production of each reaction product when a sugar chain released from ganglioside GD1a (upper row) and GD1b (lower row) is subjected to lactonization reaction and amidation reaction.

[0047] Figure 7 is a mass spectrum obtained by subjecting a sugar chain released from fetuin, which is a glycoprotein, to lactonization reaction and amidation reaction, and then subjecting the reaction product to mass spectrometric analysis.

[0048] Figure 8 is a mass spectrum obtained by subjecting a sugar chain containing α2,8-sialic acid released from ganglioside GD1a (upper row) and GD1b (lower row) to lactonization reaction and amidation reaction, and then subjecting the reaction product to mass spectrometric analysis.

[0049] Figure 9 is a graph showing the ratio of the production of each reaction product when a sugar chain released from α2,3-sialic acid glycopeptide is subjected to lactonization reaction, binding to a HILIC carrier, and amidation reaction before and after elution. DETAILED DESCRIPTION

[0050] Hereinafter, a specific embodiment of the present application will be described with reference to the accompanying drawings. The inventors have found that by adding an amidation reaction solution containing at least one compound selected from the group consisting of ammonia, amines, and salts thereof, which react with lactonized sialic acid, to a sample in which sialic acid is lactonized, rapid amidation can be induced.

[0051] Figure 1 is a flowchart showing the flow of an analysis method of a preparation method of a sample of the present embodiment. In step S1001, a sample containing a sugar chain is prepared.

[0052] The sample containing a sugar chain is not particularly limited, and can contain at least one molecule selected from the group consisting of a released sugar chain, a glycopeptide, a glycoprotein, and a glycolipid. The preparation method of the sample of the present embodiment is used for modification of a lactone formed on a sugar chain, and is particularly suitable for analysis of the binding mode of sialic acid, and therefore, the sugar chain in the sample preferably contains a sugar chain that can have sialic acid at the terminal, such as an N-bound type sugar chain, an O-bound type sugar chain, a glycolipid type sugar chain, and the like. The sugar chain in the sample preferably contains at least one of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid.

[0053] In the case where the sample contains free sugar chains, the sugar chains that are free from glycoproteins, glycopeptides, and glycolipids can be used. As a method for liberating sugar chains from glycoproteins, glycopeptides, and glycolipids, methods such as enzymatic treatment using N-glycosidase, O-glycosidase, endoglycoceramidase, and the like, hydrazine decomposition, β elimination based on alkali treatment, and the like can be used. In the case where N-bound sugar chains are liberated from the peptide chains of glycopeptides and glycoproteins, it is appropriate to use enzymatic treatment based on peptide-N-glycosidase F (PNGase F), peptide-N-glycosidase A (PNGase A), endo-β-N-acetylglucosaminidase (Endo M), and the like. Furthermore, modification such as pyridylamination (PA) of the reducing end of the sugar chain can be appropriately performed. The cleavage of the peptide chain of the glycopeptide or glycoprotein described later can also be performed before the enzymatic treatment.

[0054] In the case where the sample contains glycopeptides and / or glycoproteins, as described in the section "Regarding side reactions of glycopeptides and glycoproteins" described later, treatment for inhibiting side reactions of the peptide portion can be appropriately performed. Furthermore, for a sample in which the number of residues of amino acids of the peptide chain of the glycopeptide or glycoprotein is large, it is preferable to cleave the peptide chain by enzymatic cleavage or the like and then use it. For example, in the case of preparing a sample for mass spectrometric analysis, the number of amino acid residues of the peptide chain is preferably 30 or less, more preferably 20 or less, and further preferably 15 or less. On the other hand, in the case where it is required to specify the origin of the peptide to which the sugar chain is bound, the number of amino acid residues of the peptide chain is preferably 2 or more, and more preferably 3 or more.

[0055] As the digestive enzyme at the time of cleaving the peptide chain of the glycopeptide or glycoprotein, trypsin, lysine C-terminal endopeptidase (Lys-C), arginine endopeptidase, chymotrypsin, pepsin, thermolysin, protease K, protease E, and the like can be used. Two or more of these digestive enzymes can also be used in combination. The conditions at the time of cleaving the peptide chain are not particularly limited, and an appropriate experimental protocol can be used in accordance with the digestive enzyme used. Denaturation treatment or alkylation treatment of the proteins and peptides in the sample can also be performed before this cleavage. The conditions of the denaturation treatment or alkylation treatment are not particularly limited.

[0056] In addition, the cleavage treatment of the peptide chain described above can also be performed after the lactone amide contained in the sugar chain is lactonized by the sample preparation method of the present embodiment. Furthermore, the peptide chain can also be cleaved by chemical cleavage or the like without being cleaved by an enzyme.

[0057] If the step S1001 ends, the process proceeds to step S1003.

[0058] (Lactonization reaction)

[0059] In step S1003, the sample is brought into contact with a reaction solution for lactonization (hereinafter, referred to as a lactonization reaction solution), and a lactonization reaction of lactonizing at least a part of the sialic acid contained in the sugar chain is performed. In the lactonization reaction, a2, 3-sialic acid, a2, 8-sialic acid, and a2, 9-sialic acid are suitably lactonized. The lactonization reaction solution contains a dehydration condensing agent.

[0060] In the following description, the case where the binding mode of sialic acid is analyzed is exemplified. In this case, the lactonization reaction solution contains, in addition to the dehydration condensing agent, a nucleophile containing at least one selected from the group consisting of an alcohol, an amine, and a salt thereof.

[0061] The kind and concentration of the dehydration condensing agent and the nucleophile are adjusted so that a dehydration reaction or a nucleophilic reaction selectively occurs depending on the binding mode of sialic acid. The lactone produced by intramolecular dehydration of the carboxyl group of a2, 3-sialic acid is a six-membered ring, and the lactone that can be produced by intramolecular dehydration of the carboxyl group of a2, 6-sialic acid is a seven-membered ring. Therefore, a2, 3-sialic acid, which produces a six-membered ring that is more stable than a seven-membered ring, is more easily lactonized than a2, 6-sialic acid. In addition, the carboxyl group of a2, 3-sialic acid is located at a position having a larger steric hindrance than the carboxyl group of a2, 6-sialic acid, and therefore, a large molecule is less likely to react with a2, 3-sialic acid than with a2, 6-sialic acid. Based on such differences in molecular structure due to the binding mode of sialic acid, the kind and concentration of the dehydration condensing agent and the nucleophile can be adjusted so that different modifications are made depending on the binding mode of sialic acid.

[0062] (Dehydration condensing agent in lactonization reaction)

[0063] The dehydration condensing agent preferably contains a carbodiimide. This is because, if a carbodiimide is used, the carboxyl group present at a position having a large steric hindrance is less likely to be amidated than when a phosphonium-based dehydration condensing agent (so-called BOP reagent) or a uronium-based dehydration condensing agent is used as the dehydration condensing agent. As examples of the carbodiimide, 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-tolylcarbodiimide, bis(2,6-diisopropylisopropylphenyl)carbodiimide, bis(trimethylsilyl)carbodiimide, 1,3-bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)carbodiimide (BDDC), or a salt thereof can be listed.

[0064] (Additive in lactonization reaction)

[0065] In order to promote the dehydration condensation caused by the dehydration condensing agent, and to inhibit the side reaction, it is preferable to use an additive having high nucleophilicity in addition to the carbodiimide. As the additive having high nucleophilicity, it is preferable to use 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt), 4-dimethylaminopyridine (DMAP), 2-cyano-2-(hydroxyimino)acetic acid ethyl ester (CHA), N-hydroxysuccinimide (Hosu), 6-chloro-l-hydroxy-benzotriazole (Cl-HoBt), N-hydroxy-3,4-dihydro-4-oxo-l,2,3-benzotriazine (HOOBt), and the like.

[0066] (Nucleophile in lactonization reaction)

[0067] The amine used as the nucleophile preferably contains a primary alkyl amine and / or a secondary alkyl amine having 2 or more carbon atoms. The primary alkyl amine is preferably ethylamine, propylamine, isopropylamine, butylamine, sec-butylamine, tert-butylamine, and the like. The secondary alkyl amine is preferably dimethylamine, ethylmethylamine, diethylamine, propylmethylamine, isopropylmethylamine, and the like. From the viewpoint that a carboxyl group present in a site having large steric hindrance such as a carboxyl group of α2,3-sialic acid is not easily amidated, it is preferable to use an amine having a branched alkyl group such as isopropylamine. In the case where an amine is used as the nucleophile in the lactonization reaction solution, based on the binding mode of sialic acid, a carboxyl group of a part of sialic acid such as α2,6-sialic acid is amidated.

[0068] The alcohol used as the nucleophile is not particularly limited, and for example, methanol, ethanol, and the like can be used. In the case where an alcohol is used as the nucleophile in the lactonization reaction solution, based on the binding mode of sialic acid, a carboxyl group of a part of sialic acid such as α2,6-sialic acid is esterified.

[0069] In addition, the nucleophile can also include a salt of the above-mentioned nucleophile.

[0070] (Phase in which lactonization reaction is performed)

[0071] The lactonization reaction can be performed in a liquid phase or in a solid phase. In the case of performing the reaction in a liquid phase, the reaction is preferably performed in a non-aqueous solvent such as dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). By performing the reaction in a non-aqueous solvent, there is a tendency to suppress side reactions, and it is suitable for cases in which a glycopeptide or a glycoprotein is used as the sample. The concentration of each component in the liquid phase reaction is not particularly limited, and can be appropriately determined depending on the kind of the dehydrative condensing agent or the amine, etc. The concentration of the dehydrative condensing agent in the lactonization reaction solution is, for example, preferably 1 mM to 5 M, and more preferably 10 mM to 3 M. In the case of using a carbodiimide and an additive such as HOAt or HOBt, which have high nucleophilicity, the respective concentrations are preferably in the above ranges. The concentration of the amine in the lactonization reaction solution is preferably 0.01 M to 20 M, and more preferably 0.1 M to 10 M. The reaction temperature during the lactonization reaction is preferably about -20°C to 100°C, and more preferably -10°C to 50°C.

[0072] In the case of performing the lactonization reaction in a solid phase, the solid phase carrier is not particularly limited as long as it can immobilize a sugar chain, a glycopeptide, a glycoprotein, etc. For example, in order to immobilize a glycopeptide or a glycoprotein, a solid phase carrier having an epoxy group, a tosyl group, a carboxyl group, an amino group, etc. as a ligand can be used. Furthermore, in order to immobilize a sugar chain, a solid phase carrier having a hydrazide group or an aminooxy group, etc. as a ligand can be used. By performing the reaction in a state in which the sample is immobilized to the solid phase carrier, it becomes easier to remove the lactonization reaction solution after the lactonization reaction, etc.

[0073] If the step S1003 ends, the process proceeds to step S1005.

[0074] In addition, it is preferable to perform an operation for removing the lactonization reaction solution from the sample after the lactonization reaction. The operation for removing the lactonization reaction solution is not particularly limited as long as it can sufficiently reduce the concentration of the components required for the lactonization reaction, and, for example, the reaction solution is separated from the sugar chain bound to the solid phase carrier by centrifugation, etc., and washing is performed using a washing solution; or the sample is dried and solidified by centrifugal concentration, etc.

[0075] (Amide formation reaction)

[0076] In step S1005, an amidation reaction is performed in which the sample is brought into contact with a reaction solution for amidation of lactone (hereinafter, referred to as an amidation reaction solution) to amidate the lactone of the sialic acid subjected to lactonization. The inventors have found a method for rapidly and directly amidating lactone, which is completely different from the conventional technique of opening the ring of lactone by hydrolysis and then amidating the carboxyl group. This reaction is suitable even under anhydrous conditions as described later, and is thus considered to be a reaction different from hydrolysis, which is ammonolysis based on the interaction between an amino group and lactone. Hereinafter, the opening of the ring of lactone and amidation based on ammonia, amine, or a salt thereof, which can be performed even under anhydrous conditions, is referred to as ammonolysis.

[0077] The amidation reaction solution contains at least one selected from the group consisting of ammonia, amine, and a salt thereof. In the case of using amine, a different amine from that used in the lactonization reaction solution is used, or the mass is made different by modification based on a stable isotope or the like. A dehydration condensing agent is not required in the amidation reaction, and can not be contained. It is preferable that the amidation reaction be performed only by bringing the sample into contact with the amidation reaction solution.

[0078] In addition, although a dehydration condensing agent is not required in the amidation reaction, a dehydration condensing agent can be contained in the amidation reaction solution. For example, the lactonization reaction solution added to the sample in step S1003 can be left without being removed, and ammonia, amine, or a salt thereof can be added thereto, thereby preparing the amidation reaction solution. In this way, in the amidation reaction, lactone can be stabilized by a simple operation.

[0079] (Amine in amidation reaction)

[0080] The amine contained in the amidation reaction solution is preferably a primary amine, more preferably a primary amine having a linear hydrocarbon group, and further preferably a primary amine having a linear alkyl group. Among the amines contained in the amidation reaction solution, as the primary amine having a linear alkyl group, a primary amine having 10 or fewer carbon atoms is preferable, a primary amine having 7 or fewer carbon atoms is more preferable, and methylamine, ethylamine, propylamine, butylamine, and pentylamine are further preferable, with methylamine being most preferable. When the amine contained in the amidation reaction solution has a linear structure without a branch (hereinafter, a "branch" refers to a branch of a hydrocarbon chain) or has a small number of carbon atoms, it can be more efficiently amidated, and is thus preferable.

[0081] In the case where 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, the unsaturated chain hydrocarbon group more preferably contains an allyl group, and the amine most preferably is an allylamine. The amine contained in the amidation reaction solution can be a primary amine containing a hydroxyl group, in which case, an ethanolamine is preferred. Although examples in which the amine contained in the amidation reaction solution contains an allyl group or a hydroxyl group are shown in the Examples described later, the amine contained in the amidation reaction solution is not particularly limited to these examples, and can contain various functional groups other than an alkyl group. As a result of the sugar chain amidation reaction, the sugar chain is modified to contain such a functional group, so that the sugar chain subjected to the modification is more easily separated not only by mass spectrometry but also by chromatography and the like.

[0082] In addition, the amidation reaction solution can contain a salt of the above-described amine.

[0083] (Concentration of the amidation reaction solution)

[0084] The concentration of ammonia, amines, and salts thereof in the amidation reaction solution is preferably 0.1 M (M is mol / l) or more, more preferably 0.3 M or more, further preferably 0.5 M or more, still further preferably 1.0 M or more, and most preferably 3.0 M or more. As a suitable example, the concentration of ammonia or a primary amine, particularly a methylamine, is preferably 0.1 M or more, more preferably 0.3 M or more, further preferably 0.5 M or more, still further preferably 1.0 M or more, and most preferably 3.0 M or more. The higher the concentration of the amidation reaction solution, the more reliably the amidation of the lactone can be performed.

[0085] (Solvent of the amidation reaction solution)

[0086] The solvent of the amidation reaction solution can be an aqueous solvent or an organic solvent, but from the viewpoint of preventing the hydrolysis of the lactone and reliably causing rapid amidation, a solvent having a small amount of water content is preferred. The solvent of the amidation reaction solution is preferably a dehydrated solvent to which a dehydration operation for suppressing the water content has been applied, and further preferably is a water-free solvent. The solvent of the amidation reaction solution preferably contains at least one of methanol and acetonitrile.

[0087] In addition, the amidation reaction solution can contain an appropriate amount of water, and the solvent of the amidation reaction solution can be water.

[0088] (pH of the amidation reaction solution)

[0089] The pH of the amidation reaction solution is preferably 7.7 or higher, more preferably 8.0 or higher, further preferably 8.8 or higher, and most preferably 10.3 or higher. If the pH of the amidation reaction solution is high, the lactone is more reliably amidated, and thus this is preferred.

[0090] (Time for causing the amidation reaction)

[0091] The amidation reaction is completed within several seconds to several minutes. Therefore, in order to amidate the lactone by the amidation reaction, the time for which the sample is contacted with the amidation reaction solution (hereinafter referred to as the reaction time) is preferably less than 1 hour, more preferably less than 30 minutes, further preferably less than 15 minutes, still further preferably less than 5 minutes, and most preferably less than 1 minute. It is also suitable to merely wash the sample with the amidation reaction solution, or to temporarily pass the sample held on a carrier or the like with the solution. Furthermore, the time from the end of the contact of the sample with the lactonization reaction solution until the end of the contact of the sample with the amidation reaction solution is preferably less than 1.5 hours, more preferably less than 1 hour, and further preferably less than 30 minutes. In this way, the amidation reaction is completed in a short time, and thus it is possible to prevent the decomposition of the unstable lactone and impair the quantitative nature of the sugar chain analysis. Furthermore, by setting the reaction time of the amidation reaction to be short, it is possible to more efficiently perform the analysis of the sample.

[0092] (Regarding the state of the sample in the amidation reaction)

[0093] The amidation reaction can be performed in a liquid phase, or can be performed in a solid phase. The state of the sample at the time of causing the amidation reaction is not particularly limited as long as the sample can be contacted with the amidation reaction solution, but it is preferred that the sugar chain contained in the sample be contacted with the amidation reaction solution in a state of being bound to or adsorbed on a solid-phase carrier.

[0094] As in the case of the lactonization reaction, in the case of performing the reaction in a solid phase, as the solid-phase carrier, any carrier can be used without particular limitation as long as it is a carrier that can immobilize a sugar chain, a glycopeptide, a glycoprotein, or the like. For example, in order to immobilize a glycopeptide or a glycoprotein, a solid-phase carrier having an epoxy group, a tosyl group, a carboxyl group, an amino group, or the like as a ligand can be used. Furthermore, in order to immobilize a sugar chain, a solid-phase carrier having a hydrazide group or an aminooxy group, or the like as a ligand can be used. Furthermore, from the viewpoint of ionization efficiency after amidation and the like, it is also preferred that the sugar chain be adsorbed on a carrier for hydrophilic interaction chromatography (hereinafter referred to as HILIC), that is, a stationary phase, and the carrier for HILIC further preferably contains an amide group.

[0095] After the amidation reaction solution is allowed to act on the sample fixed to the solid phase carrier to perform amidation, the sample is only required to be released from the carrier and recovered by chemical methods or enzyme reactions, etc. For example, the glycoprotein or glycopeptide fixed to the carrier can be digested and recovered by a glycosidase such as PNGase F or a digestive enzyme such as trypsin, or the sugar chain bound to the solid phase carrier having a hydrazide group can be released and recovered by a weak acid solution. In HILIC, the amidation reaction solution using acetonitrile or the like as a solvent can be used for the amidation reaction, and a water-based solution such as water can be used for elution of the sample.

[0096] The reaction is performed in a state in which the sample is fixed to the solid phase carrier, and thus removal or desalting purification of the reaction solution becomes easier, and preparation of the sample can be simplified. In addition, in the case of using the solid phase carrier, the sample is fixed in a state of a glycoprotein or glycopeptide, and after the amidation reaction, if cleavage is performed by a glycosidase such as PNGase F or the like, the sample after the amidation reaction can also be recovered as a free sugar chain.

[0097] In addition, the sample after the lactonization can also be subjected to the amidation reaction after purification and elution by HILIC. In order not to easily cause hydrolysis, it is preferable to appropriately adjust the solvent for washing the carrier for HILIC.

[0098] As a suitable example of the sample preparation method of the present embodiment, as shown in the examples, the present inventors have found that the sugar chain adsorbed on the solid phase carrier is subjected to ammonolysis.

[0099] By the above-described preparation method, in the case where the sugar chain in the sample contains α2,3-, α2,8-, and α2,9-sialic acids, in the lactonization reaction of step S1003, each of the sialic acids is lactonized, and the lactonized sialic acids are stabilized by the amidation reaction of step S1005.

[0100] If step S1005 is ended, step S1007 is entered.

[0101] In step S1007, the sample is analyzed by mass spectrometry and / or chromatography. By the above-described lactonization reaction and amidation reaction, the mass of the sugar chain that is difficult to be lactonized like α2,6-sialic acid is different from the mass of the sugar chain that can be lactonized such as α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid. Therefore, by mass spectrometry, these sugar chains can be separated based on the binding mode of the sialic acid.

[0102] The method of ionization in the mass spectrometric analysis is not particularly limited, and matrix-assisted laser desorption ionization (MALDI) method, electrospray ionization (ESI) method, nano-ESI method, and the like can be used. The method of ionization is particularly preferably the MALDI method. In the ionization in the mass spectrometric analysis, either of the positive ion mode and the negative ion mode can be used. The mass spectrometric analysis can be performed in multiple stages, whereby the structure of the sugar chain other than the binding mode of the sialic acid, the structure of the peptide chain can be analyzed.

[0103] Further, analysis can be performed using an analysis method other than the mass spectrometric analysis, such as chromatography, based on the properties of the modified body produced as a result of the lactonization reaction and the amidation reaction. The column for liquid chromatography is not particularly limited, and a hydrophobic reversed-phase column such as C30, C18, C8, C4, a carbon column, a normal-phase column for HILIC, and the like can be appropriately used. After the liquid chromatographic analysis, in order to more precisely perform analysis of the components in the sample, it is preferable to perform measurement by mass spectrometric analysis. In this case, it is more preferable to ionize the eluate from the liquid chromatograph directly in the mass spectrometric analyzer by online control.

[0104] If the step S1007 ends, the process ends.

[0105] (Regarding side reactions of glycopeptides and glycoproteins)

[0106] In the case where the lactonization reaction solution and the amidation reaction solution are added to the glycopeptide or the glycoprotein, and the sialic acid is modified as described above, a side reaction such as intramolecular dehydration condensation can sometimes occur between the side chain of the amino acid contained in the glycopeptide or the glycoprotein, or between the amino group or the carboxyl group located at the end of the main chain. In this case, there is a problem that the peak of the mass spectrum corresponding to the sugar chain of the analysis object is separated, and the analysis becomes difficult.

[0107] The inventors and others have clarified that the side reactions of the peptide moiety mainly originate from the presence of an amino group, and have clarified that the side reactions of the peptide moiety at the time of sialic acid modification can be inhibited by end-capping the amino group in advance by chemical modification or the like before sialic acid modification. For details, refer to the following literature: Katsunori Nishikawa, Masaru Sekiya, Shinya Iwamoto, Koji Tanaka, "A Universal Approach to linkage-Specific Derivatization for Sialic Acids on Glycopeptides", American Society for Mass Spectrometry, June 2017, Vol. 28, No. 1 Supplement, Poster No. MP091. The modification based on the amidation reaction of the present embodiment can also be used for glycopeptides and glycoproteins in the same manner as this. That is, the amino group of the glycopeptide or glycoprotein is end-capped by a reaction such as dicarboxamidation or guanidination, followed by a lactonization reaction, followed by rapid amidation based on the amidation reaction. At this time, if a method of forming a lactone according to the linkage of sialic acid is used, the linkage of sialic acid can also be recognized.

[0108] In addition, there are glycopeptides in which side reactions are difficult to occur on the basis of the characteristics of the amino acid sequence. For example, glycopeptides generated by digesting the Fc region of IgG with a digestive enzyme such as trypsin do not have lysine, and the amino group at the N terminus is also rapidly cyclized and dehydrated in the presence of a dehydration condensing agent to be pyroglutamylated. As a result, since there is no amino group, end-capping of the amino group in advance such as dicarboxamidation or guanidination is not necessary. With respect to such glycopeptides, the lactonization reaction is performed without end-capping of the amino group, and the lactone generated is amidated by the amidation reaction, and a mass spectrum sufficient for analysis can be obtained.

[0109] [Examples]

[0110] Hereinafter, examples of the present embodiment will be shown, but the present application is not limited to the following examples. In addition, in the following content, unless otherwise specified, the notation of % indicates weight %.

[0111] Study of the concentration of amine in the amidation reaction

[0112] As a sample, a substance in which a sugar chain was cleaved from a sialic acid glycopeptide (a2, 3-SGP; FUJIFILM Wako Pure Chemical Corporation) to which a2, 3-sialic acid was bound by PNGase F was used. The sialic acid glycopeptide is a peptide in which a sugar chain is bound to a plurality of residues. The sample was bound to a solid-phase carrier (BlotGlyco; Sumitomo 3M) composed of magnetic beads having a hydrazide group as a ligand. The binding of the sugar chain to the solid-phase carrier was performed in accordance with the standard experimental protocol of the sugar chain purification kit BlotGlyco.

[0113] The carrier after the binding of the sugar chain is washed 3 times with 200 μL of DMSO. Then, 100 μL of a lactonization reaction solution (2 M isopropylamine hydrochloride, 500 mM EDC-HCl, 500 mM HOBt) containing isopropylamine is added, and the reaction is allowed to proceed for 1 hour with gentle stirring at 800 rpm. (By this, α2, 6-sialic acid is converted to isopropylamide, and α2, 3-sialic acid is converted to a lactone body.) After the reaction solution is removed by centrifugation, 1 wash is performed using 200 μL of methanol. Then, 3 washes are performed using 200 μL of an aqueous methylamine solution (concentration: 0.1 to 10%), whereby an amidation reaction of the lactone is performed. Subsequently, 2 washes are performed using 200 μL of methanol, and 3 washes are performed using 200 μL of water. Then, the reacted sugar chain sample is freed from the carrier by a method according to a standard experimental protocol, desalted and purified using a StageTip Carbon, and dried and solidified by centrifugal concentration (SpeedVac (Thermo Fisher Scientific)). The StageTip Carbon is a carbon column in which an Empore Disk Carbon (3M) is cut to a diameter of about 1 mm and packed into a 200-μL tip. The dried and solidified sample is redissolved in 10 μL of water, 1 μL is dropped onto a focusing plate, 0.5 μL of a 100 mM 3AQ / CA solution and 2 mM ammonium sulfate dissolved in 50% acetonitrile (ACN) are added as a matrix, and a 3AQ-based labeling of the reducing end of the sugar chain is performed by allowing the reaction to proceed for 1.5 hours at 75°C in a micro heat block. After the reaction, the plate is cooled to room temperature, and time-of-flight mass spectrometry is performed by MALDI-QIT-TOF-MS (AXIMA-Resonance, Shimadzu / Kratos) in a negative ion mode.

[0114] Figure 2 is a graph showing the mass spectrum when 1% (a) and 10% (b) of an aqueous methylamine solution is used as an amidation reaction solution to cause ammonolysis. The α2, 3-A2-glycan released from the sample α2, 3-SGP is converted to a lactone body by the intramolecular dehydration condensation with the lactonization reaction solution described above. Here, the hydrazide magnetic beads are washed only with the methylamine solution without using a dehydration condensing agent thereafter, but it is known that formamidation of the original lactone structure occurs (peak corresponding to m / z 2471.9). The peak observed at m / z 2360.9 is a peak in which methylamine is not bound and a carboxyl group remains. It is presumed that this peak corresponds to a sugar chain in which hydrolysis occurs and not ammonolysis of the lactone. In the amidation reaction using a 10% aqueous methylamine solution, the peak from the hydrolysis further decreases, and almost complete ammonolysis occurs.

[0115] Figure 3is a graph showing the ratio of sialic acid that has undergone hydrolysis to sialic acid that has undergone ammonolysis (ammonolysis efficiency) calculated from the signal intensity of peaks on a mass spectrum with respect to the concentration of methylamine in the amidation reaction solution. It is known that sufficient ammonolysis is induced even in the case where the concentration of the methylamine solution is 1%, but by using an amidation reaction solution containing methylamine at a higher concentration, more efficient ammonolysis can be induced.

[0116] <Research on the kind of amine in the amidation reaction>

[0117] Figure 4 is a graph showing the generation ratio when the amidation reaction was performed under approximately the same conditions as the above-described research on the amine concentration, using 3M aqueous ammonia or an aqueous alkylamine solution (in the case of methylamine, corresponding to a 10% concentration) as the amidation reaction solution, respectively.

[0118] If the results of Figure 4 are observed, in the case where there is no ammonia and no branched alkyl primary amine, although ammonolysis is efficiently induced, the ammonolysis efficiency of alkyl amines having a branch such as isopropylamine and tert-butylamine is low, and hydrolysis (generation of -COOH) predominates. Furthermore, in the case where a tertiary amine is used in the amidation reaction solution, it does not react even under the dehydration condensing agent, and thus only hydrolysis occurs, and in the case where a secondary amine is used, it also hardly reacts, and hydrolysis predominates. Furthermore, in the case of allylamine and ethanolamine, ammonolysis predominates. It is thus known that as long as the primary amine has no branch at least in the hydrocarbon chain portion, it can contain other functional groups, and can contain a double bond or a hydroxyl group. From the above, it is known that in order to induce ammonolysis, a primary amine having no branch on the carbon chain is particularly suitable.

[0119] <Research on the solvent in the amidation reaction>

[0120] Figure 5 is a graph showing the generation ratio when 1.2M methylamine dissolved in 90% ACN, 3M methylamine dissolved in methanol, or 3M ethanolamine dissolved in ACN, etc. were used as the amidation reaction solution under approximately the same conditions as the above-described research.

[0121] If the results of Figure 5 are observed, it is observed that in all cases, ammonolysis is induced at a high ratio, and the peak corresponding to the sugar chain that is amidated predominates. Even in the case of amine dissolved in methanol or ACN under a state where water is substantially not contained, amidation is induced without problems, and thus it is strongly suggested that the lactone is not amidated after temporary hydrolysis, but the amidation is induced by the direct action of the amine on the lactone. Under the condition where water is substantially not contained, hydrolysis is further suppressed, and even in the case where ethanolamine, which hydrolyzes sialic acid at around 5% in an aqueous solvent, is used as the amidation reaction solution, it is almost completely amidated.

[0122] <Study on pH in amidation reaction>

[0123] Figure 6 This is a graph showing the production ratio when a solution containing an aqueous solution of 3M methylamine and an aqueous solution of 3M methylamine hydrochloride in any proportion is used as the amidation reaction solution under conditions roughly the same as those described in the above studies. Figure 6 "MA" indicates an aqueous solution of methylamine, and "MA-HCl" indicates an aqueous solution of methylamine hydrochloride.

[0124] In this study, it was assumed that even after the amidation reaction solution was added to the sample, α2,3-sialic acid would remain in a lactone state under certain conditions. Therefore, to further quantitatively evaluate the unstable lactone, after the amidation reaction solution was added to the sample for amidation, it was washed twice with 200 μL of H2O and twice with 200 μL of ACN. Then, the amidation reaction was carried out again using an amidation reaction solution containing 3M ethanolamine dissolved in ACN. Under the conditions of the amidation reaction carried out in these two stages, it was possible to clearly distinguish the amidated substance (detected as the formamide), the hydrolyzed substance (detected as -COOH), and the substance remaining in the lactone state (detected as the amidated substance with ethanolamine) in the first stage of the amidation reaction. In addition, the amidation reaction in this study was not carried out by washing three times with 200 μL of amidation reaction solution as in the previous studies, but by adding 100 μL of amidation reaction solution and stirring at 700 rpm for 2 minutes.

[0125] If observe Figure 6 As a result, without the addition of an amidation reaction solution (“without aminolysis”) or using a 3M methylamine hydrochloride solution (pH 4.7) as the amidation reaction solution, almost no ammonolysis occurs, and sialic acid remains essentially entirely in the lactone state. If the proportion of methylamine solution in the preparation of the amidation reaction solution is increased to raise the pH, sialic acid undergoes slow hydrolysis and ammonolysis. At pH 8.8, approximately 90% of the sialic acid is amidated, and in amidation reaction solutions with pH values ​​above 10.3, virtually all of the sialic acid is amidated.

[0126] <Study on amidation reaction using fetuin-free glycans as samples>

[0127] The fetuin, which is a glycoprotein, was denatured / reduced by dissolving it in 20 mM ammonium bicarbonate, 10 mM DTT, 0.02% SDS, and treating it at 100°C for 3 minutes. Then, after cooling to room temperature, the sugar chain was liberated by adding PNGase F and incubating overnight at 37°C. The next day, the PNGase F was inactivated by heat treatment at 100°C for 3 minutes, thereby stopping the enzymatic reaction.

[0128] The liberated sugar chain was subjected to the same binding on hydrazide magnetic beads and specific modification using a lactonization reaction solution containing isopropylamine as in the above-described study, followed by an amide reaction based on 10% aqueous methylamine. Elution from the magnetic beads or detection using mass spectrometric analysis was performed using the same method as in the above-described study.

[0129] Figure 7 The mass spectrum is of the sugar chain liberated from fetuin. In the numbers indicated in correspondence with each peak, the number on the left indicates the number of α2, 3-sialic acids contained in the molecule corresponding to the peak, and the number on the right indicates the number of α2, 6-sialic acids contained in the molecule. It is known that: no product or unreacted body resulting from hydrolysis was detected, and the sialic acid subjected to lactonization was efficiently formamidated. If the mass spectrum is compared with the mass spectrum reported in Patent Literature 1 or Non-Patent Literature 1, it is known that even without performing an amide reaction using a dehydration condensing agent, the lactone is efficiently directly formamidated by ammonolysis.

[0130] <Study of the Amide Reaction Using a Sugar Lipid-Type Sugar Chain as a Test Sample>

[0131] As a sample, Human Disialoganglioside (bisanionyl ganglioside) GD1a and GD1b (HyTest) were used as glycosphingolipids. After dissolving the above glycolipids in 45 μL of 50 mM sodium acetate buffer (pH 5.5) containing 0.2% Triton x100, and leaving them at 60°C for 20 minutes, Endoglycoceramidase I (a substance purified from Actinomyces based on the following reference; Ishibashi Y, Nakasone T, Kiyohara M, Horibata Y, Sakaguchi K, Hijikata A, Ichinose S, Omori A, Yasui Y, Imamura A, Ishida H, Kiso M, Okino N, and Ito M. A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. Journal of Biological Chemistry, 2007, Vol. 282, pp. 11386-11396) was added, and a sugar chain elimination reaction was performed at 37°C for 16 hours.

[0132] The free sugar chain of the glycolipid type was bound to a hydrazide carrier, the remaining hydrazide group was capped, and then a sialic acid binding mode-specific modification was performed using isopropylamine, in the same manner as the above study. After the amidation reaction, the hydrazide carrier was washed 3 times with 200 μL of 1% aqueous methylamine, 3 times with 200 μL of DMSO, 3 times with 200 μL of methanol, and 3 times with 200 μL of water. Then, the reducing end of the sugar chain was labeled using a high-sensitivity reagent, aoWR, while being freed from the carrier. Then, the excess reagent was removed using a HILIC carrier, 2,5-dihydroxybenzoic acid (2,5-DHB) was used as a matrix, the sample was ionized by MALDI, and mass spectrometry was performed by time-of-flight mass spectrometry (MALDI-TOF MS) in the positive ion mode.

[0133] Figure 8is a graph showing the mass spectrum of the obtained glycolipid-type sugar chain. The upper section is the mass spectrum of the sugar chain released from the glycosphingolipid disialo ganglioside GD1a, and the lower section is the mass spectrum of the sugar chain released from the disialo ganglioside GD1b. In the case of the GD1a sugar chain having α2,3-sialic acid at two places, a signal can be observed at m / z 1746. This corresponds to the mass of both of the α2,3-sialic acids being formylated. It is thus known that, by ammonolysis, the glycolipid-type sugar chain is also formylated via the lactone in the same manner. Furthermore, in the case of the GD1b type, a signal can also be observed at m / z 1746, both of the sialic acids being formylated, in the same manner as in the case of the GD1a type. The GD1b type has a linear polysialic acid structure having α2,3-sialic acid and α2,8-sialic acid bound to the α2,3-sialic acid, but since both of the sialic acids are formylated, it is suggested that even the lactone generated from the α2,8-sialic acid can be subjected to the ammonolysis of the present application without problems.

[0134]

[0135] The α2,3-SGP was dissolved in 20 mM ammonium bicarbonate, PNGase F was added, and incubation was performed at 37°C overnight, thereby releasing the sugar chain. The next day, heat treatment was performed at 100°C for 3 minutes to inactivate the PNGase F, thereby stopping the enzyme reaction. Then, desalting was performed using StageTip Carbon, and drying and solidification were performed in an Eppendorf tube using a SpeedVac.

[0136] Then, a lactonization reaction solution (2 M isopropylamine hydrochloride, 500 mM EDC-HCl, 500 mM HOBt) containing 20 μL of isopropylamine was added, and reaction was performed with stirring at 2000 rpm for 1 hour. (By this, the α2,6-sialic acid is converted to isopropylamide, and the α2,3-sialic acid is converted to a lactone body.) After that, dilution was performed using 120 μL of ACN, and addition was performed to a GLTip Amide (GL science), and centrifugation was performed at 4000 x g, thereby performing liquid passage, and the sugar chain was adsorbed to the carrier for HILIC containing an amide group. Then, 20 to 200 μL of a 90% ACN 4% methylamine solution was used as an amidation reaction solution, and liquid passage was performed, and thereby amidation reaction was performed. Further, washing was performed by passing 100 μL of 90% ACN 0.1% TFA twice, and finally, 20 μL of H2O was passed twice to elute the sugar chain, and the eluate was dried and solidified using a SpeedVac. Then, further desalting was performed using StageTip Carbon, on-target 3AQization was performed as in the above-described study, and mass spectrometric analysis was performed. ​

[0137] Figure 9 Figures (a) to (d) are graphs showing the amount of each amidation reaction solution and the ratio of products. In all cases (a) to (d), almost complete ammonolysis was confirmed. With the amidation reaction solution reduced to 20 μL, the contact time between the support and the amidation reaction solution was considered to be as long as tens of seconds, suggesting that the ammonolysis reaction occurred extremely rapidly.

[0138] <Study on the amidation reaction after purification using HILIC>

[0139] Following the same procedures as in the previous study, the sugar chain was cleaved from α2,3-SGP and released. A lactone reaction solution containing isopropylamine was added to the sugar chain to induce a reaction. The mixture was then diluted with ACN and adsorbed onto a HILIC support. The sugar chain was then eluted by passing 100 μL of 90% ACN and 0.1% TFA solution through the buffer twice, followed by two passes of 20 μL of H₂O. 6.7 μL of a 40% methylamine aqueous solution was added to prepare an amidation reaction solution with a final concentration of 10% methylamine. The solution was gently stirred and allowed to stand at room temperature for 2 minutes to initiate the amidation reaction. The solvent was then removed using a SpeedVac, and further desalted using a StageTipCarbon. Mass spectrometry analysis was performed using on-target 3AQ oxidation, as described in the previous study.

[0140] The results are shown in Figure 9 (e) The lactones in the sample were predominantly formamided, but products from hydrolysis were also detected. This can be explained by the fact that some of the lactones hydrolyzed during the washing of the support after the lactonization reaction solution containing isopropylamine was removed via the HILIC support. Therefore, while the amidation reaction solution can also be added to the sample eluted from the HILIC support, from the viewpoint of substantially causing ammonolysis of all lactones and maximizing reaction efficiency, it is preferable to add the amidation reaction solution while the sample is adsorbed onto the HILIC support.

[0141] This invention is not limited to the embodiments described above. Other methods conceived within the scope of the technical concept of this invention are also included within the scope of this invention.

Claims

1. A method for preparing a sample, characterized in that, Includes the following steps: A lactone-forming reaction is carried out, wherein at least a portion of the sialic acid contained in the sugar chain is lactone-formed. and An amidation reaction is carried out by adding an amidation reaction solution to the sample, the amidation reaction solution containing ammonia that reacts with the lactone-modified sialic acid, thereby utilizing ammonolysis based on the interaction between amino and lactone to lactone amidate the lactone-modified sialic acid. The pH of the amidation reaction solution is above 7.

7. In the amidation reaction solution, the concentration of ammonia is 3M or higher. In the lactonelation reaction, at least one of the sialic acids selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid and α2,9-sialic acid is lactonelated.

2. The method for preparing the sample as described in claim 1, characterized in that, After the lactoneation reaction, the lactoneation reaction solution is removed from the sample.

3. The method for preparing the sample as described in claim 1 or 2, characterized in that, The amidation reaction is carried out solely by contacting the sample with the amidation reaction solution.

4. The method for preparing the sample as described in claim 1 or 2, characterized in that, The amidation reaction solution does not contain a dehydrating condensing agent that reacts with the lactone.

5. The method for preparing the sample as described in claim 1 or 2, characterized in that, To carry out the amidation reaction, the contact time between the sample and the amidation reaction solution shall be less than 30 minutes.

6. The method for preparing the sample as described in claim 1 or 2, characterized in that, The pH of the amidation reaction solution is above 8.

0.

7. The method for preparing the sample as described in claim 1 or 2, characterized in that, During the lactonization reaction, a lactonization reaction solution is added to the sample to lactonize a portion of the sialic acid contained in the sugar chain. The lactonization reaction solution contains a dehydrating condensing agent that reacts with the sialic acid contained in the sugar chain.

8. The method for preparing the sample as described in claim 7, characterized in that, The lactone reaction solution also contains a nucleophile that reacts with the sialic acid contained in the sugar chain. The mass of the nucleophile differs from the mass of the ammonia used in the amidation reaction. Based on the binding mode of the sialic acid, a portion of the sialic acid is lactone-ized, causing a portion of the nucleophile to bind to another portion of the sialic acid.

9. The method for preparing the sample as described in claim 8, characterized in that, α2,3-sialic acid is esterified, thereby binding a portion of the nucleophile to α2,6-sialic acid.

10. The method for preparing the sample as described in claim 1 or 2, characterized in that, When the sample comes into contact with the amidation reaction solution, the sample binds to or adsorbs onto the solid support.

11. The method for preparing the sample as described in claim 1 or 2, characterized in that, The amidation reaction solution contains an organic solvent.

12. An analytical method, characterized in that, Includes the following steps: The sample is prepared by the sample preparation method according to any one of claims 1 to 11; and the prepared sample is analyzed.

13. The analytical method as described in claim 12, characterized in that, The prepared sample was analyzed by at least one of mass spectrometry and chromatography.

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