JP2022122455A - Method and kit for labeling sugar chain - Google Patents

Abstract

To provide a method capable of obtaining analysis results equivalent to those obtained when sodium cyanoborohydride is used as a reducing agent, even in a case where picoline borane is used as a reducing agent, in labeling a sugar chain.SOLUTION: The present invention relates to: a method for labeling a sugar chain, comprising a step (A) of contacting the sugar chain with a labeling reaction solution containing a labeling compound, picoline borane and an alkali metal salt to obtain a labeled sugar chain; and a kit for labeling a sugar chain, comprising a labeling compound, picoline borane and an alkali metal salt.SELECTED DRAWING: None

Description

The present invention relates to a method and kit for labeling sugar chains.

Biopolymers such as sugar chains, glycoproteins, glycopeptides, peptides, oligopeptides, proteins, nucleic acids, and lipids play important roles in biotechnology fields such as medicine, cell engineering, and organ engineering. Elucidation of the control mechanism of biological reactions by this will lead to the development of the biotechnology field.

A sugar chain is a general term for molecules in which monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and derivatives thereof are linked in a chain by glycosidic bonds. Among biopolymers, sugar chains are extremely diverse and are deeply involved in various functions of naturally occurring organisms, such as intercellular communication, regulation of protein functions and interactions, etc. is becoming clear.

Sugar chains often exist in vivo as complex carbohydrates bound to proteins, lipids, and the like. Biopolymers with sugar chains include, for example, proteoglycans in the cell walls of plant cells that contribute to cell stabilization; glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc.; intercellular interactions and cell recognition and glycoproteins involved in The mechanisms by which the sugar chains contained in these biopolymers act as substitutes, assist, amplify, regulate, or inhibit the functions of these biopolymers and control highly precise biological reactions are being gradually clarified.

If the relationship between cell differentiation and proliferation, cell adhesion, immunity, canceration of cells, and sugar chains is clarified, new developments will be made by closely linking sugar chain engineering, medicine, cell engineering, and organ engineering. can be expected.

In addition, for example, in glycoprotein pharmaceuticals, the sugar chain often plays an important role in expression of biological activity and the like. Therefore, evaluation of sugar chains is extremely important as a quality control parameter for glycoprotein drugs. In particular, regarding antibody drugs, it has been reported that the sugar chain structure affects antibody-dependent cellular cytotoxicity (ADCC activity), and the importance of sugar chain structure analysis is increasing.

Under these circumstances, there is a demand for a method for rapid, simple, and highly accurate analysis of sugar chain structures. Sugar chains are analyzed by various methods such as analytical methods and lectin array methods.

In order to analyze sugar chains using these various methods, it is necessary to separate and purify sugar chains from proteins, peptides, lipids, nucleic acids, etc. contained in biological samples in advance. Purification and labeling of sugar chains require time and man-hours, making it difficult to prepare a large variety of samples at one time.

As a technique for solving this problem, for example, Patent Document 1 describes a method for preparing a sugar chain sample using polymer particles having functional groups for trapping sugar chains.

WO2008/018170

By the way, in order to label a sugar chain, it is necessary to bring the sugar chain into contact with a labeling compound and reduce it with a reducing agent. As a reducing agent, sodium cyanoborohydride is often used. However, since sodium cyanoborohydride is a highly toxic compound, it is desired to use a safer reducing agent such as picoline borane.

However, the inventors of the present application have found that, in conventional sugar chain labeling methods, when the reducing agent is changed from sodium cyanoborohydride to picoline borane, peak pattern fluctuation may occur during sugar chain analysis by HPLC or the like. Found it.

Therefore, the present invention provides a method for obtaining analytical results equivalent to those obtained when sodium cyanoborohydride is used as a reducing agent, even when picoline borane is used as a reducing agent, in labeling sugar chains. With the goal.

［（式（１）中、Ｒ^１，Ｒ^２は－Ｏ－，－Ｓ－，－ＮＨ－，－ＣＯ－，－ＣＯＮＨ－で中断されていてもよい炭素数１以上２０以下の炭化水素鎖を表し、Ｒ^３，Ｒ^４，Ｒ^５はＨ，ＣＨ_３又は炭素数２以上５以下の炭化水素鎖を表し、ｍ：ｎ＝９９：１～７０：３０である。］
［５］前記工程（Ａ）の後に、標識された前記糖鎖を糖鎖親和性担体に吸着させる工程（Ｂ）を更に含む、［１］～［４］のいずれかに記載の方法。
［６］標識化合物、ピコリンボラン及びアルカリ金属塩を含む、糖鎖を標識するためのキット。 The present invention includes the following aspects.
[1] A method for labeling a sugar chain, comprising the step (A) of contacting the sugar chain with a labeling reaction solution containing a labeling compound, picoline borane, and an alkali metal salt to obtain the labeled sugar chain. ,Method.
[2] The method according to [1], wherein the concentration of the alkali metal salt in the labeling reaction solution is 0.00001 mM or more and 0.1 mM or less.
[3] The method according to [1] or [2], further comprising a step (A′) of trapping the sugar chain released from the biomolecule on a carrier having a hydrazide group on its surface before the step (A). Method.
[4] The method according to [3], wherein the carrier having a hydrazide group on its surface is a carrier made of a compound having a crosslinked polymer structure represented by the following formula (1).

[(In the formula (1), R ¹ and R ² are -O-, -S-, -NH-, -CO-, -CONH-, a hydrocarbon chain having 1 or more and 20 or less carbon atoms which may be interrupted by -CONH- and R ³ , R ⁴ , and R ⁵ represent H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms, and m:n=99:1 to 70:30.]
[5] The method according to any one of [1] to [4], further comprising a step (B) of adsorbing the labeled sugar chain to a sugar chain affinity carrier after the step (A).
[6] A kit for labeling a sugar chain, containing a labeling compound, picoline borane and an alkali metal salt.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for obtaining analysis results equivalent to those obtained when sodium cyanoborohydride is used as a reducing agent even when picoline borane, which is highly safe, is used as a reducing agent in labeling sugar chains. can do.

1 is an HPLC chromatogram showing the results of Experimental Examples 1 to 5. FIG.

[Method for Labeling Sugar Chain]
In one embodiment, the present invention is a method for labeling a sugar chain, comprising contacting the sugar chain with a labeling reaction solution containing a labeling compound, picoline borane, and an alkali metal salt to obtain the labeled sugar chain. A method is provided comprising step (A).

As will be described later in Examples, according to the method of the present embodiment, even when picoline borane, which is highly safe, is used as a reducing agent in sugar chain labeling, sodium cyanoborohydride is used as a reducing agent. Analysis results equivalent to those obtained when using

In the method of this embodiment, a sugar chain is brought into contact with a labeling reaction solution containing a labeling compound, picoline borane, and an alkali metal salt to obtain the labeled sugar chain. Methods for labeling sugar chains include reductive amination methods and exchange reaction methods, and it is preferable to use reductive amination methods.

In the reductive amination method, sugar chains are labeled with a labeling compound having an amino group. In the reaction system, an organic solvent or a mixed solvent containing water and an organic solvent can be used. As an organic solvent, for example, dimethylsulfoxide (DMSO) is preferably used.

In the case of a mixed solvent containing water and an organic solvent, it is preferable that the result obtained when the pH is measured regardless of the ratio of the organic solvent is weakly acidic to basic, preferably pH 2 or more and 9 or less. It is preferably 2 or more and 8 or less, more preferably 2 or more and 7 or less.

The reaction temperature for labeling sugar chains is preferably 4° C. or higher and 90° C. or lower, more preferably 25° C. or higher and 90° C. or lower, and even more preferably 40° C. or higher and 90° C. or lower.

The labeling compound having an amino group preferably has ultraviolet-visible absorption properties or fluorescence properties. －ｔｒｉｓｕｌｐｈｏｎａｔｅ、７－ａｍｉｎｏ－１，３－ｎａｐｈｔａｌｅｎｅｄｉｓｕｌｆｏｎｉｃａｃｉｄ、２－Ａｍｉｎｏ－９（１０Ｈ）－ａｃｒｉｄｉｎｏｎｅ、５－Ａｍｉｎｏｆｌｕｏｒｅｓｃｅｉｎ、Ｄａｎｓｙｌｅｔｈｙｌｅｎｅｄｉａｍｉｎｅ、２－Ａｍｉｎｏｐｙｒｉｄｉｎｅ、７－Ａｍｉｎｏ－４－ｍｅｔｈｙｌｃｏｕｍａｒｉｎ、２－Ａｍｉｎｏｂｅｎｚａｍｉｄｅ、２－Ａｍｉｎｏｂｅｎｚｏｉｃ ａｃｉｄ , 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3-(Acetylamino)-6-aminoacridine, 2-Amino-6-cyanoethylpyridine, Ethyl p-aminobenzoate, p-Aminobenzonitrile, 7-Aminobenzoic-3, 7-Aminobenzoate At least one selected from acid and procainamide is preferable, and 2-aminobenzamide is particularly preferable.

The concentration of the labeling compound in the labeling reaction solution is preferably 1 mM or more and 10 M or less, more preferably 10 mM or more and 10 M or less, and even more preferably 100 mM or more and 3 M or less.

In the method of this embodiment, picoline borane is used as a reducing agent in the sugar chain labeling reaction. Picoline borane is safer than the conventionally used sodium cyanoborohydride.

The concentration of picoline borane in the labeling reaction solution is preferably 1 mM or more and 10 M or less, more preferably 10 mM or more and 10 M or less, and even more preferably 100 mM or more and 2 M or less.

The reaction time for sugar chain labeling is 10 minutes to 24 hours, preferably 10 minutes to 8 hours, and more preferably 10 minutes to 3 hours.

As will be described later in Examples, the inventors of the present application found that changing the reducing agent from sodium cyanoborohydride to picoline borane in the labeling of sugar chains caused variations in the peak pattern during sugar chain analysis by HPLC or the like. found that there is From the results of mass spectrometry, it was considered that sugar chains having a lactone structure were produced as by-products as a cause of peak pattern fluctuations.

Furthermore, the inventors added an alkali metal salt to the labeling reaction solution brought into contact with the sugar chain to dissolve the lactone structure of the sugar chain. It was clarified that analytical results equivalent to those obtained when sodium cyanoborohydride was used as the The inventors further clarified that when an alkaline earth metal salt is used instead of an alkali metal salt, the effect of suppressing peak pattern fluctuation tends to be less noticeable.

Alkali metal salts include lithium salts, sodium salts, potassium salts and the like. More specific examples include lithium chloride, lithium carbonate, lithium hydrogen carbonate, sodium chloride, sodium carbonate, sodium hydrogen carbonate, potassium chloride, potassium carbonate, potassium hydrogen carbonate, sodium hydrogen phosphate and the like. The alkali metal salt may be either an anhydride or a hydrate. Alkali metal salts may be used singly or in combination of two or more.

The concentration of the alkali metal salt in the labeling reaction solution is preferably 0.00001 mM or more and 0.1 mM or less. As will be described later in Examples, within the above range, even when picoline borane is used as a reducing agent, the effect of obtaining the same analysis results as when sodium cyanoborohydride is used as a reducing agent is obtained. can get.

In the method of this embodiment, the sugar chain to be labeled may be a sugar chain contained in a biological sample (biological sample). Biological samples include, for example, whole blood, serum, plasma, urine, saliva, cells, tissues, viruses, plant tissues, and the like. As a biological sample, purified glycoproteins or unpurified glycoproteins may be used. The biological sample may be pretreated by methods such as degreasing, desalting, protein fractionation, and heat denaturation.

Sugar chains can be released from biomolecules (eg, glycoproteins) containing sugar chains in a biological sample using the sugar chain releasing means. As means for releasing sugar chains, methods such as glycosidase treatment using N-glycosidase or O-glycosidase, hydrazinolysis, and β-elimination by alkali treatment can be used. When analyzing N-type sugar chains, a method using N-glycosidase is preferred. Protease treatment using trypsin, chymotrypsin, or the like may be performed prior to glycosidase treatment.

In the method of this embodiment, the sugar chain to be labeled may be captured using a carrier having a hydrazide group on its surface. That is, the method of the present embodiment may further include a step (A') of capturing the sugar chain released from the biomolecule on a carrier having a hydrazide group on its surface before step (A).

In this case, the method of the present embodiment comprises a step (A') of capturing the sugar chain released from the biomolecule on a carrier having a hydrazide group on its surface, and adding a labeling compound, picoline borane and an alkali metal salt to the sugar chain. and a step (A) of obtaining the labeled sugar chain by contacting the labeling reaction solution containing the labeling reaction solution.

Sugar chains are in equilibrium between the cyclic hemiacetal type and the non-cyclic aldehyde type in a solution such as an aqueous solution. A hydrazide group reacts with an aldehyde group of an aldehyde-type sugar chain to form a specific and stable bond. As a result, the sugar chain can be trapped on the carrier.

The compound or carrier having a hydrazide group “—C(=O)—NHNH ₂ ” is not particularly limited as long as the sugar chain to be brought into contact can interact with the hydrazide group. It may be in any form such as micelles, liposomes, soluble polymers and the like.

Among these, solid particles and gel particles can be suitably used as the polymer particles. When such polymer particles are used, the polymer particles can be easily collected by means of centrifugation, filtration, or the like after the sugar chains are captured by the polymer particles.

It is also possible to use polymer particles packed in a column. The method of using it by packing it in a column is particularly important from the viewpoint of continuous operation. Moreover, by using a filter plate (for example, MultiScreen Solvinert Filter Plate manufactured by Millipore) as a reaction container, it is possible to process a plurality of samples simultaneously. In this case, for example, the throughput of sugar chain purification is greatly improved compared to conventional purification means by column operation typified by gel filtration. In addition, if magnetic beads are used as the polymer particles, the beads can be accumulated on the wall surface of the container or the like using magnetic force, so that the beads can be washed easily.

The shape of the polymer particles is not particularly limited, but spherical or similar shapes are preferred. When the polymer particles are spherical, the average particle diameter is preferably 0.05 μm or more and 1000 μm or less, more preferably 0.05 μm or more and 200 μm or less, further preferably 0.1 μm or more and 200 μm or less. .1 μm or more and 100 μm or less is particularly preferable.

When the average particle diameter of the polymer particles is at least the lower limit, when the polymer particles are packed in a column and used, good liquid permeability can be maintained, eliminating the need to apply a large pressure. In addition, it becomes easy to collect the polymer particles by centrifugation or filtration. In addition, since the average particle diameter of the polymer particles is equal to or less than the upper limit, the contact area between the polymer particles and the sample solution can be ensured, and the efficiency of sugar chain capture can be enhanced. In addition, it becomes easy to suck up the polymer particles with a pipette or the like.

Examples of carriers having hydrazide groups on their surfaces include carriers composed of compounds having a crosslinked polymer structure represented by the following formula (1).

In formula (1), R ¹ and R ² represent a hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted by -O-, -S-, -NH-, -CO-, -CONH-. , R ³ , R ⁴ and R ⁵ represent H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms. Each of R ¹ and R ² has 1 or more 20 carbon atoms, which may be interrupted by substitution of -C- with -O-, -S-, -NH-, -CO-, -CONH- It can be the following hydrocarbon chains: Also, m and n represent the number of monomer units, and the ratio of m and n is m:n=99:1 to 70:30.

A more specific example of the carrier composed of the compound represented by the above formula (1) is a carrier composed of the compound represented by the following formula (2). As the carrier (polymer particles) composed of the compound represented by the following formula (2), a commercially available carrier (BlotGlyco (registered trademark), product number “BS-45407”, manufactured by Sumitomo Bakelite Co., Ltd.) can be suitably used. can.

In formula (2), m and n represent the numbers of monomer units, and the ratio of m and n is m:n=99:1 to 70:30.

The pH of the reaction system when the sugar chain is trapped on the carrier having a hydrazide group on its surface is preferably 2 or more and 9 or less, more preferably 2 or more and 7 or less, and 2 or more and 6 or less. More preferred. Various buffers can be used for pH adjustment.

A mixed solvent of an acid and an organic solvent can also be used as a solvent for trapping sugar chains. When using a mixed solvent of an acid and an organic solvent, the acid is not particularly limited, and examples thereof include acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid, and sulfuric acid. Among them, acetic acid, formic acid, trifluoroacetic acid and phosphoric acid are preferred, and acetic acid and trifluoroacetic acid are more preferred.

Moreover, the organic solvent is not particularly limited as long as it dissolves the sugar chain and the above acid. Drying up the sugar chain during capture often increases the capture efficiency, so an organic solvent with a relatively low boiling point such as acetonitrile is preferably used.

The mixing ratio (volume ratio) of the acid and the organic solvent is preferably 0.1:99.9 to 10:90, more preferably 0.5:99.5 to 5:95, and 1: More preferably 99-5:95.

The temperature at which the sugar chain is captured is preferably 4°C or higher and 90°C or lower, more preferably 30°C or higher and 90°C or lower, even more preferably 30°C or higher and 80°C or lower, and 40°C or higher and 80°C or higher. °C or less is particularly preferred. The reaction time for capturing sugar chains can be set appropriately. A sugar chain may be captured by filling a column with a carrier having a hydrazide group on its surface and allowing a sample solution containing the sugar chain to pass through.

Subsequently, the carrier bound to the sugar chain (capturing the sugar chain) is washed. As a result, substances other than sugar chains (non-specifically adsorbed substances) can be removed from the substances captured by the carrier.

Methods for removing substances other than sugar chains include washing with an aqueous solution of guanidine, which is a chaotropic reagent capable of dissociating hydrophobic bonds, and pure water or water-soluble buffers (e.g., phosphate buffer, Tris buffer, etc.). ) can be used.

As for the washing conditions, the temperature is preferably 4° C. or more and 40° C. or less, and the washing time is preferably 10 seconds or more and 30 minutes or less. Examples of the washing method include immersing the carrier in a washing liquid and repeating exchange of the washing liquid.

Specifically, the carrier is placed in a centrifuge tube or tube, a washing solution is added, the carrier is shaken, the carrier is precipitated by centrifugation, and the supernatant is removed. Precipitation of the carrier can be carried out by natural sedimentation or by centrifugation. It is preferable to perform the washing operation 3 times or more and 6 times or less. When magnetic beads are used as a carrier, centrifugation is not required, which is convenient.

Moreover, it is also possible to use a filter tube which is a tube-shaped container and has a filter with a pore size that is permeable to the liquid but impermeable to the carrier at the bottom. By putting the carrier in a filter tube and using it, it is possible to remove the washing solution required for washing through the filter, and the process of removing the supernatant after the above-mentioned centrifugation operation is unnecessary, resulting in workability. can be improved.

In addition, various 6- to 384-well multiwell plates with filters attached to the bottom are commercially available, and high throughput can be achieved by using these plates. In particular, the 96-well multi-well plate is most suitable for high-throughput, for which a solution pipetting device, an aspiration removal system, a plate transfer system, etc. have been developed.

After carrying out the sugar chain-capturing reaction, a washing solution may be passed through the column to perform washing treatment continuously from the sugar chain-capturing reaction. In addition, when a multiwell plate is used, substances other than the carrier that captures sugar chains may be removed by filtration or centrifugation.

Excess hydrazide groups on the carrier having hydrazide groups on its surface can be capped using, for example, acetic anhydride.

When step (A') is carried out, it is preferable to liberate the sugar chains captured by the carrier having hydrazide groups on its surface after step (A') and before step (A). Sugar chains can be released by acid treatment. The acid treatment can be carried out by contacting the sugar chain-entrapped carrier with a mixed solvent of an acid and an organic solvent, or a mixed solvent of an acid, water and an organic solvent.

In the case of a mixed solvent of an acid, water and an organic solvent, the proportion (volume ratio) of water is preferably 0.1% or more and 90% or less, more preferably 0.1% or more and 80% or less, More preferably, it is 0.1% or more and 50% or less.

Aqueous buffers may be used in place of water. The concentration of the buffering agent in the aqueous buffer is preferably 0.1 mM or more and 1 M or less, more preferably 0.1 mM or more and 500 mM or less, and even more preferably 1 mM or more and 100 mM or less.

The pH of the mixed solvent of acid and organic solvent or the mixed solvent of acid, water and organic solvent is preferably 2 or more and 9 or less, more preferably 2 or more and 7 or less, and 2 or more and 6 or less. is more preferred. By carrying out the sugar chain release reaction at weakly acidic to near neutral conditions, the release reaction of the sugar chain, such as elimination of the sialic acid residue, can be improved compared with the release reaction of the sugar chain by, for example, treatment with 10% trifluoroacetic acid, which is a strong acid. can suppress the hydrolysis of

Examples of the acid in the mixed solvent of acid and organic solvent or the mixed solvent of acid, water and organic solvent include acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid and sulfuric acid. Among them, acetic acid, formic acid, trifluoroacetic acid and phosphoric acid are preferred, and acetic acid and trifluoroacetic acid are more preferred.

The acid treatment temperature is preferably 4° C. or higher and 90° C. or lower, more preferably 25° C. or higher and 90° C. or lower, and even more preferably 40° C. or higher and 90° C. or lower. The acid treatment time is preferably 10 minutes or more and 24 hours or less, more preferably 10 minutes or more and 8 hours or less, and even more preferably 10 minutes or more and 3 hours or less.

From the viewpoint of efficiently liberating sugar chains, the acid treatment is preferably carried out in an open system to completely evaporate the solvent.

In the method of the present embodiment, the reaction product after step (A) includes unreacted labeled compounds, reducing agents, salts, etc., in addition to the labeled sugar chains. Therefore, it is preferable to remove these substances before analysis of the labeled sugar chain.

Examples of the removal method include removal by solid phase extraction using a carrier with affinity for sugar chains such as silica columns, removal by gel filtration, removal by ion exchange resin, and the like. Any method may be used, but sialic acid The pH of the solvent used to prevent the separation of is preferably near neutral. As a result, it becomes possible to prepare a labeled sugar chain sample from which substances other than the labeled sugar chain have been removed.

Among them, it is preferable to adsorb a solution containing a labeled sugar chain to a sugar chain affinity carrier, wash the carrier, and then elute the labeled sugar chain from the sugar chain affinity carrier.

That is, the method of the present embodiment further includes a step (B) of adsorbing the labeled sugar chain to the sugar chain affinity carrier after step (A), and the sugar chain is captured by the sugar chain affinity carrier. It is preferable to wash the sugar chain in a state where the sugar chain is washed, and then to elute the labeled sugar chain from the sugar chain affinity carrier.

The sugar chain affinity carrier includes, for example, a carrier containing silica, which may be composed of silica alone or may be of a silica-organic hybrid type. Also, the shape thereof is not particularly limited, and may be particulate, plate-like, fibrous, porous, or the like. Further, the silica may be silica having a silanol group, or may be one whose surface is modified with an aminopropyl group or the like.

The sample solution for adsorbing the labeled sugar chain to the sugar chain affinity carrier is preferably a solution containing more than 95% by volume of an organic solvent, preferably a solution containing 96% by volume or more of an organic solvent. A solution containing 98% by volume or more of an organic solvent is more preferable, and a solution containing 99% by volume or more of an organic solvent is particularly preferable.

As the organic solvent, acetonitrile, hexane, ethyl acetate, methylene chloride, tetrahydrofuran, etc. can be used, and acetonitrile is particularly preferred.

After the labeled sugar chain is adsorbed on the sugar chain affinity carrier by the above operation, the labeled sugar chain is purified by washing and removing unnecessary substances other than the labeled sugar chain by passing a washing solution through the carrier. be able to. The cleaning liquid preferably contains 90% by volume or more of the organic solvent and 10% by volume or less of water, and more preferably contains 95% by volume or more of the organic solvent and 5% by volume or less of water. As the organic solvent contained in the washing liquid, acetonitrile, methanol, ethanol, 2-propanol, hexane, ethyl acetate, methylene chloride, tetrahydrofuran, etc. can be used, and acetonitrile is particularly preferred. A plurality of types of cleaning solutions having different compositions may be used, for example, after cleaning with 100% acetonitrile, further cleaning may be performed with a mixed solution of acetonitrile:water=95:5 (v/v).

After washing, an eluate is added to the sugar chain affinity carrier to elute the labeled sugar chain. The eluate preferably contains 10% by volume or more of water, more preferably 50% by volume or more of water, and particularly preferably water. Components other than water contained in the eluate include acetonitrile and organic solvents selected from alcohols represented by methanol, ethanol, propanol, and butanol.

Through the above steps, a labeled sugar chain can be obtained. Labeled sugar chains can be analyzed by known methods such as mass spectrometry (eg, LC-MS, MALDI-TOF MS, etc.), chromatography (eg, HPLC, HPAE-PAD, etc.), electrophoresis (eg, capillary electrophoresis, etc.). can be analyzed by In the analysis of sugar chains, various databases (eg, GlycoMod, Glycosuite, SimGlycan, etc.) can be referred to as necessary.

[kit]
In one embodiment, the present invention provides a kit for labeling sugar chains comprising a labeling compound, picoline borane and an alkali metal salt.

In the kit of this embodiment, the labeling compound, picoline borane, and alkali metal salt are the same as those described above. The kit of the present embodiment can be suitably used for the purpose of carrying out the above-described sugar chain labeling method.

EXAMPLES The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples.

[Experimental example 1]
(Glycan Analysis 1 of Fetuin Derived from Calf Serum)
The sugar chain of fetuin derived from calf serum was labeled with 2-aminobenzamide (2-AB) and analyzed by HPLC. A labeling reaction solution containing sodium cyanoborohydride as a reducing agent was used. Further, as described later, the labeled sugar chain was adsorbed to a cleanup column, washed, and then recovered by elution from the cleanup column with pure water.

<<Pretreatment of sample>>
After 1 mg of calf serum-derived fetuin was dissolved in 50 μL of 100 mM ammonium bicarbonate, 5 μL of 120 mM dithiothreitol (DTT) was added and reacted at 60° C. for 30 minutes. After completion of the reaction, 10 μL of 123 mM iodoacetamide (IAA) was added and allowed to react for 1 hour at room temperature in the dark. Subsequent protease treatment with 400 U of trypsin resulted in peptide fragmentation of the protein portion. Subsequently, after treating the reaction solution at 90° C. for 5 minutes, 5 U of N-glycosidase was reacted to cleave the N-type sugar chain from the peptide.

《Trapping and Washing of Sugar Chain》
A commercially available sugar chain purification labeling kit (BlotGlyco (registered trademark), product number “BS-45407”, manufactured by Sumitomo Bakelite Co., Ltd.) was used. The kit included a carrier with hydrazide groups on its surface (BlotGlyco® beads). BlotGlyco (registered trademark) beads were compounds having a crosslinked polymer structure represented by the following formula (2).

［式（２）中、ｍ，ｎはモノマーユニット数を表し、ｍとｎの比はｍ：ｎ＝９９：１～７０：３０である。］

[In formula (2), m and n represent the number of monomer units, and the ratio of m and n is m:n=99:1 to 70:30. ]

First, 20 μL of pretreated calf serum-derived Fetuin suspension and 180 μL of 2% acetic acid/acetonitrile solution were added to a disposable column containing 5 mg of BlotGlyco (registered trademark) beads, and reacted at 80° C. for 1 hour. . The reaction was carried out in an open system, and it was visually confirmed that the solvent had completely evaporated and the content of the column had dried up at the end of the reaction.

Subsequently, after washing the column with guanidine solution, pure water, methanol and 1% triethylamine-methanol solution, 10% acetic anhydride/methanol was added and allowed to react at room temperature for 30 minutes to cap unreacted hydrazide groups. After capping, the column was washed with pure water.

《Liberation of sugar chains》
Subsequently, 20 μL of pure water and 180 μL of a 2% acetic acid/acetonitrile solution were added to the column and reacted at 70° C. for 1.5 hours. The reaction was carried out in an open system, and at the end of the reaction, it was visually confirmed that the solvent had completely evaporated and the contents of the column were in a dry state.

《Glycan labels》
Subsequently, 2-AB and sodium cyanoborohydride were added to the column at final concentrations of 1 M and 350 mM, respectively, in a mixed solvent of acetic acid, dimethyl sulfoxide (DMSO) and water (acetic acid: DMSO: water (volume ratio )=3:1:2) was added thereto, and reacted at 50° C. for 1 hour.

《Removal of surplus reagent》
Subsequently, the column was centrifuged to collect 50 μL of the reaction solution containing the labeled sugar chain and unreacted 2-AB. Subsequently, the collected reaction solution was diluted 20-fold with acetonitrile, and the cleanup column (silica gel) included in the sugar chain purification labeling kit (BlotGlyco (registered trademark), product number “BS-45407”, manufactured by Sumitomo Bakelite Co., Ltd.) was used. carrier), and the labeled sugar chain was adsorbed on the cleanup column.

Subsequently, the cleanup column was washed twice with washing solution. A mixed solution of acetonitrile and water (acetonitrile:water (volume ratio)=95:5) was used as a washing liquid. Subsequently, the labeled sugar chain was eluted with 50 μL of pure water and collected.

《Analysis of glycans》
Subsequently, the labeled sugar chains were analyzed by HPLC. The labeled sugar chain was applied to an amide column (ACQUITY UPLC(R) Glycan BEH Amide, 1.7 μm, 2.1×150 mm) and measured at an excitation wavelength of 330 nm and a fluorescence wavelength of 420 nm. FIG. 1 shows an HPLC chromatogram in which 2-AB-labeled sugar chains were detected. The horizontal axis indicates elution time, and the vertical axis indicates signal intensity (relative value).

[Experimental example 2]
(Glycan Analysis 2 of Fetuin Derived from Calf Serum)
The sugar chain of fetuin derived from calf serum was labeled with 2-AB and analyzed by HPLC. The labeled sugar chains were analyzed by HPLC in the same manner as in Experimental Example 1, except that a labeling reaction solution containing picoline borane instead of sodium cyanoborohydride was used as a reducing agent.

Specifically, as the labeling reaction solution, 2-AB and picoline borane were added to a final concentration of 2.6 M and 1.7 M, respectively, in a mixed solvent of acetic acid, dimethyl sulfoxide (DMSO) and water (acetic acid: DMSO : water (volume ratio) = 3:1:2).

FIG. 1 shows an HPLC chromatogram in which 2-AB-labeled sugar chains were detected. As a result, the peak pattern indicated by the arrow was different from the result of Experimental Example 1. Mass spectrometric analysis revealed that the peak indicated by the arrow in FIG. 1 was a sugar chain having a lactone structure.

[Experimental example 3]
(Glycan Analysis of Calf Serum-Derived Fetuin 3)
The sugar chain of fetuin derived from calf serum was labeled with 2-AB and analyzed by HPLC. The labeled sugar chain was analyzed by HPLC in the same manner as in Experimental Example 2, except that an alkali metal salt was added to the labeling reaction solution.

Specifically, as a labeling reaction solution, 2-AB, picoline borane, and sodium chloride were added to final concentrations of 2.6 M, 1.7 M, and 0.01 mM, respectively, in acetic acid, dimethyl sulfoxide (DMSO), and water. was dissolved in a mixed solvent (acetic acid: DMSO: water (volume ratio) = 3:1:2) to prepare a labeling reaction solution.

FIG. 1 shows an HPLC chromatogram in which 2-AB-labeled sugar chains were detected. As a result, it was found that the lactone ring peak disappeared from the peak pattern of Experimental Example 3, and a pattern equivalent to the peak pattern of Experimental Example 1 was obtained.

Even when picoline borane, a safe reducing agent, is used, the addition of an alkali metal salt to the labeling reaction solution is superior to the conventional reducing agent, sodium cyanoborohydride. It shows that equivalent peak patterns can be obtained.

[Experimental example 4]
(Glycan Analysis of Calf Serum-Derived Fetuin 4)
The sugar chain of fetuin derived from calf serum was labeled with 2-AB and analyzed by HPLC. The labeled sugar chain was analyzed by HPLC in the same manner as in Experimental Example 2, except that an alkali metal salt was added to the labeling reaction solution.

Specifically, as a labeling reaction solution, 2-AB, picoline borane, and potassium chloride were added to final concentrations of 2.6 M, 1.7 M, and 0.01 mM, respectively, in acetic acid, dimethyl sulfoxide (DMSO), and water. was dissolved in a mixed solvent (acetic acid: DMSO: water (volume ratio) = 3:1:2) to prepare a labeling reaction solution.

FIG. 1 shows an HPLC chromatogram in which 2-AB-labeled sugar chains were detected. As a result, it was found that the peak of the lactone ring disappeared from the peak pattern of Experimental Example 4, and a pattern equivalent to the peak pattern of Experimental Example 1 was obtained.

By adding an alkali metal salt to the labeling reaction solution, even when picoline borane, a safe reducing agent, was used, the results were significantly lower than when using a conventional reducing agent, sodium cyanoborohydride. It further supports that comparable peak patterns can be obtained.

[Experimental example 5]
(Glycan Analysis of Calf Serum-Derived Fetuin 5)
The sugar chain of fetuin derived from calf serum was labeled with 2-AB and analyzed by HPLC. The labeled sugar chain was analyzed by HPLC in the same manner as in Experimental Example 2, except that an alkali metal salt was added to the labeling reaction solution.

Specifically, as a labeling reaction solution, 2-AB, picoline borane, and lithium chloride were added to final concentrations of 2.6 M, 1.7 M, and 0.01 mM, respectively, in acetic acid, dimethyl sulfoxide (DMSO), and water. was dissolved in a mixed solvent (acetic acid: DMSO: water (volume ratio) = 3:1:2) to prepare a labeling reaction solution.

FIG. 1 shows an HPLC chromatogram in which 2-AB-labeled sugar chains were detected. As a result, it was found that the peak of the lactone ring disappeared from the peak pattern of Experimental Example 5, and a pattern equivalent to the peak pattern of Experimental Example 1 was obtained.

By adding an alkali metal salt to the labeling reaction solution, even when picoline borane, a safe reducing agent, was used, the results were significantly lower than when using a conventional reducing agent, sodium cyanoborohydride. It further supports that comparable peak patterns can be obtained.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for obtaining analysis results equivalent to those obtained when sodium cyanoborohydride is used as a reducing agent even when picoline borane, which is highly safe, is used as a reducing agent in labeling sugar chains. can do.

Claims (6)

A method for labeling a sugar chain, comprising:
A method comprising a step (A) of contacting the sugar chain with a labeling reaction solution containing a labeling compound, picoline borane and an alkali metal salt to obtain the labeled sugar chain. 2. The method according to claim 1, wherein the concentration of said alkali metal salt in said labeling reaction solution is 0.00001 mM or more and 0.1 mM or less. 3. The method according to claim 1 or 2, further comprising a step (A') of capturing the sugar chains released from the biomolecules on a carrier having a hydrazide group on its surface before step (A). 4. The method according to claim 3, wherein the carrier having a hydrazide group on its surface is a carrier comprising a compound having a crosslinked polymer structure represented by the following formula (1).

[(In the formula (1), R ¹ and R ² are -O-, -S-, -NH-, -CO-, -CONH-, a hydrocarbon chain having 1 or more and 20 or less carbon atoms which may be interrupted by -CONH- and R ³ , R ⁴ , and R ⁵ represent H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms, and m:n=99:1 to 70:30.] The method according to any one of claims 1 to 4, further comprising a step (B) of adsorbing the labeled sugar chain to a sugar chain affinity carrier after the step (A). A kit for labeling sugar chains, comprising a labeling compound, picoline borane and an alkali metal salt.

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