阅读说明：本技术 分析用试样的制备方法、分析方法和分析用试样的制备用试剂盒 (Method for preparing sample for analysis, method for analysis, and kit for preparing sample for analysis ) 是由 西风隆司 于 2019-08-23 设计创作，主要内容包括：提供分析用试样的制备方法、分析方法和分析用试样的制备用试剂盒。本发明的课题在于更准确地进行连接方式特异性的唾液酸的修饰。一种分析用试样的制备方法,其用于进行试样中含有的糖链的分析,该制备方法具备：进行第1反应的步骤,所述第1反应用于在糖链结合有唾液酸的情况下对第1连接方式的唾液酸进行内酯化,并对与第1连接方式不同的第2连接方式的唾液酸进行与内酯化不同的修饰；进行第2反应的步骤,对第1反应中生成的内酯进行开环；以及,再次进行第1反应的步骤。(Provided are a method for preparing an analysis sample, an analysis method, and a kit for preparing an analysis sample. The present invention addresses the problem of more accurately modifying sialic acid specifically for the mode of ligation. A method for preparing a sample for analysis, which is used for analyzing sugar chains contained in the sample, the method comprising: a step of performing a1 st reaction for lactonizing sialic acid in a1 st linkage form when sialic acid is bonded to a sugar chain, and modifying sialic acid in a 2 nd linkage form different from the 1 st linkage form differently from lactonization; a step of carrying out a 2 nd reaction for ring-opening the lactone produced in the 1 st reaction; and, a step of carrying out the reaction 1 again.)

1. A method for preparing a sample for analysis,

which is used for analyzing sugar chains contained in a sample,

the preparation method comprises the following steps:

a step of performing a1 st reaction for lactonizing sialic acid in a1 st linkage form when sialic acid is bonded to the sugar chain, and modifying sialic acid in a 2 nd linkage form different from the 1 st linkage form differently from lactonization;

a step of carrying out a 2 nd reaction for ring-opening the lactone produced in the 1 st reaction; and the number of the first and second groups,

the step of the 1 st reaction is carried out again.

2. The method for preparing a sample for analysis according to claim 1, wherein,

in the 2 nd reaction, the lactone formed by lactonization of the sialic acid of the 2 nd linkage is subjected to ring opening.

3. The method for preparing an analysis sample according to claim 1 or 2, further comprising:

and a step of repeating the operation of performing the 2 nd reaction and the 1 st reaction alternately only 1 or more times.

4. The method for preparing a sample for analysis according to claim 1 or 2, wherein,

the 2 nd reaction is performed by contacting the sample after the 1 st reaction with a basic solvent having a pH of 8 or more.

5. The method for preparing a sample for analysis according to claim 1 or 2, wherein,

at least one of the plurality of reactions including the 1 st reaction and the 2 nd reaction is carried out in a state where the sugar chain is bound or adsorbed to a solid phase carrier.

6. The method for preparing a sample for analysis according to claim 1 or 2, wherein,

the sample contains O-type sugar chains.

7. The method for preparing a sample for analysis according to claim 1 or 2, wherein,

the modification is esterification or amidation.

8. The method for preparing a sample for analysis according to claim 1 or 2, wherein,

subjecting the lactone produced by said 1 st reaction to a modification different from said modification.

9. The method for preparing a sample for analysis according to claim 1 or 2, wherein,

the sialic acid in the 1 st connection mode is at least one of α 2, 3-sialic acid, α 2, 8-sialic acid and α 2, 9-sialic acid, and the sialic acid in the 2 nd connection mode is α 2, 6-sialic acid.

10. An analysis method comprising:

a step of preparing an analysis sample by the method for preparing an analysis sample according to any one of claims 1 to 9; and the number of the first and second groups,

and a step of analyzing the prepared sample for analysis.

11. The assay of claim 10, wherein,

the prepared sample for analysis is analyzed by at least one of mass spectrometry and chromatography.

12. A kit for preparing a sample for analysis,

which comprises an alkaline solvent having a pH of 8 or more,

the method for preparing an analysis sample according to claim 4.

Technical Field

The present invention relates to a method for preparing an analysis sample, an analysis method, and a kit for preparing an analysis sample.

Background

Sialic acid is a sugar that is present in large amounts in organisms. Sialic acid is contained in a sugar chain bound to a protein in a living body, and is often present at a non-reducing end of the sugar chain. Therefore, sialic acid is disposed on the outside of the molecule in such a glycoprotein molecule and is directly recognized by other molecules, and therefore it plays an important role.

For example, it is known that human N-linked sugar chains (N-type sugar chains) are mainly α 2, 3-and α 2, 6-linked, and that O-linked sugar chains (O-type sugar chains) and glycosphingolipids are α 2, 8-and α 2, 9-linked, in addition to the aforementioned linkage systems.

However, mass spectrometry-based analysis of sialic acid-containing sialylated sugar chains is not easy because sialic acid has a negative charge and is difficult to ionize in a positive ion mode, and sialic acid is easily decomposed. Patent document 1 describes that sialic acid on a sugar chain is amidated and stabilized in a nonaqueous solvent. However, the method of patent document 1 cannot modify sialic acid linkage modes differently, and the mass of the sugar chain is not originally changed depending on the sialic acid linkage modes, so that the linkage modes cannot be distinguished and analyzed by mass spectrometry.

A chemical modification method for specifically modifying the mode of linkage of sialic acid is proposed, which utilizes the property that α 2, 3-sialic acid is more easily intramolecular dehydrated by a dehydration condensation agent than α 2, 6-sialic acid, and which involves lactonization of α 2, 3-sialic acid by intramolecular dehydration and reaction of α 2, 6-sialic acid with a nucleophilic reagent such as alcohol or amine, whereby molecules of different mass are produced depending on the mode of linkage of sialic acid, and therefore, the mode of linkage of sialic acid can be distinguished and analyzed by mass spectrometry, and patent document 2 and non-patent document 1 add a solution containing isopropylamine and a dehydration condensation agent to a free sugar chain, lactonize α 2, 3-sialic acid, and amidate α 2, 6-sialic acid.

Disclosure of Invention

Problems to be solved by the invention

The inventors have found that when the methods described in patent document 2 and non-patent document 1 are carried out, the reaction specificity is lowered particularly for an O-linked sugar chain, and α 2, 6-sialic acid may be partly lactonized.

Means for solving the problems

A method for preparing an analysis sample according to a preferred embodiment of the present invention is a method for analyzing sugar chains contained in a sample, the method including: a step of performing a1 st reaction for lactonizing sialic acid in a1 st linkage system when sialic acid is bonded to the sugar chain, and modifying sialic acid in a 2 nd linkage system different from the 1 st linkage system differently from lactonization; a step of carrying out a 2 nd reaction for ring-opening the lactone produced in the 1 st reaction; and a step of repeating the reaction 1.

In a more preferred embodiment, in the 2 nd reaction, the lactone formed by lactonization of the sialic acid of the 2 nd linkage is subjected to ring opening.

In a more preferred embodiment, the apparatus further comprises: the operations for carrying out the 2 nd reaction and the 1 st reaction are alternately repeated 1 or more times.

In a more preferred embodiment, the 2 nd reaction is carried out by contacting the sample subjected to the 1 st reaction with a basic solvent having a pH of 8 or more.

In a more preferred embodiment, at least one of the reactions including the 1 st reaction and the 2 nd reaction is carried out in a state where the sugar chain is bonded or adsorbed to a solid carrier.

In a more preferred embodiment, the sample contains an O-type sugar chain.

In a further preferred embodiment, the modification is esterification or amidation.

In a further preferred embodiment, the lactone produced by the reaction 1 is modified differently from the modification.

In a more preferred embodiment, the sialic acid at the 1 st linkage is at least one of α 2, 3-sialic acid, α 2, 8-sialic acid and α 2, 9-sialic acid, and the sialic acid at the 2 nd linkage is α 2, 6-sialic acid.

An analysis method according to a preferred embodiment of the present invention includes: a step of preparing an analysis sample by the method for preparing an analysis sample; and analyzing the prepared sample for analysis.

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

The kit for preparing an analysis sample according to a preferred embodiment of the present invention includes an alkaline solvent having a pH of 8 or more, and is used in the method for preparing an analysis sample.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, modification of sialic acid specific to the ligation format can be performed more accurately.

Drawings

FIG. 1 is a flow chart illustrating the flow of an analysis method of one embodiment.

Fig. 2 is a mass spectrum of an analysis sample obtained by a conventional method for preparing an analysis sample (upper half) and a method for preparing an analysis sample according to an embodiment (lower half).

Detailed Description

Hereinafter, specific embodiments of the present invention will be described with reference to the drawings.

Fig. 1 is a flowchart showing a flow of an analysis method in the method for preparing an analysis sample according to the present embodiment. In step S1001, a sample containing a sugar chain is prepared.

The present inventors have found that, in the modification of conventional linkage-specific sialic acid which selectively amidates α 2, 6-sialic acid, α 2, 6-sialic acid may be lactonized in an O-linked sugar chain, and thus the present invention has been made, and therefore, it is preferable that the sugar chain in the sample contains an O-linked sugar chain having α 2, 6-sialic acid even when other sugar chains have been unexpectedly lactonized in the modification of linkage-specific sialic acid.

When a sample contains a free sugar chain, a sugar chain released from a glycoprotein, a glycopeptide, or a glycolipid can be used, and as a method for releasing a sugar chain from a glycoprotein, a glycopeptide, or a glycolipid, a chemical release method such as enzyme treatment using O-glycosidase, N-glycosidase, or glycosphingolipid endoglycosidase (Endoglycoceramidase), hydrazine decomposition, or β dissociation by alkali treatment can be used.

When the sample contains a glycopeptide or glycoprotein, the glycopeptide or glycoprotein may be appropriately subjected to a treatment for initiating a reaction for blocking an amino group, such as dimethylamide or guanidination. This can suppress side reactions such as intramolecular dehydration condensation with an amino group or a carboxyl group at the end of the main chain. Further, a glycopeptide or a glycoprotein having a large number of amino acid residues in a peptide chain is preferably used by cleaving the peptide chain by an enzyme or the like. For example, in the case of preparing a sample for mass spectrometry, the number of amino acid residues in the peptide chain is preferably 30 or less, more preferably 20 or less, and still more preferably 15 or less. On the other hand, in the case where it is required to clarify the origin of the peptide to which the sugar chain is bonded, the number of amino acid residues in the peptide chain is preferably 2 or more, more preferably 3 or more.

As the digestive enzyme for cleaving a peptide chain of a glycopeptide or glycoprotein, trypsin, Lys-C, arginine endopeptidase, chymotrypsin, pepsin, thermolysin, proteinase K, pronase E, and the like can be used. More than 2 of these digestive enzymes may be used in combination. The conditions for cleaving the peptide chain are not particularly limited, and an appropriate protocol depending on the digestive enzyme to be used can be employed. Before the cleavage, a modification treatment or an alkylation treatment of the protein or peptide in the sample may be performed. The conditions of the modification treatment and the alkylation treatment are not particularly limited.

The cleavage treatment of the peptide chain may be performed before or after the lactonization of sialic acid contained in the sugar chain by the method for preparing an analysis sample according to the present embodiment, after the stabilization of the lactonized sialic acid. Further, the peptide chain may be cleaved by chemical cleavage or the like, instead of enzymatic cleavage.

After step S1001 ends, the process proceeds to step S1003.

(Selective lactonization)

In step S1003, a reaction (selective lactonization reaction) for bringing a sample into contact with a reaction solution for selective lactonization (hereinafter referred to as lactonization reaction solution) is performed to lactonize α 2, 3-sialic acid when sialic acid is bonded to a sugar chain and to modify α 2, 6-sialic acid differently from lactonization, and the selective lactonization reaction can suitably lactonize α 2, 8-sialic acid and α 2, 9-sialic acid in addition to α 2, 3-sialic acid.

An operation for removing the lactonization reaction solution from the sample after the selective lactonization reaction was performed. The procedure for removing the lactonization reaction solution is not particularly limited as long as the concentration of the reagent necessary for the selective lactonization reaction is sufficiently low, and the lactonization reaction solution can be separated from the sugar chains bound to the solid phase carrier by centrifugation or the like and washed with a washing solution, or the sample can be dried and solidified by centrifugal concentration.

When the removal of the reaction solution is performed or performed several times, there is no particular limitation as long as a reaction product that is selectively lactonized with sufficient reaction specificity can be obtained after step S1009 described below. The removal of the reaction solution may be appropriately performed after at least one of the steps S1003 to S1011.

The lactonization reaction solution contains a dehydration condensation agent and a nucleophile containing an alcohol, an amine, or a salt thereof. The species and concentration of these dehydration-condensing agents and nucleophiles are adjusted so as to selectively initiate dehydration or nucleophilic reactions depending on the manner of sialic acid attachment.

The lactone produced by intramolecular dehydration of the carboxyl group of α 2, 3-sialic acid is a six-membered ring, and the lactone produced by intramolecular dehydration of the carboxyl group of α 2, 6-sialic acid is a seven-membered ring, therefore α 2, 3-sialic acid which produces a six-membered ring more stable than a seven-membered ring is more easily lactonized than α 2, 6-sialic acid, furthermore, the carboxyl group of α 2, 3-sialic acid is located at a position more sterically hindered than the carboxyl group of α 2, 6-sialic acid, and therefore a larger molecule is less likely to react with α 2, 3-sialic acid than α 2, 6-sialic acid, and the species and concentrations of the dehydration condensation agent and the nucleophilic agent are adjusted based on this difference in molecular structure due to the connection mode of sialic acid, so that different modifications are made depending on the connection mode of sialic acid.

(dehydration condensation agent in Selective lactonization reaction)

The dehydration condensation agent preferably contains carbodiimide. This is because if carbodiimide is used, amidation of a carboxyl group present at a site having a large steric hindrance is less likely to proceed, as compared with the case where a phosphonium based dehydration condensation agent (so-called BOP agent) or a uronium based dehydration condensation agent is used as the dehydration condensation agent. Examples of carbodiimides include: n, N '-Dicyclohexylcarbodiimide (DCC), N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC), N '-Diisopropylcarbodiimide (DIC), 1-tert-butyl-3-ethylcarbodiimide (BEC), N' -di-tert-butylcarbodiimide, 1, 3-di-p-toluoyl-carbodiimide, bis (2, 6-diisopropylphenyl) carbodiimide, bis (trimethylsilyl) carbodiimide, 1, 3-bis (2, 2-dimethyl-1, 3-dioxolan-4-ylmethyl) carbodiimide (BDDC), salts thereof.

(additive in Selective lactonization)

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

(nucleophiles in Selective lactonization)

The amine used as the nucleophile preferably contains a primary or secondary alkylamine having 2 or more carbon atoms, the primary alkylamine is preferably ethylamine, propylamine, isopropylamine, butylamine, sec-butylamine, tert-butylamine, and the like, the secondary alkylamine is preferably dimethylamine, ethylmethylamine, diethylamine, propylmethylamine, isopropylmethylamine, and the like, and an amine having a branched alkyl group such as isopropylamine is preferably used from the viewpoint of preventing a carboxyl group present at a site having a large steric hindrance, such as a carboxyl group of α 2, 3-sialic acid, from being easily amidated, and in the case of using an amine as the nucleophile in the lactonization reaction solution, a part of the carboxyl group of sialic acid such as α 2, 6-sialic acid is amidated depending on the mode of linkage of sialic acid.

The alcohol used as the nucleophile is not particularly limited, and for example, methanol, ethanol and the like can be used, and when the alcohol is used as the nucleophile in the lactonization reaction solution, some of the carboxyl groups of sialic acid such as α 2, 6-sialic acid are esterified depending on the mode of linkage of sialic acid.

The nucleophile may contain a salt of the above nucleophile.

(concentration of dehydration condensing agent and nucleophilic agent)

The concentration of the dehydration condensation agent in the lactonization reaction solution is, for example, preferably 1 mM-5M, more preferably 10 mM-3M. When carbodiimide is used in combination with an additive having high nucleophilicity such as Oaxyma, HOAt, or HOBt, the concentration of each is preferably within the above range. The concentration of the nucleophilic reagent in the lactonization reaction solution is preferably 0.01 to 20M, and more preferably 0.1 to 10M. The reaction temperature in the selective lactonization reaction is preferably about-20 ℃ to 100 ℃, more preferably-10 ℃ to 50 ℃.

(phase for carrying out Selective lactonization)

The selective lactonization reaction can be carried out in the liquid or solid phase. When the reaction is carried out in a liquid phase, the reaction is preferably carried out in a nonaqueous solvent such as Dimethylsulfoxide (DMSO) or Dimethylformamide (DMF). By carrying out the reaction in a nonaqueous solvent, side reactions tend to be suppressed, and it is preferable to use glycopeptides and glycoproteins as samples. The concentration of each component in the liquid phase reaction is not particularly limited, and may be appropriately determined depending on the dehydration condensation agent, the kind of amine, and the like.

When the selective lactonization reaction is carried out in a solid phase, the solid phase carrier is not particularly limited as long as it can immobilize a sugar chain, a glycopeptide, a glycoprotein, and the like. For example, a solid support having an epoxy group, a tosyl group, a carboxyl group, an amino group, or the like as a ligand can be used for immobilizing glycopeptides or glycoproteins. In addition, for immobilizing sugar chains, a solid phase carrier having a hydrazide group, an aminoxy group, or the like as a ligand can be used. The sugar chain is preferably adsorbed to a stationary phase which is a carrier for hydrophilic interaction chromatography (hereinafter referred to as HILIC), and more preferably the carrier for HILIC contains an amide group. By carrying out the reaction in a state where the sample is immobilized on the solid carrier, the reaction solution after the reaction can be easily removed, and sialic acid can be efficiently modified.

The sample after the selective lactonization reaction may be subjected to purification, desalting, solubilization, concentration, drying, and other treatments by known methods, if necessary. The same applies to the front and back of each reaction shown in the flowchart of FIG. 1.

After step S1003 is completed, the process proceeds to step S1005.

(Ring opening reaction)

In step S1005, a reaction (ring-opening reaction) is performed in which a sample is brought into contact with a solvent (hereinafter, referred to as a solvent for ring-opening reaction) to open a ring of a lactone generated in a selective lactonization reaction (the ring-opening reaction in step S1005 is referred to unless otherwise mentioned.) As described above, when a selective lactonization reaction is performed, at least α 2, 6-sialic acid is lactonized in an O-linked sugar chain.

(solvent for Ring opening reaction)

The solvent for the ring-opening reaction is not particularly limited, and any solvent may be used. Even if water is used as the solvent for the ring-opening reaction, the ring-opening reaction of the lactone may be initiated by hydrolysis, although it may take several tens of hours. In order to efficiently produce a sample for analysis, the solvent for ring-opening reaction is preferably used in which lactone is ring-opened in a shorter time. From this viewpoint, the solvent for ring-opening reaction is preferably an alkaline solvent, more preferably an alkaline solvent having a pH of 8 or more, and still more preferably an alkaline solvent having a pH of 10 or more. Although a basic solvent is more preferable than an acidic solvent for hydrolysis of the lactone, the solvent for ring-opening reaction is also preferably an acidic solvent, more preferably an acidic solvent having a pH of 6 or less, and still more preferably an acidic solvent having a pH of 4 or less, from the viewpoint of rapid progress of the ring-opening reaction.

The solvent for the ring-opening reaction is preferably free of alcohol, amine or a salt thereof from the viewpoint of preventing initiation of an accidental reaction with sialic acid. However, the alcohol, amine or salt thereof may be contained as long as a reaction product selectively lactonized with sufficient reaction specificity can be obtained after step S1009 described later. In the case of containing an amine, an amine which does not form a salt is more preferable. As a suitable example, the solvent for the ring-opening reaction is preferably an aqueous sodium hydroxide solution from the viewpoint of availability, belonging to strong bases, and the like. The concentration of the aqueous sodium hydroxide solution is more preferably 5% by weight or less from the viewpoint of the risk of handling and the like. As another example, the solvent for ring-opening reaction preferably contains a quaternary alkylammonium cation or tetramethylguanidine, which is a strong base.

(time for initiating Ring opening reaction)

The time for bringing the sample subjected to the selective lactonization reaction into contact with the solvent for ring-opening reaction is not particularly limited, and depends on the kind of the solvent for ring-opening reaction, and is preferably 10 minutes or less, and a short time of several seconds or less is preferable for efficiently preparing the sample for analysis. When a sample is immobilized on a solid phase as described later, the ring-opening reaction can be initiated by, for example, flowing a solvent for the ring-opening reaction for several seconds.

(phase for carrying out Ring opening reaction)

The ring-opening reaction can be carried out in the liquid phase or in the solid phase. In the case where the selective lactonization reaction is performed in a state where the sample is immobilized on the solid phase, the ring-opening reaction may be performed while maintaining the sample subjected to the selective lactonization reaction in a state where the sample is immobilized on the solid phase. Alternatively, the ring-opening reaction may be carried out by subjecting the sample to a selective lactonization reaction and then immobilizing the sample on a solid phase.

When the selective lactonization reaction is carried out in a solid phase, the same substances as those mentioned for the selective lactonization reaction can be used as the solid phase carrier. For the immobilization of the sample on the solid support, the conditions described for the selective lactonization reaction can be used. The conditions described for the stabilization reaction described later can be applied to the release of the sample from the solid phase carrier. By carrying out the reaction in a state where the sample is immobilized on the solid phase carrier, removal of a solvent for ring-opening reaction after the ring-opening reaction and the like become easy, and sialic acid can be efficiently modified.

After step S1005 ends, the process proceeds to step S1007.

In step S1007, selective lactonization is performed again, and sialic acid accidentally lactonized in the selective lactonization performed in step S1003 is at least partially modified by the selective lactonization performed in step S1007, differently from lactonization, and sialic acid having a specific linkage such as α 2, 3-sialic acid intentionally lactonized in the selective lactonization is lactonized in each selective lactonization and is ring-opened in each ring-opening reaction.

After step S1007 ends, the process proceeds to step S1009.

In step S1009, step S1005 and step S1007 are further alternately repeated only a predetermined number of times. The lactone of an accidentally lactonized sialic acid is opened by a ring opening reaction and a selective lactonization reaction followed by a ring opening reaction and is modified again on the basis of the reaction specificity of the primary selective lactonization reaction. Since sialic acid modified in addition to lactonization remains as it is in the subsequent reaction, the amount of lactonized sialic acid is unexpectedly decreased by repeating the reaction. Therefore, by repeatedly performing the combination of the ring-opening reaction and the selective lactonization reaction, the ligation-specific selective lactonization reaction can be accurately performed.

The number of times of repeating the selective lactonization reaction (hereinafter referred to as the number of repetitions) in step S1009 is not particularly limited. The more the number of repetitions, the higher the reaction specificity of the entire multiple selective lactonization reaction, and therefore the number of repetitions is preferably 1 or more, more preferably 2 or more. If the number of repetitions is too large, the efficiency of preparing the sample for analysis is deteriorated, and therefore the number of repetitions may be appropriately set to 100 or less, 10 or less, or the like.

After step S1009 is finished, the process proceeds to step S1011.

When a sample selectively lactonized with the desired reaction specificity is obtained in step S1007, step S1009 may be omitted.

(for stabilization reaction)

In step S1011, a reaction (hereinafter referred to as stabilization reaction) is performed in which a sample is brought into contact with a reaction solution (hereinafter referred to as stabilization reaction solution) for carrying out a modification for stabilizing lactone-formed sialic acid to stabilize lactonized sialic acid, and α 2, 3-sialic acid or the like having lactone formed in a selective lactonization reaction is modified differently from the modification performed on α 2, 6-sialic acid in step S1003 to obtain a sample for analysis.

Step S1011 may be omitted, and the sample subjected to selective lactonization may be analyzed by mass spectrometry, chromatography, a combination thereof, or the like.

In the stabilization reaction, the type of the reaction for modifying sialic acid, which forms a lactone in the selective lactonization reaction, is not particularly limited, and amidation, esterification, and the like may be performed. The amidation and esterification are preferably performed on a carboxyl group composed of carbon contained in the lactone. In the case of modification by amidation, the stabilization reaction solution may contain ammonia, an amine or a salt thereof. In the case of modification by esterification, an alcohol is contained in the stabilization reaction solution.

In other words, in step S1003, the lactone is modified from the 1 st linkage type sialic acid to the lactone to form a modified form A different from the lactone, and in this case, the modified form B having a mass different from that of the modified form A is generated from the 1 st linkage type sialic acid, and here, the difference in mass between the modified form A and the modified form B is set to be large to the extent that the modified form B can be sufficiently distinguished from the modified form A by the mass resolution in the mass spectrometry of the analysis sample, and in the above, it is preferable that the 1 st linkage type sialic acid contains α 2, 3-sialic acid, α 2, 8-sialic acid, and α 2, 9-sialic acid, and the 2 nd linkage type sialic acid contains α 2, 6-sialic acid.

When the sample for analysis obtained after the stabilization reaction is analyzed by chromatography, it is preferable that a modified form having a different substituent is formed in step S1011 depending on the mode of linkage of sialic acid, since separation by chromatography can be facilitated.

Examples of the stabilization reaction include ring opening of a lactone and subsequent amidation described in patent document 2. In this case, the lactone is opened by, for example, contacting the sample with a basic solvent, an acidic solvent, or the like. Then, amidation of the ring-opened sialic acid can be performed by bringing a stabilization reaction solution containing an amine such as a primary alkylamine such as methylamine or ethylamine or a salt thereof and a dehydration-condensation agent into contact with the sample. The dehydration condensation agent may contain a phosphonium dehydration condensation agent such as phosphonium hexafluorophosphate (PyBOP) or a uronium dehydration condensation agent. In addition, from the viewpoint of improving the amidation efficiency, N-methylmorpholine (NMM) may be added to the stabilization reaction solution.

As another example of the stabilization reaction, a stabilization reaction solution is brought into contact with a sample to convert sialic acid from a lactonized form to an amidated form at a time. In this case, a stabilization reaction solution containing an amine such as a primary alkylamine such as methylamine or ethylamine or a salt thereof is brought into contact with the sample. The above-mentioned conversion can be easily initiated by setting the pH to an alkaline condition of 8 or more, preferably 10 or more. The dehydration condensation agent is not particularly essential in this case, and may be contained in the stabilization reaction solution.

(phase for carrying out stabilization reaction)

The stabilization reaction can be carried out in the liquid phase or in the solid phase. The state of the sample when the stabilization reaction is initiated is not particularly limited as long as the sample can be brought into contact with the stabilization reaction solution, but the stabilization reaction solution is preferably brought into contact with the sample in a state where a sugar chain contained in the sample is bonded or adsorbed to a solid carrier.

When the reaction is carried out in a solid phase, the same substances as those mentioned for the selective lactonization reaction can be used as the solid phase carrier. For the immobilization of the sample on the solid support, the conditions described for the selective lactonization reaction can be used.

The sample immobilized on the solid phase carrier may be subjected to lactone modification with the stabilization reaction solution, and then the sample may be isolated from the carrier by a chemical method, an enzymatic reaction, or the like and collected. For example, glycoproteins or glycopeptides immobilized on a carrier can be enzymatically or chemically cleaved by glycosidase such as PNGase F or digestive enzyme such as trypsin, or sugar chains bound to a solid carrier having a hydrazide group can be released and recovered by a weakly acidic solution. HILIC can be prepared by subjecting a stabilization reaction solution containing acetonitrile or the like as a solvent to a stabilization reaction, and eluting a sample with an aqueous solution such as water.

By carrying out the reaction in a state where the sample is immobilized on the solid carrier, the removal of the reaction solution and the desalting and purification are facilitated, and the preparation of the sample can be simplified. In the case of using a solid carrier, when a sample is immobilized in the state of glycoprotein or glycopeptide and cleavage with glycosidase such as PNGase F is performed after stabilization reaction, the sample after lactonization reaction may be collected as free sugar chains.

After step S1011 ends, the process proceeds to step S1013.

In step S1013, a sample is analyzed by mass spectrometry, chromatography, or a combination thereof by the selective lactonization reaction described above, so that sugar chains such as α 2, 6-sialic acid which are not easily lactonized differ in mass from sugar chains such as α 2, 3-sialic acid, α 2, 8-sialic acid, α 2, 9-sialic acid which are easily lactonized.

The method of ionization in mass spectrometry is not particularly limited, and a matrix-assisted laser desorption ionization (MALDI) method, an Electrospray (ESI) method, a nano-electrospray ionization (nano-LSI) method, or the like can be used. The ionization method is particularly preferably MALDI method. Ionization in mass spectrometry may use any of a positive ion mode and a negative ion mode. Mass spectrometry can be performed in multiple stages, and thus the structure of a sugar chain or the structure of a peptide chain can be appropriately analyzed, except for the linkage form of sialic acid.

Further, the modified form produced as a result of the selective lactonization reaction and stabilization reaction can be analyzed by an analysis method other than mass spectrometry such as chromatography. The column used for liquid chromatography is not particularly limited, and a hydrophobic reverse phase column such as C30, C18, C8, and C4, a carbon column, a normal phase column for HILIC, and the like can be suitably used. For precise analysis of components in a sample by multiple separations, it is preferable to perform measurement by mass spectrometry after performing liquid chromatography. In this case, it is more preferable to ionize the eluate from the liquid chromatogram directly by ESI or the like in a mass spectrometer with on-line control.

After step S1013 is completed, the process ends.

(reagent kit for preparation of sample for analysis)

Provided is a kit for preparing an analysis sample (hereinafter referred to as a preparation kit) which is suitable for use in the method for preparing an analysis sample according to the present embodiment. The preparation kit is not particularly limited as long as it contains the above ring-opening reaction solvent such as a basic solvent having a pH of 8 or more, and may contain a reagent and any consumable used for mass spectrometry other than the reagent. By preparing the sample for analysis using the preparation kit, the sample for analysis can be prepared more efficiently.

According to the above embodiment, the following operational effects can be obtained.

(1) The method for producing an analysis sample according to the present embodiment includes a step of performing a selective lactonization reaction for lactonizing sialic acid in a1 st linkage system (α 2, 3-and the like) when sialic acid is bonded to a sugar chain and performing a modification different from lactonization on sialic acid in a 2 nd linkage system (α 2, 6-and the like) different from the 1 st linkage system, a step of performing a ring-opening reaction for ring-opening a lactone produced in the selective lactonization reaction, and a step of performing the selective lactonization reaction again.

(2) In the method for preparing an analysis sample according to the present embodiment, in the ring-opening reaction, a lactone produced by lactonization of sialic acid of the 2 nd linkage system is subjected to ring-opening. This prevents the sialic acid of the 2 nd linkage from being supplied to the stabilization reaction, mass spectrometry, and other analyses as a lactone form.

(3) The method for preparing an analysis sample according to the present embodiment further includes: the step of alternately repeating the operation of the ring-opening reaction and the selective lactonization reaction only 1 or more times. This makes it possible to reduce the proportion of sialic acid that is accidentally lactonized, and to more accurately modify sialic acid that is specific to the mode of linkage.

(4) In the method for preparing an analysis sample according to the present embodiment, the ring-opening reaction is performed by bringing a sample subjected to the selective lactonization reaction into contact with an alkaline solvent or the like having a pH of 8 or more. Thus, the preparation of the sample for analysis can be efficiently performed by utilizing the alkaline condition under which the lactone is likely to undergo ring opening.

(5) In the method for preparing an analysis sample according to the present embodiment, at least one of the reactions including the selective lactonization reaction and the ring-opening reaction can be performed in a state where the sugar chains are bound or adsorbed to the solid-phase carrier. This makes it possible to easily and efficiently prepare a sample for analysis by removing a reaction solution, desalting, purifying, and the like.

(6) In the method for preparing an analysis sample according to the present embodiment, the modification of sialic acid in the 2 nd linkage mode is esterification or amidation. This enables preparation of a sample for analysis in which sialic acid of the 2 nd linkage system is stabilized.

(7) In the method for preparing an analysis sample according to the present embodiment, a lactone produced by a selective lactonization reaction is modified differently from the modification of sialic acid in the 2 nd linkage. This makes it possible to prepare a sample for analysis in which sialic acid of the 1 st linkage system is stabilized.

(8) The analysis method of the present embodiment includes: a step of preparing an analysis sample by the method for preparing an analysis sample according to the present embodiment; and analyzing the prepared sample for analysis. This makes it possible to accurately distinguish the linkage form of sialic acid and analyze the sugar chain.

(9) In the analysis method of the present embodiment, the prepared sample for analysis can be analyzed by at least one of mass spectrometry and chromatography. This makes it possible to analyze the sugar chain by distinguishing the sialic acid linkage type from the difference in mass specifically caused by the linkage type and the influence on the separation in the chromatogram.

(10) The kit for preparing an analysis sample according to the present embodiment may include an alkaline solvent having a pH of 8 or more. This enables the solvent for ring-opening reaction to be supplied and produced quickly.

The present invention is not limited to the contents of the above embodiments. Other ways that can be conceived within the scope of the technical idea of the invention also fall within the scope of the invention.