Method for preparing analytical sample, analytical method, and kit for preparing analytical sample

Abstract

The present invention relates to a method for preparing a sample for analysis, an analysis method, and a kit for preparing a sample for analysis, and provides a method for modifying sialic acid by a new mechanism. The method for preparing a sample for analysis is a method for preparing a sample for analysis from a sample containing a sugar chain, and the preparation method comprises the following operations: performing an esterification reaction, wherein the esterification reaction subjects at least a portion of the sialic acid contained in the sugar chain to an esterification other than lactonization; contacting the sample with an amidation reaction solution, and performing an amidation reaction, wherein the amidation reaction solution contains at least one compound selected from the group consisting of ammonia, amines, hydrazine, hydrazine derivatives, and hydroxylamine and salts thereof that reacts with the sialic acid modified by esterification, and the amidation reaction converts the esterification modification of the sialic acid modified by esterification into an amidation modification.

Description

Technical Field

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

[0002] Sialic acid is a sugar that exists in large quantities in living organisms. Sialic acid is also contained in sugar chains that bind to proteins in living organisms, and is mostly present at the non-reducing ends of sugar chains. Therefore, sialic acid plays an important role because it is located outside the molecule in such glycoprotein molecules and can be directly recognized by other molecules.

[0003] The linkage type between sialic acid and adjacent sugars may be different. For example, it is known that human N-linked sugar chains (N-type sugar chains) are mainly α2,3- and α2,6-linked, and O-linked sugar chains (O-type sugar chains) and glycosphingolipids have α2,8- and α2,9-linked in addition to the aforementioned linkage types. Depending on the difference in this linkage type, sialic acid can be recognized by different molecules and has different effects.

[0004] In mass spectrometry, sialic acid modification is performed as a pretreatment for sugar chains containing sialic acid. The carboxyl group of sialic acid with a negative charge is neutralized by esterification or amidation, thereby overcoming the shortcomings of inhibiting ionization and sialic acid detachment. Regarding the lactonization of sialic acid, the stability of the lactone generated by the connection mode is different, so the difference in stability can be used to modify and analyze sialic acid specifically by the connection mode.

[0005] Here, lactone is extremely unstable, is also easily hydrolyzed in water, and is hydrolyzed more rapidly under acidic or alkaline conditions. Therefore, it is reported that the lactone generated by the modification in the pre-treatment is stabilized by amidation (with reference to patent documentation 1, non-patent documentation 1 and non-patent documentation 2). Suitably, the cyclic structure in the molecule generated by lactonization is referred to as lactone structure. Lactone structure is also present in the sugar chain of the sugar chain of the sugar chain of the antibody drug in the organism, and can also be stabilized when performing their analysis. In addition, the direct amidation of the lactone recorded in non-patent documentation 2 can carry out the rapid modification of lactone, and future utilization is expected.

[0006] Lactonization is a type of esterification. In the modification of sialic acid, esterification other than lactonization is also performed. In non-patent document 3, a dehydration condensation agent 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium hydrochloride (DMT-MM) is added to the free sugar chain dissolved in methanol. Thus, α2,6-sialic acid is methylated and α2,3-sialic acid is lactonized.

[0007] In Non-Patent Document 4, a dehydration condensation agent such as N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) dissolved in ethanol or the like is added to free sugar chains, thereby α2,6-sialylation and α2,3-sialic acid lactonization.

[0008] In non-patent document 5, a solution containing ethanol and a dehydrating agent is added to a glycoprotein bound to a solid phase carrier to esterify α2,6-sialylate and lactonize α2,3-sialic acid. Then, as shown in Scheme 1(b) of non-patent document 5, in order to hydrolyze the lactone, a Tris buffer at pH 10 is added to the sample and reacted for 1 hour, and then a solution containing methylamine hydrochloride is added to the sample, and a dehydrating agent is further added thereto and reacted for 30 minutes.

[0009] In Non-Patent Document 6, ethanol containing EDC hydrochloride and HOBt is added to a glycoprotein bound to a solid phase carrier to acylate α2,6-sialylate, and then a p-toluidine solution is added to the sample together with a dehydrating condensation agent to amidate α2,3-sialic acid.

[0010] In Non-Patent Document 7, ethanol containing EDC hydrochloride and HOBt is added to a glycoprotein bound to a solid phase carrier to acylate α2,6-sialylate, and then an ethylenediamine solution is added to the sample together with a dehydrating condensation agent to amidate α2,3-sialic acid.

[0011] In Non-Patent Document 8, ethanol containing EDC and HOBt is added to sugar chains to esterify α2,6-sialylate and lactonize α2,3-sialylolactonize, and then aqueous ammonia is added and reacted in the presence of a dehydrating condensation agent to hydrolyze the lactone and amidate it.

[0012] Prior art literature

[0013] Patent Literature

[0014] Patent Document 1: Japanese Patent No. 6135710

[0015] Non-patent literature

[0016] Non-patent document 1: Nishikaze T, Tsumoto H, Sekiya S, Iwamoto S, Miura Y, Tanaka K. "Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling" Analytical Chemistry, (USA), ACS Publications, February 21, 2017, Volume 89, Issue 4, pp. 2353-2360

[0017] Non-patent document 2: Hanamatsu H, Nishikaze T, Miura N, Piao J, Okada K, Sekiya S, Iwamoto S, Sakamoto N, Tanaka K, Furukawa JI. "Sialic Acid Linkage Specific Derivatization of Glycosphingolipid Glycans by Ring-Opening Aminolysis of Lactones" Analytical Chemistry, (USA), ACS Publications, October 29, 2018, Volume 90, Issue 22, pp. 13193-13199

[0018] Non-patent literature 3: Wheeler SF, Domann P, Harvey DJ. "Derivatization of sialicacids for stabilization in matrix-assisted laser desorption / ionization massspectrometry and concomitant differentiation of alpha(2-->3)-and alpha(2-->6)-isomers" Rapid communications in mass spectrometry, (UK), John Wiley AndSons Ltd., January 2009, Volume 23,Issue 2,pp.303-12

[0019] Non-patent literature 4: Reiding KR, Blank D, Kuijper DM, Deelder AM, Wuhrer M. "High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification" Analytical Chemistry, (USA), ACSPublications, June 2014, Volume 86, Issue 12,pp.5784-93

[0020] Non-patent document 5: Li H, Gao W, Feng X, Liu BF, Liu X. "MALDI-MS analysis of sialylated N-glycan linkage isomers using solid-phase two step derivatization method," Analytica Chimica Acta, (Netherlands), Elsevier BV, June 14, 2016, Volume 924, pp. 77-85

[0021] Non-patent document 6: Yang S, Jankowska E, Kosikova M, Xie H, Cipollo J. "Solid-Phase Chemical Modification for Sialic Acid Linkage Analysis: Application to Glycoproteins of Host Cells Used in Influenza Virus Propagation" Analytical Chemistry, (USA), ACS Publications, September 2017, Volume 89, Issue 17, pp. 9508-17

[0022] Non-patent document 7: Yang S, Wu WW, Shen RF, Bern M, Cipollo J "Identification of Sialic Acid Linkages on Intact Glycopeptides via Differential Chemical Modification Using Intact GIG-HILIC" Journal of the American Society for Mass Spectrometry., (USA), Springer, April 12, 2018, Volume 29, Issue 6, pp. 1273-1283

[0023] Non-patent document 8: Lageveen-Kammeijer GSM, de Haan N, Mohaupt P, Wagt S, FiliusM, Nouta J, Falck D, Wuhrer M. "Highly sensitive CE-ESI-MS analysis of N-glycans from complex biological samples" Nature communications, (UK), NaturePub.Group, May 13, 2019, Volume 10, Issue 1,p.2137 Summary of the invention

[0024] Problem that the invention aims to solve

[0025] It is desired to propose a new method for modifying sialic acid contained in a sugar chain in order to analyze sialic acid contained in a sugar chain.

[0026] Solutions for solving problems

[0027] A first aspect of the present invention relates to a method for preparing an analytical sample, which is a method for preparing an analytical sample from a sample containing sugar chains, the method comprising the following operations: performing an esterification reaction, wherein at least a portion of sialic acid contained in the sugar chains is subjected to esterification other than lactonization; and contacting the sample with an amidation reaction solution to perform an amidation reaction, wherein the amidation reaction solution contains at least one compound selected from the group consisting of ammonia, amines, hydrazine, hydrazine derivatives, hydroxylamine, and salts thereof that reacts with the sialic acid modified by the esterification, wherein the amidation reaction converts the esterification modification of the sialic acid modified by the esterification into an amidation modification.

[0028] A second aspect of the present invention relates to an analysis method comprising the steps of: preparing a sample by the analysis sample preparation method of the first aspect; and analyzing the prepared analysis sample.

[0029] A third aspect of the present invention relates to a kit for preparing an analytical sample, which is used for the method for preparing an analytical sample according to the first aspect.

[0030] Effects of the Invention

[0031] According to the present invention, a method for modifying sialic acid by a new mechanism for analyzing sialic acid contained in a sugar chain can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a flowchart showing the flow of an analysis method according to one embodiment.

[0033] Figure 2 This is a conceptual diagram showing a kit for preparing an analytical sample.

[0034] Figure 3 It is a flowchart which shows the flow of the analysis method of a modification example.

[0035] Figure 4A It is a conceptual diagram showing the structure of sugar chain GD1a.

[0036] Figure 4B It is a conceptual diagram showing the structure of sugar chain GD1b.

[0037] Figure 4C It is a conceptual diagram showing the structure of sugar chain A2GN1.

[0038] Figure 5 It is a flowchart which shows the flow of the analysis method of a modification example.

[0039] Figure 6 This is a conceptual diagram showing the structure of sugar chains used in Examples.

[0040] Figure 7 The figures show the mass spectrum of the analytical sample prepared by performing a methyl esterification reaction but not performing an amidation reaction (a); and the mass spectrum of the analytical sample prepared by performing an amidation reaction after a methyl esterification reaction using a 1% (b), 10% (c) and 40% (d) methylamine aqueous solution, respectively.

[0041] Figure 8 The diagram shows mass spectra of analytical samples prepared by performing methyl esterification reaction followed by amidation reaction using 5.6% ammonia water (a), 10% methylamine solution (b), 17.5% ethylamine solution (c), 16.7% dimethylamine solution (d), and 20% trimethylamine solution (e).

[0042] Fig. 9 The figures show the mass spectrum of the analytical sample (a) prepared by performing an amidation reaction using a 5% methylamine aqueous solution after a methyl esterification reaction; and the mass spectrum of the analytical sample prepared by performing an amidation reaction using a 6% methylamine solution using methanol (b), dimethyl sulfoxide (c) and acetonitrile (d) as solvents, respectively.

[0043] Fig.10 The diagram shows a mass spectrum (a) of an analytical sample prepared by performing an amidation reaction using a 17.5% ethylamine solution after a methyl esterification reaction, and a mass spectrum (b) of an analytical sample prepared by performing an amidation reaction using a 10% methylamine solution after a ethyl esterification reaction.

[0044] Fig.11 The diagram shows a mass spectrum of a sample for analysis prepared by subjecting a sample containing GD1a sugar chains to a methyl esterification reaction without subjecting it to an amidation reaction (a), a mass spectrum of a sample for analysis prepared by subjecting a sample containing GD1a sugar chains to a methyl esterification reaction and then subjecting it to an amidation reaction using a 10% methylamine solution (b), a mass spectrum of a sample for analysis prepared by subjecting a sample containing GD1b sugar chains to a methyl esterification reaction without subjecting it to an amidation reaction (c), and a mass spectrum of a sample for analysis prepared by subjecting a sample containing GD1b sugar chains to a methyl esterification reaction and then subjecting it to an amidation reaction using a 10% methylamine solution (d).

[0045] Fig.12 This is a conceptual diagram showing the structure of sugar chains used in Examples.

[0046] Fig.13 The figures show the mass spectrum of the analytical sample (a) prepared by methyl esterification reaction of N-type sugar chains derived from serum glycoproteins without amidation reaction; and the mass spectrum of the analytical sample prepared by amidation reaction using 5% (b), 10% (c), 20% (d) and 40% (e) ethylamine solutions (liquid passing by vacuum suction) respectively after methyl esterification reaction.

[0047] Fig.14 The figures show the mass spectrum of the analytical sample (a) prepared by methyl esterification reaction of N-type sugar chains derived from serum glycoproteins without amidation reaction; and the mass spectrum of the analytical sample prepared by amidation reaction using 5% (b), 10% (c), 20% (d) and 40% (e) ethylamine solutions (through natural falling) respectively after methyl esterification reaction.

[0048] Fig.15The figures show the mass spectrum of the analytical sample prepared by methyl esterification reaction without amidation reaction on N-type sugar chains derived from serum glycoprotein (a); and the mass spectrum of the analytical sample prepared by amidation reaction in liquid phase using 2.5% (b), 5% (c), 10% (d), 20% (e) and 40% (f) ethylamine solutions after methyl esterification reaction.

[0049] Fig.16 The figures are a mass spectrum of the analytical sample prepared by performing an ethyl esterification reaction on the GD1a sugar chain and the A2GN1 sugar chain without performing an amidation reaction (a); and a mass spectrum of the analytical sample prepared by performing an ethyl esterification reaction, setting the solvent to acetonitrile, and performing an amidation reaction using a methylamine solution (b), an ethylamine solution (c), and a propylamine solution (d), respectively.

[0050] Fig.17 The mass spectrum of the analytical sample (e) prepared by performing an ethyl esterification reaction on the GD1b sugar chain and the A2GN1 sugar chain without performing an amidation reaction; and the mass spectrum of the analytical sample prepared by performing an ethyl esterification reaction, setting the solvent to acetonitrile, and performing an amidation reaction using a methylamine solution (f), an ethylamine solution (g), and a propylamine solution (h), respectively.

[0051] Fig.18 The diagram shows the MS / MS spectrum of an analytical sample prepared by subjecting the sugar chains of GD1a(a) and GD1b(b) to ethyl esterification and then subjecting the sugar chains to amidation reaction using acetonitrile as the solvent and a propylamine solution.

[0052] Fig.19 The diagram shows mass spectra of analytical samples prepared by subjecting GD1a sugar chains to ethyl esterification reaction and then subjecting the GD1a sugar chains to amidation reaction using acetonitrile as the solvent and propylamine solutions of 0 M, 0.125 M, 0.25 M, 0.5 M, 1 M, and 3 M concentrations, respectively.

[0053] Fig. 20 The diagram shows mass spectra of analytical samples prepared by subjecting GD1b sugar chains to ethyl esterification reaction and then subjecting the GD1b sugar chains to amidation reaction using acetonitrile as the solvent and propylamine solutions of 0 M, 0.125 M, 0.25 M, 0.5 M, 1 M, and 3 M concentrations, respectively.

[0054] Description of Reference Numerals

[0055] 10 ... an esterifying agent, 11 ... a container, 100 ... a kit for preparing an analytical sample. DETAILED DESCRIPTION

[0056] Hereinafter, the mode for carrying out the present invention will be described with reference to the drawings. In the following embodiments, when "esterification" is described, it means esterification other than lactonization unless otherwise specified.

[0057] -First Embodiment-

[0058] Figure 1 The flowchart of the analysis method of the method for preparing an analysis sample of the present embodiment is shown. In the method for preparing an analysis sample of the present embodiment, after the sugar chains contained in the sample are sialylated, the esterified sialic acid linkage is specifically amidated. In step S1001, a sample containing sugar chains is prepared.

[0059] (About the sample)

[0060] The sample containing sugar chains is not particularly limited, and may contain at least one molecule selected from the group consisting of sugar chains, glycopeptides, glycoproteins, and glycolipids. Peptides and glycopeptides may have a peptide backbone formed by 2 or more and less than 50 amino acids, and proteins and glycoproteins may have a peptide backbone formed by more than 50 amino acids. However, there are also conventional exceptions, and the limits of the range of peptides and proteins and the limits of the range of glycopeptides and glycoproteins are not limited to this. In the method for preparing the analytical sample of the present embodiment, the connection mode of the sialic acid contained in the sugar chain is specifically modified. The sugar chain in the sample preferably contains N-linked sugar chains (N-type sugar chains) or O-linked sugar chains (O-type sugar chains), or glycolipid-type sugar chains, etc., which may have sialic acid at the end or other than the end. In addition, the sugar chain in the sample more preferably contains or may contain at least one of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid, and further preferably contains or may contain α2,6-sialic acid in addition to this.

[0061] In the case where the sample contains free sugar chains, sugar chains freed from glycoproteins, glycopeptides or glycolipids can be used. As the method for the freeing, enzyme treatment using N-glycosidase, O-glycosidase, or endoglycosphingolipid glycosidase (Endoglycoceramidase), hydrazine decomposition, β-detachment based on alkali treatment, etc. can be adopted. In the case where the N-linked sugar chains are freed from the peptide chains of glycopeptides and glycoproteins, enzyme treatment using peptide-N-glycosidase F (PNGase F), peptide-N-glycosidase A (PNGase A), or endo-β-N-acetylglucosaminidase (Endo M) can be appropriately used. In addition, modifications such as pyridine amination (PA labeling) of the reducing end of the sugar chain can be appropriately performed. Before the enzyme treatment, the peptide chain of the glycopeptide or glycoprotein described later can also be cut.

[0062] When the sample contains glycopeptides or glycoproteins, as described in the section "About the inhibition of side reactions of glycopeptides and glycoproteins" described later, a treatment for inhibiting side reactions of the peptide portion can be appropriately performed. In addition, glycopeptides or glycoproteins with a large number of amino acid residues in the peptide chain are preferably used by cutting the peptide chain by enzyme cutting or the like. For example, when preparing a sample for mass spectrometry analysis, the number of amino acid residues in the peptide chain is preferably 30 or less, more preferably 20 or less, and further preferably 15 or less. On the other hand, when it is required to clarify the source of the peptide to which the sugar chain is bound, the number of amino acid residues in the peptide chain is preferably 2 or more, more preferably 3 or more.

[0063] As the digestive enzyme when the peptide chain of glycopeptide or glycoprotein is cut, trypsin, lysine endopeptidase, arginine endopeptidase, chymotrypsin, pepsin, thermolysin, proteinase K, or pronase E etc. can be used. Two or more of these digestive enzymes can be used in combination. The conditions when cutting the peptide chain are not particularly limited, and appropriate regulations corresponding to the digestive enzyme used can be adopted. Before the cutting, the protein and peptide in the sample can be modified or alkylated. The conditions of the modification or alkylation are not particularly limited. The peptide chain can be cut by chemical cutting, etc. instead of enzyme cutting.

[0064] After step S1001 is completed, proceed to step S1003.

[0065] (Esterification reaction)

[0066] In step S1003, the sample is contacted with a reaction solution for esterification (hereinafter referred to as esterification reaction solution), and an esterification reaction (hereinafter recorded as esterification reaction, when not specifically mentioned, refers to the esterification reaction of step S1003) is performed to esterify at least a portion of the sialic acid contained in the sugar chain. The esterification reaction is a reaction in which the sialic acid contained in the sugar chain is supplied to an esterification other than lactonization. However, in the esterification reaction, as long as esterification other than lactonization occurs to a part of the sialic acid, lactonization of the sialic acid of the other part will not be hindered. In the following embodiments, "esterification other than lactonization" refers to that a part of the components of the esterification reaction solution is bonded to the carboxylic acid of sialic acid to form a carboxylate. That is, a part of the components equivalent to the esterification reaction solution is introduced into the R part of -COOR representing the carboxylate. In the esterification reaction, it is preferred that the sialic acid contained in the sugar chain is non-specifically esterified in a connection mode. In the esterification reaction, the carboxyl groups of α2,3-sialic acid, α2,6-sialic acid, α2,8-sialic acid and α2,9-sialic acid are preferably esterified.

[0067] The composition of the esterification reaction solution is not particularly limited as long as at least a portion of the sialic acid contained in the sugar chain, in particular, sialic acid such as α2,3-sialic acid, other than at least α2,6-sialic acid, is esterified. From the viewpoint of modification specific to the connection mode, it is preferred that α2,6-sialic acid is also esterified. The esterification reaction solution preferably contains at least one of an alcohol and an esterifying agent. However, the esterification method is not limited to a method using an alcohol or an esterifying agent, and any esterification method can be used.

[0068] (When the esterification reaction solution contains alcohol)

[0069] When the esterification reaction solution contains alcohol, the esterification reaction solution preferably further contains a condensing agent, and more preferably contains an additive in addition to the condensing agent. The esterification reaction solution may contain an alcohol such as methanol or ethanol and an acid such as hydrochloric acid.

[0070] The alcohol contained in the esterification reaction solution is not particularly limited, and any alcohol with any valence number may be used. The alcohol contained in the esterification reaction solution is preferably a monovalent alcohol, more preferably methanol, ethanol, propanol, butanol, pentanol or hexanol, and further preferably methanol or ethanol.

[0071] The condensing agent contained in the esterification reaction solution is not particularly limited and may include at least one of carbodiimide, carbonyldiimidazole, phosgene derivatives, phosphonium condensing agents, ureonium condensing agents, formamidinium condensing agents and trifluoromethanesulfonate reagents.

[0072] Examples of the carbodiimide in the condensing agent include: 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate metho-p-toluenesulfonate)(CMC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC), 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-diisopropylphenyl)carbodiimide, bis(trimethylsilyl)carbodiimide, and 1,3-bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)carbodiimide (BDDC).

[0073] Examples of the carbonyldiimidazole in the condensing agent include 1,1′-carbonyldiimidazole (CDI), 1,1′-carbonylbis(1,2,4-triazole) (CDT), and 1,1′-oxalyldiimidazole.

[0074] Examples of the phosgene derivative in the condensing agent include bis(pentafluorophenyl) carbonate, di-2-pyridyl carbonate, di(N-succinimidyl) carbonate, thiophosgene, triphosgene and O,O'-di-2-pyridylthiocarbonate.

[0075] Examples of the phosphonium dehydration condensation agent in the condensation agent include: (benzotriazol-1-yloxy) tris-(dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxy tris(pyrrolidinyl) phosphonium hexafluorophosphate (PyBOP), bromotris(dimethylamino) phosphonium hexafluorophosphate (BroP), bromotris(pyrrolidinyl) phosphonium hexafluorophosphate (PyBroP), (7-azabenzotriazol-1-yloxy) tris(pyrrolidinyl) phosphonium hexafluorophosphate (PyAOP) and chlorotrispyrrolidinyl phosphonium hexafluorophosphate (PyCloP). These are collectively referred to as "BOP reagents".

[0076] Examples of the uronium dehydration condensation agent in the condensation agent include: (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbonium hexafluorophosphate (COMU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) and O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU).

[0077] Examples of the trifluoromethanesulfonate reagent in the condensation agent include: 2-[N,N-bis(trifluoromethanesulfonyl)amino]-5-chloropyridine, N-(2-pyridine)bis(trifluoromethanesulfonimide), 4-nitrophenyl trifluoromethanesulfonate, N-phenylbis(trifluoromethanesulfonimide), trifluoromethanesulfonyl chloride, trifluoromethanesulfonic anhydride, trifluoromethanesulfonylaniline and 1-(trifluoromethanesulfonyl)imidazole.

[0078] In order to promote condensation using a condensing agent and suppress side reactions, it is preferred to use a highly nucleophilic additive in addition to the condensing agent. Examples of highly nucleophilic additives include 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt), 4-(dimethylamino)pyridine (DMAP), ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), N-hydroxy-succinimide (HOSu), 6-chloro-1-hydroxy-benzotriazole (Cl-HoBt), and N-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt).

[0079] (When the esterification reaction solution contains an esterifying agent)

[0080] The esterifying agent contained in the esterification reaction solution is not particularly limited. Among the esterifying agents, an alkylating agent is preferred, and a methylating agent, an ethylating agent, a propylating agent such as an isopropylating agent, a butylating agent such as a tert-butylating agent, or a benzylating agent is more preferred, and a methylating agent or an ethylating agent is further preferred.

[0081] Examples of the methylating agent in the esterifying agent include methyl bromide, dimethyl carbonate, dimethyl sulfate, N,N-dimethylformamide dimethyl acetal, N,N'-diisopropyl-O-methylisourea, methyl fluorosulfonate, methyl iodide, methyl methanesulfonate, 1-methyl-3-p-tolyltriazene, trimethyl orthoformate, tetramethylammonium chloride, methyl p-toluenesulfonate, tetramethylammonium hydroxide, 3-(trifluoromethyl)phenyltrimethylammonium hydroxide, trimethyloxonium tetrafluoroborate, trimethylsulfonium hydroxide and methyl trifluoromethanesulfonate.

[0082] Examples of the ethylating agent in the esterifying agent include ethyl bromide, diethyl carbonate, diethyl sulfate, N,N-dimethylformamide diethyl acetal, 1-ethyl-3-p-tolyltriazene, O-ethyl-N,N'-diisopropylisourea, ethyl iodide, ethyl methanesulfonate, triethyl orthoformate, ethyl trifluoromethanesulfonate and triethyloxonium tetrafluoroborate.

[0083] Examples of the propylating agent in the esterifying agent include: 1-bromopropane, 2-bromopropane, N,N-dimethylformamide dipropyl acetal, dipropyl sulfate, diisopropyl sulfate, 1-iodopropane, 2-iodopropane, 1-isopropyl-3-p-tolyltriazene, isopropyl methanesulfonate, triisopropyl orthoformate, propyl methanesulfonate and O,N,N'-triisopropylisourea.

[0084] Examples of the butylating agent in the esterifying agent include: 1-bromobutane, 2-bromo-2-methylpropane, 2-iodo-2-methylpropane, tert-butyl 2,2,2-trichloroacetimidate, O-tert-butyl-N,N'-diisopropylisourea, N,N-dimethylformamide dibutyl acetal, N,N-dimethylformamide di-tert-butyl acetal, dibutyl sulfate, 1-iodobutane and tributyl orthoformate.

[0085] Examples of the benzylating agent in the esterifying agent include benzyl bromide, benzyl chloride, 1-benzyl-3-p-tolyltriazene, benzyl 2,2,2-trichloroacetimidate, benzyl 2,2,2-trifluoro-N-phenylacetimidate, and O-benzyl-N,N′-diisopropylisourea.

[0086] Examples of the esterifying agent may include any alkylating agents other than those mentioned above.

[0087] The esterification reaction solution preferably contains a triazene derivative. The triazene derivative contained in the esterification reaction solution is preferably 1-methyl-3-p-tolyltriazene (MTT), 1-ethyl-3-p-tolyltriazene (ETT), 1-isopropyl-3-p-tolyltriazene, 1-benzyl-3-p-tolyltriazene or 1-(4-nitrobenzyl)-3-p-tolyltriazene, more preferably MTT or ETT.

[0088] The esterification reaction solution is not limited to the above-mentioned esterification agent, and may contain any esterification agent such as 2-chloroethyl methanesulfonate, N,N-dimethylformamide dineopentyl acetal, diamyl sulfate, etc. In addition, the esterification reaction solution may contain a salt of the above-mentioned esterification agent.

[0089] (Concentration of alcohol or esterification agent in esterification reaction solution)

[0090] In the esterification reaction, it is preferred to adjust the concentration of the alcohol or esterifying agent used so that at least one of α2,3-sialic acid, α2,8-sialic acid and α2,9-sialic acid is esterified. From the viewpoint of modifying the connection mode specifically, it is preferred to adjust the concentration of the alcohol or esterifying agent used so that α2,6-sialic acid is esterified. In addition, even if all the sialic acids of the sugar chains in the sample are esterified, the connection mode-specific modification can be performed, so as long as there is no obstacle in other aspects, the concentration of the alcohol, condensing agent or additive, or esterifying agent in the esterification reaction solution can be set higher.

[0091] As an example, when an esterification reaction is carried out using an esterification agent, the concentration of the esterification agent such as MTT or ETT is preferably 10mM to 10M, more preferably 50mM to 5M, and further preferably 100mM to 1M. When an alcohol is used for the esterification reaction, the concentration of the condensing agent can be, for example, 1mM to 5M. When a condensing agent and an additive are used in combination, the concentration of each can be set to the above range. The concentration of the alcohol is preferably 0.01 to 20M, more preferably 0.1M to 10M. The reaction temperature is preferably about -20°C to 100°C, more preferably -10°C to 50°C.

[0092] (Phase for esterification reaction)

[0093] The esterification reaction can be carried out in a liquid phase or a solid phase. The state of the sample during the esterification reaction is not particularly limited as long as the sample can be brought into contact with the esterification reaction solution.

[0094] When the reaction is carried out in a solid phase, as a solid phase carrier, as long as it can fix sugar chains, glycopeptides or glycoproteins, etc., it can be used without particular restrictions. For example, in order to fix glycopeptides or glycoproteins, a solid phase carrier having an epoxy group, a tosyl group, a carboxyl group, an amino group, etc. as a ligand can be used. In addition, in order to fix sugar chains, a solid phase carrier having a hydrazide group, an aminooxy group, etc. as a ligand can be used. In addition, it is also preferred that the sugar chain is adsorbed on a carrier, i.e., a stationary phase, for hydrophilic interaction chromatography (HILIC), and it is further preferred that the carrier for HILIC contains an amide group.

[0095] By reacting the sample while it is fixed on a solid phase carrier, the removal and desalting purification of the reaction solution become easier, and the preparation of the sample can be simplified. In addition, when the sample is fixed on a solid phase carrier in the state of a glycoprotein or a glycopeptide, if the sample is cut by a glycosidase such as PNGase F after the esterification reaction, the sample after the esterification reaction can also be recovered in the form of a free sugar chain. When the sugar chain is fixed on a solid phase carrier having a hydrazide group, an aminooxy group, etc. as a ligand, the sample after the esterification reaction can be freed by acid treatment, etc. to be recovered.

[0096] The sample after the esterification reaction may be purified, desalted, solubilized, or treated by a known method, etc., as necessary. The same applies before and after the amidation reaction described below.

[0097] When the sample is freed from the solid support after the esterification reaction, the conditions described for the amidation reaction described below can be used. By performing the reaction while the sample is fixed to the solid support, the removal of the esterification reaction solution after the esterification reaction becomes easy, and sialic acid modification can be performed efficiently.

[0098] After step S1003 is completed, proceed to step S1005.

[0099] (Amidation reaction)

[0100] In step S1005, the sample is contacted with a reaction solution (hereinafter referred to as an amidation reaction solution) to perform an amidation reaction, wherein the amidation reaction amidates the sialic acid modified by the esterification of step S1003 (hereinafter referred to as an amidation reaction, unless otherwise specified, it refers to the amidation reaction of step S1005). In the amidation reaction, the esterification modification of the sialic acid esterified in step S1003 is converted into an amidation modification (hereinafter appropriately referred to as esteramide conversion). In the amidation reaction, at least one of α2,3-sialic acid, α2,8-sialic acid and α2,9-sialic acid, in particular at least one of α2,3-sialic acid and α2,8-sialic acid is amidated.

[0101] The inventors have discovered a method for rapidly and directly amidating the ester formed in sialic acid, which is completely different from the common technical knowledge of amidating the carboxyl group of sialic acid by adding a solution containing a dehydrating condensation agent and an amine to a sample. Furthermore, surprisingly, the efficiency of the direct amidation is different when it is performed on sialic acids of a certain connection mode such as α2,3-sialic acid and α2,8-sialic acid and when it is performed on sialic acids of other connection modes such as α2,6-sialic acid. Therefore, by performing an amidation reaction on the sugar chains of these connection modes of sialic acids including esterification, it is possible to perform connection mode-specific modification.

[0102] In addition, as shown in Non-Patent Document 2, lactonized sialic acid can also be rapidly amidated using the amidation reaction solution of this embodiment. Therefore, even if α2,3-, α2,8- or α2,9-sialic acid is lactonized by the esterification reaction of step S1003, amidation occurs in the same manner as when esterification is performed using the amidation reaction solution. Therefore, before the amidation reaction, it is preferred not to perform an operation for cleaving the lactone structure generated by the esterification reaction.

[0103] For example, in the method of non-patent document 4, when methanol is used instead of ethanol in the esterification of α2,6-sialic acid in the first stage, the reaction rate of esterification is increased, so for α2,3-sialic acid, not only lactonization but also methyl esterification occurs, and it becomes indistinguishable from α2,6-sialic acid. In the method of this embodiment, in this case, both methyl esterified or lactonized sialic acid can be amidated. Therefore, in the method of this embodiment, the connection mode-specific modification of sialic acid can be performed more reliably.

[0104] In the following embodiments, when referring to a lactone formed in sialic acid, it refers to a lactone formed inside sialic acid, etc., in addition to a lactone formed between sialic acid and a monosaccharide adjacent to the sialic acid.

[0105] The amidation reaction solution contains: a reactant containing ammonia, amine or a salt thereof that reacts with the sialic acid modified by esterification (hereinafter referred to as an amidation reactant). The amidation reactant is used to perform amidation-based modification by bonding at least a portion of the amidation reactant to the sialic acid. The amidation reactant is, for example, a nucleophilic reagent. Preferably, the amidation reaction is performed by simply contacting the sample with the amidation reaction solution, and the lactone is stabilized by a simple operation.

[0106] It should be noted that, in the amidation reaction, the dehydration condensation agent is not necessary and may not be included, but the dehydration condensation agent may also be included in the amidation reaction solution. For example, ammonia, amine or their salts may be added to the sample without removing the esterification reaction solution added to the sample in step S1003, thereby preparing the amidation reaction solution. In this way, the amidation reaction can be carried out with a simple operation. Alternatively, after the operation for removing the esterification reaction solution added to the sample in step S1003, the amidation reaction is carried out only by contacting the sample with the amidation reaction solution. In addition, in the amidation reaction, after contacting the sample with the amidation reaction solution, the operation of reacting the sample with the dehydration condensation agent may not be carried out, but it may also be carried out for, for example, other purposes.

[0107] When the analytical sample obtained by the method for preparing the analytical sample of the present embodiment is analyzed by mass spectrometry, the alcohol or esterifying agent and the amidating agent contained in the esterification reaction solution are selected so that the mass of the modified body of the ester formed by the esterification reaction is different from that of the modified body of the amide formed by the amidation reaction. The alcohol or esterifying agent and the amidating agent contained in the esterification reaction solution are selected so that the mass separation of the two modified bodies obtained is performed with good accuracy according to the mass resolution of the mass spectrometry. In order to clearly distinguish the two modified bodies by a single mass spectrometry analysis, it is preferred to sufficiently set the mass difference by using a combination of methyl ester / acetamide, ethyl ester / formamide, etc., or to expand the mass difference by stable isotope labeling.

[0108] The combination of the modified body of the ester formed by the esterification reaction and the modified body of the amide formed by the amidation reaction can be appropriately changed according to the sample used. For example, in sialic acid, in addition to the representative Neu5Ac, there is also Neu5Gc, which is 16Da larger than it. These sialic acids can be mixed, so for example, in the case of a disialyl double-chain sugar chain, there are three types of sialic acids, Neu5Ac x2, Neu5Ac+Neu5Gc, and Neu5Gc x2 (hereinafter, x2 means containing 2 identical sialic acids) as a combination of sialic acids. When the sample in which they are mixed is subjected to methyl esterification and acetamido, 9 peaks shown in the following Table 1 can be detected. (In the following Tables 1 and 2, the mass difference based on the mass in the case of Neu5Ac x2 and α2,6-x2 is shown)

[0109] Table 1

[0110]

[0111] Here, +13 and +16, +26 and +29, +29 and +32, +42 and +45 have similar masses, and when isotope distribution is also considered, peaks are partially detected repeatedly, so there is a possibility that data analysis cannot be performed correctly. In this case, by using propylamine as an amidation reagent to perform amidation after methyl esterification, it is possible to prevent peak duplication and perform data analysis correctly. When methyl esterification and propionamidation are performed on the three mixed samples of Neu5Ac x2, Neu5Ac+Neu5Gc, and Neu5Gc x2, 9 peaks shown in the following Table 2 can be detected.

[0112] Table 2

[0113]

[0114] In this case, the peaks are separated appropriately. The same is true for sugar chains containing three or more sialic acids. In this way, the combination of esterification and amidation can be appropriately selected according to the complexity of the sugar chains contained in the sample.

[0115] When the analytical sample obtained by the method for preparing an analytical sample of this embodiment is analyzed by chromatography, the alcohol or esterification agent and the amidation reaction agent contained in the esterification reaction solution are selected so that the two modified forms obtained can be separated with good accuracy according to the resolution of the chromatography.

[0116] (Amine in the amidation reaction)

[0117] In the following embodiments, the term "amine" includes hydrazine, hydrazine derivatives and hydroxylamine, but does not include ammonia and ammonia salts. When an amine is used in the amidation reaction, the amine contained in the amidation reagent is preferably at least one compound selected from primary amines, hydrazine, hydrazine derivatives and hydroxylamine and their salts. The carboxyl group of α2,3-sialic acid and the like is in a position with greater steric hindrance than the carboxyl group of α2,6-sialic acid, so it is believed that primary amines are more likely to selectively react with α2,3-sialic acid and the like than other amines.

[0118] When a primary amine is used as an amidation reagent, a primary amine having one or less carbon atoms directly bonded to the carbon atom bonded to the amino group is more preferred. This is because, in this case, even if there is a branch on the carbon chain, if the branch exists at a position far from the amino group, the reduction in the efficiency of the amidation reaction can be suppressed.

[0119] The amidation reaction agent is more preferably a primary amine having a straight-chain hydrocarbon group, and more preferably a primary amine having a straight-chain alkyl group. As for the amidation reaction agent, as a primary amine having a straight-chain alkyl group, a primary amine having a carbon number of 10 or less is preferred, and a primary amine having a carbon number of 6 or less, i.e., methylamine, ethylamine, propylamine, butylamine, pentylamine and hexylamine is further preferred, and methylamine is most preferred. When the amine contained in the amidation reaction solution has a straight-chain structure without a branch (hereinafter, "branch" means a branch of a hydrocarbon chain), or when the carbon number is small, the esterified sialic acid is amidated more efficiently, so it is preferred.

[0120] The hydrazine derivatives included in the amidation reaction agent are not particularly limited. In the following embodiments, hydrazides such as acetic hydrazide, acetic hydrazide, benzohydrazide and benzoic acid hydrazide are also included in the hydrazine derivatives and can be used as amidation reaction agents. The hydrazine derivatives included in the amidation reaction agent can be selected from at least one compound in the group consisting of methylhydrazine, ethylhydrazine, propylhydrazine, butylhydrazine, phenylhydrazine and benzylhydrazine, and acetic hydrazide, acetic hydrazide, benzohydrazide and benzoic acid hydrazide. From the viewpoint of improving or maintaining the efficiency of the amidation reaction, hydrazine or its derivatives as the amidation reaction agent are preferably hydrazine or methylhydrazine.

[0121] In the case where the amidation reagent is a primary amine having an unsaturated chain hydrocarbon group, the unsaturated chain hydrocarbon group preferably contains a double bond, and the unsaturated chain hydrocarbon group more preferably contains an allyl group (Allyl), and the amine is further preferably allylamine (Allylamine). The amidation reagent can be a primary amine containing a hydroxyl group, and ethanolamine is preferred in this case. The amine contained in the amidation reaction solution can also contain various functional groups other than alkyl. As a result of the amidation reaction, the sugar chain is modified in a manner containing such a functional group, so that the modified sugar chain not only becomes easier to separate by mass spectrometry, but also becomes easier to separate by chromatography, etc.

[0122] The amidation reagent may contain ammonia or a salt of an amine described above as an amidation reagent. As the salt of ammonia or amine contained in the amidation reagent, inorganic or organic acid salts of ammonia or amine may be cited, preferably inorganic salts such as carbonates, hydrochlorides, nitrates, sulfates, phosphates or methanesulfonates, more preferably carbonates, hydrochlorides and nitrates, and further preferably hydrochlorides. As the hydrochloride of a primary amine having a straight-chain hydrocarbon group, methylamine hydrochloride, ethylamine hydrochloride, propylamine hydrochloride, butylamine hydrochloride, and amylamine hydrochloride are more preferred, and methylamine hydrochloride or ethylamine hydrochloride is further preferred.

[0123] (Concentration of amidation reaction solution)

[0124] The concentration of the amidating agent such as methylamine or ethylamine in the amidation reaction solution is preferably 0.01% or more, more preferably 0.1% or more, and further preferably 1% or more in weight / volume %. The higher the concentration of the amidating agent in the amidation reaction solution, the more reliably the amidation of the esterified sialic acid can be performed. The concentration of the amidating agent such as methylamine or ethylamine in the amidation reaction solution is preferably less than 40% in weight / volume %, more preferably less than 20%. Thus, it is possible to suppress undesirable reactions caused by high concentrations, or to suppress the amidation of α2.6-sialic acid, thereby facilitating modification of the specificity of the connection mode.

[0125] (Solvent for the amidation reaction solution)

[0126] From the viewpoint of reliable occurrence of amidation, the solvent of the amidation reaction solution is preferably an aqueous solvent, an organic solvent or a mixed solvent of an aqueous solvent and an organic solvent. The solvent of the amidation reaction solution can be, for example, water, an alcohol such as methanol or ethanol, dimethyl sulfoxide (DMSO) or acetonitrile or a mixture thereof. In the case where the connection method specifically modifies α2,6-sialic acid and other sialic acids, from the viewpoint of inhibiting the amidation of α2,6-sialic acid, the solvent of the amidation reaction solution preferably includes an organic solvent.

[0127] (pH of amidation reaction solution)

[0128] The pH of the amidation reaction solution is 7.7 or higher. The pH of the amidation reaction solution is preferably 8.0 or higher, more preferably 8.8 or higher, and further preferably 10.3 or higher. When the pH of the amidation reaction solution is increased, side reactions such as hydrolysis are suppressed, or the esterified sialic acid is amidated more reliably by using various amidation reaction agents, so it is preferred.

[0129] (Time for the amidation reaction to occur)

[0130] The amidation reaction is completed within a few seconds to a few minutes. Therefore, in order to amidate the esterified sialic acid by the amidation reaction, the time (hereinafter referred to as the reaction time) for contacting the sample with the amidation reaction solution is preferably less than 1 hour, more preferably less than 30 minutes, further preferably less than 15 minutes, further preferably less than 5 minutes, and most preferably less than 1 minute. It is appropriate to only wash the sample with the amidation reaction solution, or temporarily pass the liquid through the sample held on the carrier, etc. The time for the sample to contact the amidation reaction solution is not particularly limited, and from the viewpoint of fully completing the reaction, it can be appropriately set to more than 0.1 seconds or more than 1 second, etc. In addition, the sample can also be mixed with the amidation reaction solution and directly dried without setting the reaction time. By setting the reaction time of the amidation reaction relatively short, the analysis of the sample can be carried out more efficiently.

[0131] (Phase for Amidation Reaction)

[0132] As long as the sample can be contacted with the amidation reaction solution, the state of the sample when the amidation reaction occurs is not particularly limited, and can be either a solid phase or a liquid phase. In the case of carrying out the amidation reaction in the liquid phase, the amidation reaction solution can be added to the sample under the state of the solution after the residual esterification reaction as described above, and the known pre-treatments such as purification, desalination, and solubilization can also be carried out after the esterification reaction. In the case of carrying out the amidation reaction under the state fixed to the solid phase, the sample supplied to the esterification reaction in the solid phase can be maintained in the state fixed to the solid phase and the amidation reaction can be carried out. In addition, it is also possible to fix the sample to the solid phase and carry out the amidation reaction after the sample is supplied to the esterification reaction.

[0133] When the amidation reaction is carried out in the solid phase, as a solid phase carrier, the same solid phase carrier as that described above for the esterification reaction can be used. Regarding the fixation of the sample on the solid phase carrier, the conditions described above for the esterification reaction can be used. When the amidation reaction is carried out in the solid phase, the sample fixed on the solid phase carrier is allowed to act with the amidation reaction solution to be amidated, and then the sample is freed from the carrier and recovered by chemical methods or enzyme reactions. For example, glycosidases such as PNGase F or digestive enzymes such as trypsin can be used to enzymatically cut and recovered glycoproteins and glycopeptides fixed to the carrier, and weakly acidic solutions can be used to free and recover sugar chains bonded to the solid phase carrier having hydrazide groups. In HILIC, an amidation reaction solution using acetonitrile as a solvent can be used to carry out an amidation reaction, and the sample can be eluted with an aqueous solution such as water.

[0134] After step S1005 is completed, step S1007 is started. In step S1007, an analytical sample is prepared. The method for preparing the analytical sample is not particularly limited as long as an analytical sample containing the sugar chain subjected to the amidation reaction in step S1005 and used for the analysis performed in step S1009 is obtained.

[0135] For example, in the case of mass spectrometry using matrix-assisted laser desorption / ionization (Matrix Assisted Laser Desorption / Ionization; MALDI) in step S1009, the sample for analysis is prepared, for example, as follows. A solution containing the sample obtained after the amidation reaction is added dropwise to a MALDI sample plate, and then a solution containing a matrix is ​​added dropwise to the solution on the plate and dried. Thus, a mixed crystal of the sample and the matrix can be obtained as a sample for mass spectrometry. In the preparation of the sample for mass spectrometry, the solution containing the sample can be added to the MALDI sample plate after mixing with the solution containing the matrix, the order of adding these solutions dropwise can be changed, and an additive of the matrix can be further used. In the case of other analyses such as chromatography, the sample for analysis is also prepared by a known method.

[0136] In the analytical sample obtained by the above preparation method, α2,6-sialic acid is esterified by esterification reaction. α2,3-, α2,8- and α2,9-sialic acid, especially α2,3- and α2,8-sialic acid, are esterified by esterification reaction, and then converted to amidation by amidation reaction.

[0137] After step S1007 is completed, step S1009 begins.

[0138] In step S1009, the analytical sample is analyzed. The analytical sample is preferably analyzed by at least one of mass spectrometry and chromatography.

[0139] The sugar chains modified by the esterification reaction and the amidation reaction described above have different masses from each other, and therefore, these sugar chains can be separated according to the sialic acid attachment pattern by mass spectrometry.

[0140] The method of ionization in mass spectrometry is not particularly limited, and MALDI, electrospray ionization (ESI) method, nano-electrospray ionization (nano-ESI) method, etc. can be used. The method of ionization is particularly preferably MALDI. In the ionization in mass spectrometry, any one of the positive ion mode and the negative ion mode can be used. Mass spectrometry can be a single mass spectrometry, or it can be carried out in multiple stages. In the multi-stage mass spectrometry, the structure of the sugar chain or the structure of the peptide chain other than the connection mode of sialic acid can be appropriately analyzed. As a mass spectrometer, at least one or more quadrupole type, ion trap type and time-of-flight type can be used in combination. It is also suitable to carry out the dissociation of ions or the addition of atoms or atomic groups to ions.

[0141] When the analysis is performed by chromatography, liquid chromatography is preferred. The column used for liquid chromatography is not particularly limited, and a hydrophobic reverse phase column or carbon column such as C30, C18, C8, or C4, a normal phase column for HILIC, etc. can be suitably used. When the measurement is performed by liquid chromatography-mass spectrometry (LC / MS), it is preferred to precisely analyze the components in the sample by multiple separations.

[0142] The data obtained by mass spectrometry or chromatography are analyzed to analyze sialic acid in the sugar chain contained in the sample, etc. In the data analysis, the structure of the sugar chain including the connection mode of sialic acid can be estimated, etc. The analysis method of the data obtained by mass spectrometry or chromatography is not particularly limited.

[0143] After step S1009 is completed, the processing ends.

[0144] (Regarding the inhibition of side effects of glycopeptides and glycoproteins)

[0145] When the esterification reaction solution and the amidation reaction solution are added to the glycopeptide or glycoprotein, when the sialic acid is modified as described above, side reactions such as intramolecular dehydration condensation sometimes occur between the amino group and the carboxyl group at the side chain or the main chain terminal of the amino acid contained in the glycopeptide or glycoprotein. In this case, the amino group is blocked by chemical modification before the sialic acid modification, so that the side reaction of the peptide part can be suppressed during the sialic acid modification. For details, refer to the following literature: Takashi Nishikaze, Sadanori Sekiya, Shinichi Iwamoto, Koichi Tanaka. "A Universal Approach to linkage-Specific Derivatization for Sialic Acids on Glycopeptides," Journal of The American Society for Mass Spectrometry, June 2017, Volume 28, Issue 1 Supplement, Poster No. MP091. For example, a reaction such as dimethyl labeling or guanidation to block the amino group of the glycopeptide or glycoprotein can be performed, and then an esterification reaction and an amidation reaction can be performed.

[0146] (About the kit for preparing analytical samples)

[0147] A kit for preparing an analytical sample (hereinafter referred to as a preparation kit) which is preferably used in the method for preparing an analytical sample according to the present embodiment is provided.

[0148] Figure 2 This is a conceptual diagram showing a preparation kit 100 according to the present embodiment. Figure 2 In the example, the preparation kit 100 includes an esterifying agent 10 in the esterification reaction. The esterifying agent 10 is contained in a container 11. The capacity and shape of the container 11 are not particularly limited. The preparation kit 100 may include reagents, or any consumables used in the above-mentioned analysis other than reagents, or a file recording a procedure for preparing the analytical sample in this embodiment or a URL of a website recording the procedure. For example, the preparation kit 100 may include: a reagent contained in the esterification reaction solution such as an alcohol, a condensing agent, an additive or an esterifying agent; and at least one compound selected from the group consisting of ammonia, amines, hydrazine, hydrazine derivatives and hydroxylamine and their salts that reacts with sialic acid modified by esterification. By using the preparation kit 100 to prepare the analytical sample, the analytical sample can be prepared more efficiently.

[0149] The following modifications are also within the scope of the present invention and can be combined with the above-mentioned embodiment. In the following modifications, parts having the same structure and function as the above-mentioned embodiment are referred to with the same reference numerals, and the description thereof is appropriately omitted.

[0150] (Variant 1)

[0151] In the above embodiment, in addition to the analysis of the sample obtained by the amidation reaction, the analysis of the sample obtained by the esterification reaction can also be performed. The analysis of the sample obtained by the amidation reaction is referred to as the first analysis, and the analysis of the sample obtained by the esterification reaction is referred to as the second analysis.

[0152] Figure 3 This is a flow chart showing the flow of the analysis method of the analysis sample preparation method of this modification. Steps S2001 and S2003 are respectively the same as steps S1001 and S1003 in the above embodiment, and thus descriptions thereof are omitted. After step S2003 is completed, step S2005 is started.

[0153] In step S2005, a portion of the sample is contacted with an amidation reaction solution to perform an amidation reaction. The solution containing the sample obtained in step S2003 can be divided by fractionation, etc., and the amidation reaction is performed on a portion of the solution using the same amidation reaction solution as in the above embodiment under the same conditions. After step S2005 is completed, step S2007 is started.

[0154] In step S2007, an analysis sample is prepared for the sample obtained in step S2003 and the sample obtained in step S2005. The analysis sample for the first analysis prepared using the sample after the amidation reaction is set as the first analysis sample, and the analysis sample for the second analysis prepared using the sample after the esterification reaction is set as the second analysis sample. The first analysis sample and the second analysis sample are prepared by a known method, etc., similarly to the preparation method of the analysis sample in the above embodiment. After step S2007 is completed, step S2009 is started.

[0155] In step S2009, the sample for analysis is analyzed. The first analysis of the first analysis sample and the second analysis of the second analysis sample are performed. The first analysis and the second analysis are preferably performed by the same analysis method. For example, the first analysis and the second analysis are both performed by mass spectrometry, or both are performed by chromatography, or both are performed by LC / MS. Thus, data analysis can be performed based on the comparison of the data obtained by the first analysis with the data obtained by the second analysis. For example, in the case where the first analysis and the second analysis are performed by mass spectrometry, the peak of the sugar chain containing α2,3-sialic acid in the mass spectrum obtained by the second analysis is offset in the mass spectrum obtained by the first analysis by the amount of m / z corresponding to the mass change during the conversion from esterification modification to amidation modification. In this way, based on the difference between the data obtained by the first analysis and the data obtained by the second analysis, and the information on the mass change amount accompanying the conversion from esterification to amidation, a more detailed analysis of the sugar chain such as the identification peak can be performed. After step S2009 is completed, the processing is completed.

[0156] (Variant 2)

[0157] In the above embodiment, an example is described in which esterified sialic acid is amidated by an amidation reaction to modify sialic acid specifically in a linkage mode. However, the above amidation reaction may be used to amidate sialic acid without specifically modifying the linkage mode.

[0158] (Variant 3)

[0159] The inventors unexpectedly found that in the amidation reaction described above, the amidation of sialic acid in the sugar chain can be carried out structure-specifically depending on the composition of the solvent of the amidation reaction solution. This means that, for example, in the above Figure 1 Step S1005 of the flowchart or Figure 3 When the amidation reaction is performed in step S2005 of the flowchart of , the sialic acid is selectively amidated for sialic acids of the same or different bonding modes, depending on the position of the sialic acid in the sugar chain or the structure of the sugar chain near the sialic acid. Here, the "structure of the sugar chain near the sialic acid" refers to the structure of the bond between the monosaccharide directly bonded to the sialic acid and other sugars.

[0160] In the amidation reaction of this variant, the amidation reaction solution preferably contains an organic solvent, more preferably acetonitrile. The concentration of acetonitrile in the solvent of the amidation reaction solution is appropriately adjusted so that the sialic acid is selectively amidated as described above. The solvent of the amidation reaction solution more preferably contains more than 90% acetonitrile in weight / volume %, further preferably contains more than 95%, and further preferably contains more than 98%. The most preferred solvent of the amidation reaction solution is acetonitrile, that is, it is essentially composed of acetonitrile. Thus, sialic acids other than α2,6-sialic acid, especially α2,3-sialic acid, α2,8-sialic acid and α2,9-sialic acid can be selectively amidated according to the structure of the sugar chain. In particular, α2,3-sialic acid and α2,8-sialic acid present at the end of the sugar chain, especially the non-reducing end, can be selectively amidated. Regarding other conditions in the amidation reaction of this variant, they can be appropriately selected from the same conditions as those in the above-mentioned embodiment.

[0161] More specifically, a sample containing a sugar chain is subjected to an amidation reaction using an amidation reaction solution containing an organic solvent, particularly acetonitrile, so that the sialic acid under the following conditions is selectively amidated. The condition refers to another monosaccharide being bonded to the 4-position of the monosaccharide directly bonded to sialic acid other than α2,6-sialic acid, particularly α2,3- or α2,8-sialic acid. It can be adjusted so that sialic acid that does not meet the condition will not be amidated by an amidation reaction solution containing an organic solvent such as acetonitrile. It can be considered that the above conditions are based on the following principle: the lactone structure of sialic acid forms a cyclic structure containing the 4-position of the monosaccharide adjacent to the sialic acid, and therefore, the lactone structure has an influence as an intermediate of the amidation reaction, etc.

[0162] Figure 4A , 4B 4C is a conceptual diagram showing an example of the selectivity in the amidation reaction of this modification. Figure 4A and 4B The structures of human disialoganglioside GD1a and GD1b, which are glycosphingolipids, are shown respectively. Figure 4A , 4BIn 4C, the sialic acid with a high possibility of being amidated is represented by the symbol of Neu5Ac (high), the sialic acid with a low possibility of being amidated is represented by the symbol of Neu5Ac (low), and the sialic acid with an extremely low possibility of being amidated is represented by the symbol of Neu5Ac (extremely low). The sialic acid with a high possibility of being amidated is amidated by an amidation reaction solution containing an organic solvent such as acetonitrile or an aqueous solvent such as water. The amino acid with a low possibility of being amidated will not be amidated by an amidation reaction solution containing an organic solvent such as acetonitrile, but will be amidated by an amidation reaction solution containing an aqueous solvent such as water. For the sialic acid with an extremely low possibility of being amidated, it is difficult to amidate unless the concentration of the amidation reaction agent is set higher than the concentration when other sialic acids are amidated.

[0163] GD1a sugar chain ( Figure 4A ) includes: a straight chain formed by glucose (Glc), two galactoses (Gal) and N-acetylgalactosamine (GalNAc), and α2,3-sialic acid bonded to each of the two Gals. For the connection mode of α2,3-, the sialic acid is schematically shown by recording the sialic acid at the lower left of the Gal to which the sialic acid is bonded (the same is true in the following figures). In the amidation reaction of this variant, in the GD1a sugar chain, the α2,3-sialic acid (Neu5Ac (high)) bonded to the Gal bonded to GalNAc at the 4th position is selectively amidated. In addition, the α2,3-sialic acid (Neu5Ac (low)) bonded to the Gal that is not bonded to the monosaccharide at the 4th position can be prevented from being amidated.

[0164] The GD1b sugar chain includes: a straight chain formed by Glc, two Gals and GalNAc; α2,3-sialic acid bonded to Gal bonded to Glc; and α2,8-sialic acid bonded to the α2,3-sialic acid. The connection mode is α2,8-, which is schematically shown by recording the sialic acid just below the monosaccharide to which the sialic acid is bonded (the same applies to the following figures). The GD1a sugar chain and the GD1b sugar chain are isomers. In the amidation reaction of this variant, the GD1b sugar chain ( Figure 4B ), α2,3-sialic acid (Neu5Ac(high)) bonded to Neu5Ac bonded to Gal at position 4 is selectively amidated. Also, α2,3-sialic acid (Neu5Ac(low)) bonded to Gal not bonded to monosaccharide at position 4 can be prevented from being amidated.

[0165] Figure 4CA conceptual diagram showing the structure of the A2GN1 sugar chain. The A2GN1 sugar chain has: a basic structure formed by N-acetyl-D-glucosamine (GlcNAc) and mannose (Man), and two side chains. GlcNAc, galactose (Gal) and sialic acid (Neu5Ac) are bonded to the two side chains, respectively. For the connection mode of α2,6-, the sialic acid is schematically shown by recording the sialic acid at the upper left of the Gal to which the sialic acid is bonded (the same applies to the following figures). The possibility of α2,6-sialic acid being amidated is extremely low, and it is not easy to be amidated by an amidation reaction.

[0166] It should be noted that, in addition to Figure 4A , 4B In addition to the sugar chains shown in 4C, it is also applicable to various sugar chains including glycolipid-type sugar chains and N-type sugar chains.

[0167] (Variant 4)

[0168] In the above-mentioned modification 3, amino acids can also be modified in a connection-specific and structure-specific manner. In this modification, α2,6-sialic acid is esterified by an esterification reaction. Among sialic acids other than α2,6-sialic acid, sialic acids that satisfy the conditions described in modification 3 are modified by the first stage of amidation. The reaction in which the amidation in the first stage occurs is referred to as the first amidation reaction, and the amidation reaction solution and the amidation reagent in the first amidation reaction are referred to as the first amidation reaction solution and the first amidation reagent, respectively. Among sialic acids other than α2,6-sialic acid, sialic acids that do not satisfy the conditions described in modification 3 are modified by the second stage of amidation. The reaction in which the amidation in the second stage occurs is referred to as the second amidation reaction, and the amidation reaction solution and the amidation reagent in the second amidation reaction are referred to as the second amidation reaction solution and the second amidation reagent, respectively. The first amidation reagent and the second amidation reagent are selected in such a manner that the modified bodies generated by their bonding to sialic acid can be distinguished and detected by mass spectrometry or the like.

[0169] Figure 5 Flowchart showing the flow of the analysis method of this modification. Step S3001 and step S3003 are Figure 1 Steps S1001 and S1003 of the flowchart are the same, so the description is omitted. Through the esterification reaction, at least α2,6-sialic acid is esterified, and other sialic acids are esterified (or lactonized). After step S3003 is completed, step S3005 is started.

[0170] In step S3005, the sample is contacted with the first amidation reaction solution to perform the first amidation reaction. The solvent of the first amidation reaction solution preferably contains an organic solvent such as acetonitrile, and is particularly preferably substantially composed of only organic solvents such as acetonitrile. After the first amidation reaction, the first amidation reaction solution is preferably removed from the sample, and the other steps are the same as after the above-mentioned esterification reaction, such as purification, desalting, solubilization, binding of sugar chains to a solid phase carrier, or release of sugar chains from a solid phase carrier. Through the first amidation reaction, sialic acids that meet the conditions of the above-mentioned variant 3, such as α2,3-, α2,8- and α2,9-sialic acids, especially α2,3- and α2,8-sialic acids, are modified by the first amidation reaction agent at the non-reducing end. After step S3005 is completed, step S3007 is started.

[0171] In step S3007, the sample is contacted with a second amidation reaction solution to perform a second amidation reaction. The solvent of the second amidation reaction solution includes an aqueous solvent such as water. Through the second amidation reaction, α2,3-, α2,8- and α2,9-sialic acids, especially α2,3- and α2,8-sialic acids, that meet the conditions of the above-mentioned modification example 3 are modified by the second amidation reagent. After step S3007 is completed, step S3009 is started. Steps S3009 and S3011 are the same as those described above. Figure 1 Steps S1007 and S1009 of the flowchart are the same, so their description is omitted.

[0172] The analysis method of this modification can distinguish and detect α2,6-sialic acid from other sialic acids in a linkage-specific manner. In addition, α2,3-, α2,8-, and α2,9-sialic acids can be distinguished and detected based on the structure of the sugar chain such as the presence of the sialic acid at the non-reducing end.

[0173] (Way)

[0174] Those skilled in the art will appreciate that the above-described exemplary embodiments or modifications thereof are specific examples of the following aspects.

[0175] (Item 1) A method for preparing a sample for analysis according to one embodiment, which is a method for preparing a sample for analysis from a sample containing a sugar chain, the method comprising the following operations: performing an esterification reaction, wherein at least a portion of the sialic acid contained in the sugar chain is subjected to esterification other than lactonization; contacting the sample with an amidation reaction solution, and performing an amidation reaction, wherein the amidation reaction solution contains at least one compound selected from the group consisting of ammonia, amines, hydrazine, hydrazine derivatives, and hydroxylamine and salts thereof that reacts with the sialic acid modified by the esterification, wherein the amidation reaction converts the esterification modification of the sialic acid modified by the esterification into an amidation modification. Thus, in order to analyze the sialic acid contained in the sugar chain, the sialic acid can be modified by a new mechanism.

[0176] (Item 2) In another embodiment of the method for preparing an analytical sample, the method for preparing an analytical sample according to Item 1 includes the step of contacting the sample with an esterification reaction solution to perform the esterification reaction, wherein the esterification reaction solution contains at least one of an alcohol and an esterifying agent. Thus, sialic acid contained in the sugar chain can be more reliably esterified.

[0177] (Item 3) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to Item 2, the esterification reaction solution contains an esterifying agent, and the esterifying agent is a triazene derivative, thereby enabling sialic acid contained in the sugar chain to be efficiently esterified.

[0178] (Item 4) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to Item 2 or Item 3, the amidation reaction is performed simply by bringing the sample into contact with the amidation reaction solution after an operation for removing the esterification reaction solution from the sample. Thus, the analytical sample can be prepared simply.

[0179] (Item 5) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 2 to 4, the amidation reaction solution does not contain a dehydration condensation agent. This allows the amidation reaction solution to be prepared more simply.

[0180] (Item 6) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 1 to 5, after the sample is brought into contact with the amidation reaction solution, the sample is not reacted with a dehydration condensation agent. This can reduce the number of steps in preparing the analytical sample, and the preparation can be performed more simply.

[0181] (Item 7) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 1 to 6, the time for which the sample is in contact with the amidation reaction solution for the amidation reaction is shorter than 30 minutes. Thus, the analytical sample can be prepared efficiently in a shorter time.

[0182] (Item 8) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 1 to 7, an operation for cleaving the lactone structure generated by the esterification reaction is not performed before the amidation reaction. This allows α2,3-sialic acid, α2,8-sialic acid, α2,9-sialic acid, etc., which are easy to form lactones, to be amidated more reliably.

[0183] (Item 9) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 1 to 8, the compound is a primary amine. Thus, sialic acid can be modified more reliably according to the reactivity of the compound.

[0184] (Item 10) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to Item 9, one or less carbon atoms are directly bonded to the carbon atom bonded to the amino group of the primary amine. Thus, sialic acid can be more reliably modified according to the reactivity of the compound.

[0185] (Item 11) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 1 to 10, the compound contains an alkyl group. This allows sialic acid to be modified more reliably based on the reactivity of the compound.

[0186] (Item 12) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 1 to 10, the pH of the amidation reaction solution is 7.7 or higher. This facilitates the amidation reaction under alkaline conditions, and enables more reliable modification of sialic acid.

[0187] (Item 13) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 2 to 5, when the sample is brought into contact with the esterification reaction solution, at least a portion of the sialic acid that has not been modified by the esterification is lactonized, and when the sample is brought into contact with the amidation reaction solution, the lactone structure of the sialic acid that has been modified by the lactonization is converted to an amidation modification. Thus, when lactonization occurs by the esterification reaction, the sialic acid can be amidated in the same manner as when esterification occurs.

[0188] (Item 14) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 1 to 13, in the amidation reaction, at least one sialic acid selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid is amidated. Thus, α2,3-sialic acid, α2,8-sialic acid, or α2,9-sialic acid can be amidated by a new mechanism.

[0189] (Item 15) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 1 to 14, in the amidation reaction, the concentration of the compound in the amidation reaction solution is adjusted so that the esterification modification of α2,6-sialic acid modified by the esterification does not convert to the amidation modification. Thus, α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid can be distinguished from α2,6-sialic acid and analyzed.

[0190] (Item 16) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 1 to 15, at least one of the esterification reaction and the amidation reaction is performed in a state where the sample is bound to or adsorbed on a solid phase carrier. This simplifies operations such as purification and enables efficient preparation of the analytical sample.

[0191] (Item 17) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to any one of Items 1 to 16, the solvent of the amidation reaction solution contains an organic solvent. This can inhibit the amidation of α2,6-sialic acid, and can perform a more precise analysis of the specificity of the linkage mode. In addition, structure-specific amidation of sialic acid can be performed.

[0192] (Item 18) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to Item 17, the organic solvent is acetonitrile. This allows for more reliable structure-specific amidation of sialic acid.

[0193] (Item 19) In another embodiment of the method for preparing an analytical sample, in the method for preparing an analytical sample according to Item 17 or Item 18, in the amidation reaction, at least one sialic acid selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid and α2,9-sialic acid is amidated according to the position of the at least one sialic acid in the sugar chain or the structure of the sugar chain. Thus, information on the position of the sialic acid in the sugar chain or the structure of the sugar chain can be obtained, or analysis using the information can be performed.

[0194] (Item 20) In another embodiment of the method for preparing a sample for analysis, the method for preparing a sample for analysis according to Item 19 further comprises the following steps: after the amidation reaction is carried out using the amidation reaction solution containing the organic solvent, the amidation reaction solution containing an aqueous solvent is brought into contact with the sample provided for the amidation reaction to amidate the sialic acid that was not amidated in the amidation reaction. Thus, three or more sialic acids with different attachment modes or positions in the sugar chain can be distinguished and detected.

[0195] (Item 21) In another embodiment of the method for preparing an analytical sample, the method for preparing an analytical sample according to any one of Items 1 to 20 comprises: preparing a first analytical sample from the sample after the amidation reaction; and preparing a second analytical sample from the sample after the esterification reaction and before the amidation reaction. Thus, by comparing the analysis of the first analytical sample with the analysis of the second analytical sample, a more detailed analysis of sugar chains can be performed.

[0196] (Item 22) An analytical method of one embodiment comprises the steps of preparing a sample by the method for preparing an analytical sample according to any one of Items 1 to 20, and analyzing the prepared sample. Thus, sialic acid contained in a sugar chain can be analyzed by utilizing modification based on a new mechanism.

[0197] (Item 23) Another method of analysis comprises the following operations: preparing the first analysis sample and the second analysis sample by the method for preparing the analysis sample of the method of Item 21; analyzing the prepared first analysis sample and the second analysis sample; and analyzing the sugar chains contained in the samples based on the difference between the data obtained by the analysis of the first analysis sample and the data obtained by the analysis of the second analysis sample. Thus, by comparing the analysis of the first analysis sample with the analysis of the second analysis sample, a more detailed analysis of the sugar chains can be performed.

[0198] (Item 24) In another embodiment of the method for preparing a sample for analysis, in the method for preparing a sample for analysis according to Item 22 or Item 23, the analysis is performed by at least one of mass spectrometry and chromatography. Thus, even if the sample contains various substances, they can be separated and analyzed.

[0199] (Item 25) An analytical sample preparation kit according to one embodiment is used in the analytical sample preparation method according to any one of Items 1 to 20. This allows the analytical sample to be prepared efficiently.

[0200] The present invention is not limited to the contents of the above-mentioned embodiment, and other embodiments that can be conceived within the technical concept of the present invention are also included in the scope of the present invention.

[0201] Example

[0202] Examples of the present embodiment are shown below, but the present invention is not limited to the following examples. It should be noted that, in the following, the description of % means volume / volume % unless otherwise specified.

[0203] (Examples 1 to 4)

[0204] In Examples 1 to 4, esterification reaction and amidation reaction were performed in a state where the N-type sugar chain was bonded to a carrier, and the obtained sugar chain samples were analyzed by mass spectrometry.

[0205] <Preparation of evaluation sugar chain samples containing α2,3-sialic acid>

[0206] Perform the following steps 1-3 in numerical order to prepare the sugar chain A2glycan (33A2) containing two α2,3-sialic acids. Use PNGase F to free A2 glycan (33A2) from α2,3-sialic acid glycopeptide (α2,3-Sialylglycopeptide (SGP, Fushimi Pharmaceutical). The free sugar chain is desalted using Stage Tip Carbon. Stage Tip Carbon is a carbon column made by cutting Empore Disc Carbon (manufactured by 3M) into a diameter of about 1 mm and loading it into a 200 μL tip.

[0207] 1. To a tube into which 20 μL of a solution containing α2,3-SGP at a concentration of 1 nmol / μL was dispensed, 10 μL of PNGase F (SIGMA) 0.25 U / μL (2.5 U / tube) was added.

[0208] 2. Gently tap and centrifuge, and incubate overnight (o / n) at 37°C.

[0209] 3. On the second day, use Stage Tip Carbon to desalt the sugar chains.

[0210] <Preparation of evaluation sugar chain samples containing α2,6-sialic acid>

[0211] A2GN1 sugar chain containing α2,6-sialic acid (Tokyo Chemical Industry) was redissolved in water and mixed to prepare a sugar chain sample for evaluation.

[0212] <Esterification reaction and amidation reaction>

[0213] The following steps 1 to 8 are performed in the order of numbers, and the prepared sugar chain sample for evaluation is subjected to esterification reaction and amidation reaction. In the esterification reaction, either 1-methyl-3-p-tolyltriazene (MTT) or 1-ethyl-3-p-tolyltriazene (ETT) is used, and the amidation reagent in the amidation reaction is described in the results of each example.

[0214] 1. The prepared sugar chain sample for evaluation was bound to hydrazide beads (BlotGlyco; Sumitomo Bakelite) according to the protocol of BlotGlyco.

[0215] 2. A solution containing 500 mM MTT or ETT in DMSO as the solvent was added to 100 μL of beads, and the beads were reacted at 60° C. for 1 hour (esterification reaction).

[0216] 3. Wash the beads three times with 200 μL of methanol.

[0217] 4. After adding 100 μL of the amidation reaction solution to the beads and stirring, the amidation reaction solution was removed by centrifugation.

[0218] 5. Wash the beads three times with 200 μL of methanol.

[0219] 6. Release the sugar chain sample from the beads. Release according to the Blotglyco protocol.

[0220] 7. The free sugar chain sample was concentrated and dried by vacuum centrifugation.

[0221] 8. Desalting of sugar chains using Stage Tip Carbon.

[0222] <Mass spectrometry analysis>

[0223] The desalted and dried sugar chains were redissolved in 20 μL of water. 0.5 μL of the solution obtained by redissolution was added dropwise to a 700 μm μFocus plate (Hudson Surface Technology). As a matrix, a matrix solution (containing 5 mM NaCl) containing 5 mg / mL 2,5-dihydroxybenzoic acid (DHB) dissolved in 50% acetonitrile (ACN) was added, and after air-drying at room temperature and pressure, 0.2 μL of ethanol was added to recrystallize it to obtain a sample for mass spectrometry. Then, MALDI-quadrupole ion trap (Quadrupole Ion Trap)-time of flight (Time of Flight) mass spectrometer (MALDI-QIT-TOF-MS) (AXIMA-Resonance, Shimadzu / Kratos) was used to measure in positive ion mode.

[0224] <Results>

[0225] Figure 6 This is a conceptual diagram showing the structure of a sugar chain sample containing α2,3-sialic acid (hereinafter referred to as α2,3-sugar chain sample) used in this example. The α2,3-sugar chain sample has a basic structure formed by N-acetyl-D-glucosamine (GlcNAc) and mannose (Man), and two side chains. GlcNAc, galactose (Gal) and sialic acid (Neu5Ac) are bonded to the two side chains, respectively.

[0226] The sugar chain sample containing α2,6-sialic acid (hereinafter referred to as α2,6-sugar chain sample) used in this example was the above-mentioned Figure 4C A2GN1 sugar chain shown. The α2,6-sugar chain sample differs from the α2,3-sugar chain sample in that, in addition to the sialic acid linkage, one GlcNAc is missing.

[0227] (Example 1)

[0228] Figure 7 The mass spectrum (a) is a mass spectrum of a sample obtained by subjecting the sugar chain to a solid phase carrier and then subjecting the sugar chain to a methyl esterification modification reaction, and then freeing the sugar chain without subjecting it to an amidation reaction. The mass spectrum shows the m / z of the detected ion on the horizontal axis and the intensity of the detection signal of the detected ion on the vertical axis, and the same is true for the following figures. The peak of m / z 2070.9 is the peak of the α2,6-sugar chain sample in which both sialic acids in the sugar chain are methyl esterified. In contrast, for the α2,3-sugar chain sample, both a peak (m / z 2273.9) in which sialic acids in two locations are methyl esterified and a peak (m / z 2241.9) in which one of the two sialic acids is methyl esterified and the other is lactonized are observed.

[0229] Figure 7The mass spectra (b), (c) and (d) are mass spectra of samples obtained by subjecting sialic acid to methyl esterification reaction while the sugar chain is bound to a solid phase carrier, and then subjecting it to amidation reaction using 1% (b), 10% (c) and 40% (d) methylamine solutions in weight / volume %, respectively. After the methyl esterification reaction, in the sample subject to the amidation reaction, not only the lactonized α2,3-sialic acid but also the methyl esterified α2,3-sialic acid is converted to formamide. Surprisingly, although both are ester structures, α2,6-sialic acid is hardly amidated and remains in the state of methyl ester. When treated with concentrated methylamine of 10% or more, α2,6-sialic acid is also slightly amidated, but its reaction rate is slow, which is sufficient to distinguish them from each other in comparison with α2,3-sialic acid which is converted to amide with extremely good efficiency. This example shows that the ester formed from α2,3-sialic acid can be selectively amidated using the amidation reaction solution in the above embodiment, indicating that this method is effective for identifying the linkage form of sialic acid.

[0230] (Example 2)

[0231] Figure 8 The figure shows the mass spectra of samples obtained by subjecting sialic acid to methyl esterification reaction in a state where the sugar chain is bound to a solid phase carrier, and then subjecting it to amidation reaction using 5.6% ammonia water (a), 10% methylamine solution (b), 17.5% ethylamine solution (c), 16.7% dimethylamine solution (d), and 20% trimethylamine solution (e) in weight / volume %. In the case of ammonia (a), no Figure 7 The peaks of m / z 2273.9 and m / z 2241.9 shown in (a) are not concentrated into a peak of m / z 2243.9 corresponding to the sugar chain in which the two sialic acids in the sugar chain are amidated. Therefore, it is known that the structure-specific amidation of α2,3-sialic acid has occurred. Figure 7 Similarly. It can be seen that there is no problem when using ethylamine (c), and the connection mode is specifically acetamidate (m / z 2300.0). On the other hand, when dimethylamine (d) or trimethylamine (e) is used, the peak of the sugar chain containing α2,3-sialic acid disappears. It can be speculated that these are amines that are difficult to undergo aminolysis of lactone (refer to non-patent document 2), so hydrolysis is promoted, and the original carboxylic acid (-COOH) structure is restored, the ionization efficiency is reduced, and it becomes undetectable in positive ion mode.

[0232] (Example 3)

[0233] Fig. 9The mass spectra of the samples obtained by subjecting sialic acid to methyl esterification reaction while the sugar chain was bound to a solid phase carrier, and then subjecting it to amidation reaction using 5% or 6% (weight / volume %) methylamine solution with water (a), methanol (b), dimethyl sulfoxide (c), and acetonitrile (d) as solvents, respectively. It is shown that α2,3-sialic acid is specifically amidated without any problems even with an organic solvent. Compared with the case where the solvent is water, the amidation of α2,6-sialic acid ester is further suppressed when the reaction is carried out under organic solvent conditions.

[0234] (Example 4)

[0235] Fig.10 The upper part (a) is a mass spectrum of a sample obtained by subjecting sialic acid to a methylesterification reaction using MTT and then subjecting it to an acetamidate reaction using a 17.5% (weight / volume%) ethylamine solution. Fig.10 The lower part (b) of is a diagram showing a mass spectrum of a sample obtained by performing an ethyl esterification reaction on sialic acid using ETT and then performing a formiamidation reaction using a 10% (weight / volume %) methylamine solution. It can be seen that even if the combination of ester and amine is changed, there is no problem, and the esterified α2,3-sialic acid is selectively converted to amide.

[0236] (Example 5)

[0237] In Example 5, esterification reaction and amidation reaction were performed in a state where the glycolipid type sugar chain was bound to a carrier, and the obtained sugar chain sample was analyzed by mass spectrometry.

[0238] <Preparation of Sugar Chain Samples for Evaluation>

[0239] As samples, human disialogangliosides GD1a and GD1b (HyTest), which are glycosphingolipids, were used. The above glycolipid was dissolved in 45 μL of 50 mM sodium acetate buffer (pH 5.5) containing 0.2% Triton x100 and allowed to stand at 60°C for 20 minutes. Then, 5 μL of Endoglycoceramidase I (purified from actinomycetes according to the following literature; 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 hydrolyzesoligogalactosylceramides to produce galactooligosaccharides and ceramides," Journal of Biological Chemistry, 2007, Volume 282, pp. 11386-11396) was added and the sugar chain removal reaction was carried out at 37°C for 16 hours.

[0240] <Esterification reaction and amidation reaction>

[0241] The following steps 1 to 7 were carried out in numerical order, and the prepared sugar chain sample for evaluation was subjected to esterification reaction and amidation reaction.

[0242] 1. The prepared sugar chain sample for evaluation is bound to a solid phase carrier (BlotGlyco) according to the protocol of BlotGlyco.

[0243] 2. The beads were added to a solution containing 500 mM MTT using DMSO as the solvent to carry out an esterification reaction.

[0244] 3. Wash the solid phase carrier three times with 200 μL of methanol.

[0245] 4. The solid phase support was washed three times with 200 μL of methylamine solution to perform an amidation reaction.

[0246] 5. Wash the solid phase carrier three times with 200 μL of water.

[0247] 6. According to the protocol of BlotGlyco, the reducing end of the sugar chain was reacted with aoWR, a high-sensitivity label for MALDI.

[0248] 7. Excess reagents were removed from the samples using HILIC plates (Waters).

[0249] <Mass spectrometry analysis>

[0250] Ultraflex II TOF / TOF-MS (Bruker) was used for the measurement in positive ion mode using DHB as the matrix.

[0251] <Results>

[0252] The structures of the GD1a sugar chain and the GD1b sugar chain used in this example are shown above. Figure 4A and 4B .

[0253] Fig.11 The figures show the mass spectra of the sample (a) that was not amidated and the sample (b) that was amidated using a 10% (weight / volume %) methylamine solution after a methylesterification reaction using a GD1a sugar chain as a sample, and the mass spectra of the sample (c) that was not amidated and the sample (d) that was amidated using a 10% (weight / volume %) methylamine solution after a methylesterification reaction using a GD1b sugar chain as a sample.

[0254] When GD1a was methyl esterified with MTT but not amidated, a peak corresponding to the methyl esterification of two α2,3-sialic acids (m / z 1748) and a peak corresponding to the lactonization of one of them (m / z 1716) were observed. After methyl esterification, when amidation reaction was performed with a methylamine solution, the methylamidated form was concentrated.

[0255] For GD1b, when methyl esterification with MTT was performed and amidation was not performed, the peak of the sugar chain that was methyl esterified and lactonized (m / z 1716) was mainly observed. After methyl esterification, when amidation reaction was performed with a methylamine solution, the methyl amidated form was concentrated. This shows that the conversion of sialic acid from ester to amide (ester amide conversion) in the above embodiment that is specific to the sialic acid linkage mode is effective not only for α2,3-sialic acid but also for α2,8-sialic acid.

[0256] (Example 6)

[0257] In the above examples, the amidation reaction was carried out in a state of being bound to a solid phase support. In this example, N-type sugar chains derived from standard serum were used as samples, and the amidation reaction was carried out in a state of being adsorbed to a solid phase support, and the obtained sugar chain samples were analyzed by mass spectrometry.

[0258] <Preparation of Sugar Chain Samples for Evaluation>

[0259] Commercially available serum was reduced and alkylated, and then digested with trypsin and the sugar chains were released using PNGase F to prepare N-type sugar chains.

[0260] <Esterification reaction>

[0261] The following steps 1 to 5 are performed in numerical order to subject N-type sugar chains derived from glycoproteins in serum to an esterification reaction, and the reducing ends are labeled with aoWR.

[0262] 1. The prepared N-type sugar chains were bound to beads (BlotGlyco). The binding was performed according to the protocol of BlotGlyco.

[0263] 2. A 500 mM MTT solution dissolved in DMSO was added to 100 μL of beads and reacted at 60° C. for 1 hour.

[0264] 3. Wash the beads three times with 200 μL of methanol.

[0265] 4. Label the sugar chains with aoWR reagent and recover them.

[0266] 5. Remove aoWR reagent using HILIC plate (Waters).

[0267] <Amidation reaction>

[0268] The N-type sugar chains derived from human serum glycoprotein after the above esterification reaction are diluted with 99% acetonitrile / 1% acetic acid to prepare a 90% acetonitrile concentration. Then, the solution containing the sugar chain sample is applied to the HILIC plate (waters), and the solution is passed through the carrier of the plate by vacuum suction from the back, so that the sugar chain is adsorbed on the HILIC carrier. Then, in order to carry out ester amide conversion, acetonitrile containing 5-40% (weight / volume %) of ethylamine is added to the plate as an amidation reaction solution, and the amidation reaction solution is passed through the carrier of the plate by vacuum suction from the back. Then, 95% acetonitrile / 1% acetic acid is added to the plate, and vacuum suction is also performed from the back to clean the plate carrier. This operation is repeated three times. Finally, 5% acetonitrile / 1% acetic acid is added to the plate to elute the sugar chain from the HILIC plate.

[0269] <Mass spectrometry analysis>

[0270] The measurement was performed using Ultraflex II TOF / TOF-MS (Bruker) in positive ion mode using DHB as a matrix.

[0271] <Results>

[0272] Fig.12 This is a conceptual diagram showing an example of the structure of an N-type sugar chain derived from human serum glycoprotein detected in this Example. Fig.12The sugar chain sample shown has a basic structure formed by GlcNAc and Man, and three side chains. GlcNAc, Gal and sialic acid (Neu5Ac) are bonded to the three side chains respectively. The sialic acid is linked in α2,3- or α2,6- ways. The same sugar chain is also detected in Example 7 described later.

[0273] Fig.13 To show the mass spectrum obtained in this example, Fig.12 The mass spectrum (a) is a mass spectrum of a sample that was subjected to a methyl esterification reaction but not to an amidation reaction. The mass spectra (b) to (e) are mass spectra of samples that were subjected to an amidation reaction using 5% (b), 10% (c), 20% (d) and 40% (e) ethylamine solutions in weight / volume % respectively after a methyl esterification reaction.

[0274] In the spectrum (a) where only methyl esterification was performed, Fig.12 A three-chain trisialo sugar chain with fucose bonded to it (+Δ schematically shows the bond of fucose to the sugar chain) was observed at m / z 3498.32. Since all the sialic acids were methylated, it was impossible to distinguish between α2,3- and α2,6-. A peak of +14 Da was also observed at m / z 3352.19, but it was equivalent to a hypermethylated peak obtained by indiscriminately methylating the hydroxyl group.

[0275] In contrast, when ester amide conversion was performed using ethylamine on the HILIC plate ((b)-(e)), a peak shift was observed according to the concentration. The three-chain trisialoyl sugar chain (m / z 3352.19) that was only methyl-esterified was a mixture of sugar chains in which all three sialic acids were α2,6- and sugar chains in which two sialic acids were α2,6- and one sialic acid was α2,3-, with an existence ratio of about 1:9. The ester amide conversion converted α2,3- from methyl ester to acetamide, so the intensity of the peak of the sugar chain with a mass increase of 13 Da increased. Fig.13 , Fig.14 and Fig.15 In the figure, the change in m / z corresponding to 13Da is schematically shown by arrow A13. In addition, the fucosylated three-chain trisialic acid sugar chain has two α2,6- and one α2,3- at an almost 100% existence ratio, so when the esteramide conversion occurs, the mass completely shifts to 13Da. Peaks of +13Da (m / z 3379.44 and 3525.57) were also detected, but these are superamidation peaks obtained by partially converting α2,6-sialic acid ester into acetamide. In summary, it can be seen that Fig.13In (bd), α2,3-sialic acid undergoes ester-amide conversion specifically depending on the concentration of ethylamine used. This experimental result shows that the ester-amide conversion specific to the connection mode can also occur in the state of adsorption to the HILIC plate.

[0276] Fig.14 In order to show that the amidation reaction solution was not vacuum-sucked from the back side but naturally dropped during the liquid passing stage, Fig.13 The mass spectrum of the sample obtained in the same experiment. Thus, the reaction time can be increased from several seconds to 20 to 30 minutes. Thus, even at low concentrations of amine, ester amide conversion can be efficiently performed.

[0277] (Example 7)

[0278] In this example, N-type sugar chains derived from standard serum were used as samples, amidated in liquid phase, and the obtained sugar chain samples were analyzed by mass spectrometry. Preparation of the evaluation sugar chain samples and esterification reaction were performed under the same conditions as in Example 6.

[0279] <Amidation reaction>

[0280] The N-type sugar chains derived from human serum glycoprotein prepared by the above method are dispensed into a tube, diluted to 10 times with an acetonitrile solution containing 2.5-40% (weight / volume%) ethylamine, and gently mixed with a vortex mixer to perform an amidation reaction. Then, the solution containing the sample is applied to a HILIC plate (Waters), and the solution is passed through the carrier of the plate by vacuum suction from the back, so that the sugar chains are adsorbed to the HILIC carrier. Then, 95% acetonitrile / 1% acetic acid is added to the plate, and vacuum suction is also performed from the back to clean the plate carrier. This operation is repeated three times. Finally, 5% acetonitrile / 1% acetic acid is added to the plate to elute the sugar chains from the HILIC plate.

[0281] <Mass spectrometry analysis>

[0282] The measurement was performed using MALDI-QIT-TOF-MS (AXIMA-Resonance, Shimadzu / Kratos) in positive ion mode using DHB as a matrix.

[0283] Fig.15 To show the mass spectrum obtained in this example, Fig.12The mass spectrum (a) is a mass spectrum of a sample that was subjected to a methyl esterification reaction but not to an amidation reaction. The mass spectra (b) to (f) are mass spectra of samples that were subjected to an amidation reaction using 2.5% (b), 5% (c), 10% (d), 20% (e) and 40% (f) ethylamine solutions in weight / volume % respectively after a methyl esterification reaction.

[0284] For the sugar chain sample, ethylamine of various concentrations was added and mixed, and it took about one minute until purification started with the HILIC plate, but it was confirmed that ester amide conversion occurred without problems even when a low concentration of ethylamine of about 2.5% was used. In addition, it was found that the amidation of α2,6-sialic acid ester was also suppressed when a high concentration of amine was used. In this example, purification was performed using a HILIC carrier, but the ester amide conversion was performed in the form of a liquid phase reaction before adsorption to the HILIC carrier, indicating that the ester amide conversion specific to the connection method was performed in the form of a liquid phase reaction without problems.

[0285] (Example 8)

[0286] In Example 8, glycosphingolipid (GSL) sugar chains obtained from GSL were subjected to ethyl esterification reaction in a state where GSL sugar chains were bound to a carrier, and the obtained sugar chain samples were subjected to amidation reaction and analyzed by mass spectrometry.

[0287] <Preparation of sugar chain samples containing α2,3-sialic acid and esterification reaction>

[0288] Sugar chains (GD1a, GD1b) containing a total of 2 α2,3- or α2,8-sialic acids were prepared by the same method as in Example 5. The free sugar chains were bonded to the solid phase support by the Glycoblotting method, ethyl esterified with 1-ethyl-3-p-tolyltriazene (ETT), and recovered as aoWR-labeled sugar chains (100 μL). For the A2GN1 sugar chain, a sample was prepared in the same manner as in Example 1 above, and subjected to the esterification reaction in the same manner as described above in this Example.

[0289] <Amidation reaction and mass spectrometry analysis>

[0290] 25 μL of the obtained aoWR labeled sugar chain solution was mixed with 250 μL of an amine solution (concentration 1 mol / L, solvent acetonitrile), and excess reagent was removed from the sample using a HILIC plate (Waters). DHB was used as a matrix and measured in positive ion mode using an Ultraflex II TOF / TOF-MS (Bruker). The A2GN1 sugar chain was also subjected to the amidation reaction and mass spectrometry analysis.

[0291] <Results>

[0292] Fig.16 The diagrams show the mass spectra of samples (a) obtained by subjecting GD1a and A2GN1 sugar chains to ethyl esterification modification reaction but not to amidation reaction, and samples (b, c, d) obtained by subjecting GD1a and A2GN1 sugar chains to amidation reaction using 1M (M is mol / L) methylamine, ethylamine, and propylamine solutions, respectively. For GD1a, when ethyl esterification using ETT was performed but not to amidation, a peak corresponding to the case where two α2,3-sialic acids were methyl esterified was detected (m / z 1776). In the following figures, the arrows shown in the mass spectra show the peaks in association with the modified forms of the products. After methyl esterification, when amidation reaction using a methylamine solution was performed, the products in which only one sialic acid was methylamidated were concentrated (m / z 1761). Similarly, when using ethylamine and propylamine solutions, the products in which only one sialic acid was amidated were concentrated (m / z 1775, 1789).

[0293] Fig.17 The diagram shows the mass spectra of samples (e) obtained by subjecting GD1b and A2GN1 sugar chains as samples to ethyl esterification modification reaction but without amidation reaction; and samples (f, g, h) obtained by subjecting acetonitrile solutions containing 1 M (M is mol / L) methylamine, ethylamine, and propylamine, respectively, to amidation reaction. For GD1b, when ethyl esterification using ETT was performed but amidation was not performed, a peak corresponding to the case where one α2,3-sialic acid and one α2,8-sialic acid were methylated was detected (m / z 1776). After methyl esterification, when amidation reaction was performed using a methylamine solution, the peaks were concentrated into products in which only one sialic acid was methylated (m / z 1761). Similarly, when ethylamine and propylamine solutions were used, the peaks were concentrated into products in which only one sialic acid was amidated (m / z 1775, 1789). Regarding Fig.16 (a)-(d) and Fig.17 In all reactions (e) to (h), the A2GN1 sugar chain having α2,6-sialic acid added as an internal standard sugar chain was detected as an ethyl ester.

[0294] Fig.18 To show that GD1a ( Fig.18 (a)) and GD1b( Fig.18Figure 2 shows the MS / MS spectra of (b) (all m / z 1779). It can be seen that in GD1a, a fragment corresponding to the ethyl-esterified GM3 sugar chain was detected at m / z 1090, and in GD1b, only α2,8-sialic acid was propionamidated. That is, it was shown that position-specific α2,3- / α2,8-sialic acid ester-amide exchange occurred under the present conditions, indicating that the above method is effective not only for determining the sialic acid connection mode, but also for analyzing the bonding position.

[0295] (Example 9)

[0296] In Example 9, a propylamine solution using acetonitrile as a solvent was used as an amidation reaction solution, and the concentration of propylamine in the propylamine solution was prepared to be a plurality of concentrations ranging from 0 M to 3 M. In addition, mass spectrometry analysis of GSL sugar chains was performed in the same manner as in Example 8.

[0297] <Results>

[0298] Fig.19 The figure shows the mass spectra of GD1a obtained when propylamine solutions of various concentrations were used as the amidation reaction solution. In the case of GD1a, when a 0.125M propylamine solution was used, ester amide exchange was hardly performed, and when a concentration of 0.25M or higher was used, only one α2,3-sialic acid present at the end of the sugar chain was propylamidated.

[0299] Fig. 20 The figure shows the mass spectra of GD1b obtained when propylamine solutions of various concentrations were used as the amidation reaction solution. In the case of GD1b, only α2,8-sialic acid, which is easy to form a lactone, was propionamidated regardless of the propylamine concentration. The results also suggest that not only the optimal concentration for amidation of α2,3- or α2,8-sialic acid esters present at the terminal is displayed, but also that α2,8-sialic acid and α2,3-sialic acid can be distinguished.

Claims

1. A method for preparing an analytical sample from a sample containing sugar chains, the method comprising the following steps: contacting the sample with an esterification reaction solution to perform an esterification reaction in which at least a portion of the sialic acid contained in the sugar chain is subjected to esterification other than lactonization, wherein the esterification reaction solution contains at least one of an alcohol and an esterification agent; contacting at least a portion of the sample subjected to the esterification reaction other than lactonization with an amidation reaction solution to perform an amidation reaction, wherein the amidation reaction solution contains at least one compound selected from the group consisting of ammonia, amines, hydrazine, hydrazine derivatives, hydroxylamine, and salts thereof that reacts with the sialic acid modified by the esterification reaction other than lactonization, wherein the amidation reaction converts the esterification modification of at least one of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid modified by the esterification reaction into an amidation modification; adjusting the concentration of the compound in the amidation reaction solution so that the esterification modification of the α2,6-sialic acid modified by esterification does not convert to the amidation modification; The hydrazine derivative is at least one compound selected from the group consisting of methylhydrazine, ethylhydrazine, propylhydrazine, butylhydrazine, phenylhydrazine and benzylhydrazine, and acetic hydrazine, acetic hydrazide, benzohydrazide and benzoic acid hydrazide.

2. The method for preparing an analytical sample according to claim 1, wherein: The esterification reaction solution comprises an esterification agent, The esterifying agent is a triazene derivative; The triazene derivative is selected from 1-methyl-3-p-tolyltriazene, 1-ethyl-3-p-tolyltriazene, 1-isopropyl-3-p-tolyltriazene, 1-benzyl-3-p-tolyltriazene or 1-(4-nitrobenzyl)-3-p-tolyltriazene.

3. The method for preparing an analytical sample according to claim 1 or 2, wherein: The amidation reaction is performed only by bringing the sample into contact with the amidation reaction solution after the operation for removing the esterification reaction solution from the sample.

4. The method for preparing an analytical sample according to claim 1 or 2, wherein: The amidation reaction solution does not contain a dehydration condensation agent.

5. The method for preparing an analytical sample according to claim 1 or 2, wherein: After the sample is brought into contact with the amidation reaction solution, an operation of reacting the sample with a dehydration condensation agent is not performed.

6. The method for preparing an analytical sample according to claim 1 or 2, wherein: The time for which the sample is in contact with the amidation reaction solution in order to perform the amidation reaction is shorter than 30 minutes.

7. The method for preparing an analytical sample according to claim 1 or 2, wherein: Prior to the amidation reaction, an operation for cleaving the lactone structure generated by the esterification reaction is not performed.

8. The method for preparing an analytical sample according to claim 1 or 2, wherein: The compound is a primary amine.

9. The method for preparing an analytical sample according to claim 8, wherein: One or less carbon atoms are directly bonded to the carbon atom bonded to the amino group of the primary amine.

10. The method for preparing an analytical sample according to claim 1 or 2, wherein: The compound comprises an alkyl group.

11. The method for preparing an analytical sample according to claim 1 or 2, wherein: The pH of the amidation reaction solution is 7.7 or higher.

12. The method for preparing an analytical sample according to claim 1 or 2, wherein: When the sample is brought into contact with the esterification reaction solution, at least a portion of the sialic acid that has not been modified by the esterification other than lactonization is lactonized, When the sample is brought into contact with the amidation reaction solution, the lactone structure of the sialic acid modified by the lactonization is converted to an amidation modification.

13. The method for preparing an analytical sample according to claim 1 or 2, wherein: At least one of the esterification reaction and the amidation reaction is performed in a state where the sample is bound to or adsorbed on a solid phase support.

14. The method for preparing an analytical sample according to claim 1 or 2, wherein: The solvent of the amidation reaction solution includes an organic solvent.

15. The method for preparing an analytical sample according to claim 14, wherein: The organic solvent is acetonitrile.

16. The method for preparing an analytical sample according to claim 14, wherein: In the amidation reaction, at least one sialic acid selected from the group consisting of α2,3-sialic acid, α2,8-sialic acid, and α2,9-sialic acid is amidated depending on the position of the at least one sialic acid in the sugar chain or the structure of the sugar chain.

17. The method for preparing an analytical sample according to claim 16, wherein: It also has the following operations: After the amidation reaction is performed using the amidation reaction solution containing the organic solvent, the amidation reaction solution containing an aqueous solvent is brought into contact with the sample used for the amidation reaction to perform amidation of sialic acid that has not been amidated in the amidation reaction.

18. The method for preparing an analytical sample according to claim 1 or 2, comprising the following steps: preparing a first analytical sample from the sample subjected to the amidation reaction; and A second analysis sample is prepared from the sample subjected to the esterification reaction other than lactonization and before the amidation reaction.

19. An analysis method comprising the following operations: A method for preparing an analytical sample according to any one of claims 1 to 17 is used to prepare a sample; and an analysis of the prepared analytical sample is performed.

20. An analysis method comprising the following operations: preparing the first analytical sample and the second analytical sample by the analytical sample preparation method according to claim 18; performing analysis of the prepared first analysis sample and the second analysis sample; and, Sugar chains contained in the sample are analyzed based on the difference between the data obtained by the analysis of the first analysis sample and the data obtained by the analysis of the second analysis sample.

21. The analysis method according to claim 19 or 20, wherein: The analyzing is performed by at least one of mass spectrometry and chromatography.

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