JP2013142566A - Preparation method of sugar chain sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method with which excessive labeling reagents can be removed efficiently even under high throughput, the method being provided for removing the excessive labeling reagents by using a column shape in which the inside of a channel is filled with monolith silica.SOLUTION: The method uses a plate in which a plurality of columns are disposed and in each column, the inside of a channel is filled with monolith silica of which the macro diameter is 1 to 20 μm. Therefore, even if a plurality of sugar chain samples are processed simultaneously, while a high sugar chain recovery rate is maintained, excessive labeling reagents can be removed effectively. Further, the sugar chain sample can also be processed promptly by passing a monolith silica part of the column through suction or the like.

Description

  The present invention relates to a method for preparing a labeled sugar chain sample, a sugar chain sample prepared by the method, and a method for analyzing a sugar chain using the sugar chain sample.

  The 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 form by glycosidic bonds.

  Sugar chains are very diverse and are substances that are involved in various functions of naturally occurring organisms. Sugar chains often exist as complex carbohydrates bound to proteins, lipids, and the like in vivo, and are one of the important components in vivo. It is becoming clear that sugar chains in the body are deeply involved in cell-to-cell information transmission, protein function and interaction regulation.

  For example, biopolymers having sugar chains include plant cell wall proteoglycans that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc., and cell-cell interactions and cells. Examples include glycoproteins involved in recognition. The mechanisms by which these high-molecular sugar chains control advanced and precise biological reactions while acting, assisting, amplifying, regulating, or inhibiting each other's functions are gradually being clarified. Furthermore, if the relationship between these sugar chains and cell differentiation / proliferation, cell adhesion, immunity, and cell carcinogenesis is clarified, sugar engineering and medicine, cell engineering, or organ engineering are closely related. Therefore, we can expect new developments in research and development.

  In order to detect diseases early and to maintain a high quality of life (QOL), biomarkers that can prevent the development of diseases and diagnose their transition are necessary. Analysis of glycosyltransferase gene-disrupted mice involved in sugar chain biosynthesis reveals that sugar chains are essential for maintaining the functions of various tissues and organs (Non-Patent Documents 1 and 2). It is also known that various diseases are caused when an abnormality is observed in sugar chain modification (Non-patent Document 3). Since the structure of the sugar chain changes markedly depending on the canceration of cells and various diseases, it is expected to be used as a biomarker for examining the transition of diseases.

  Since sugar chains themselves do not have fluorescence or ultraviolet absorptivity, they are often modified (labeled) in advance when analyzing sugar chains. When analyzing sugar chains by HPLC or HPLC-MS, it is common to apply fluorescent labeling such as 2-aminobenzamide derivatization (2AB) and 2-aminopyridine derivatization (PA) (Non-patent literature) 4). Moreover, when analyzing a sugar chain by MALDI-TOF MS, the measurement sensitivity is improved by labeling (Non-Patent Documents 5 and 6).

  When labeling a sugar chain, an excess amount of a labeling reagent is often allowed to act on the sugar chain in order to increase the labeling efficiency. If an excessive amount of labeling reagent is present in the sample solution, it will hinder HPLC measurement and mass spectrometry, so it must be removed in advance. For removal of the reagent, methods such as gel filtration (for example, using Sephadex G-15, Non-Patent Document 6) and solid phase extraction (for example, using silica gel, graphite carbon, etc.) are used. Conventionally, when these separations are performed, those in which an appropriate container (column, cartridge, etc.) is filled with a solid phase (packing material) have been mainly used. In order to improve the separation performance of these columns (cartridges), a method of either increasing the packing density of the solid phase or decreasing the number average diameter of the packing material is usually used. However, in the former, the packing density does not increase as expected due to variations in the shape and diameter of the packing material, and in the latter, the pressure load on the column and the apparatus increases, so that the processing speed tends to be limited. In a small-scale column (cartridge), the dead volume of the frit (bead blocking member) necessary to hold the packing tends to cause a reduction in separation performance and recovery rate. Therefore, in order to solve these problems, a method using a column filled with monolithic silica in the flow path has been developed (Patent Document 1).

International Publication No. 2009-150834

Ioffe E., Stanley P., Proc. Natl. Acad. Sci., 91, pp. 728-732 (1994) Metzler M., Gertz A., Sarker M., Schachter H., Schrader J.W., Marth J.D., EMBO J., 13, pp.2056-2065 (1994) Powell L.D., Paneerselvam K., Vij R., Diaz S., Manzi A., Buist N., Freeze H., Varki A., J. Clin. Invest., 94, pp. 1901-1909 (1994) Shilova N.V., Bovin N.V., Russian Journal of Bioorganic Chemistry, 29, pp.309-324 (2003) Shinohara Y., Furukawa J.-i., Niikura K., Miura Y., Nishimura S.-I., Anal. Chem., 76, pp.6989-6997 (2004) Furukawa J.-i., Shinohara Y., Kuramoto H., Miura Y., Shimaoka H., Kurogochi M., Nakano M., Nishimura S.-I., Anal. Chem., 80, pp.1094-1101 (2008)

  An object of the present invention is a method for preparing a glycan sample characterized by removing excess labeling reagent using a column packed with monolithic silica in the flow path, and is efficient even when the throughput is increased. It is an object of the present invention to provide a method capable of removing excessive labeling reagent.

  As a result of intensive studies to solve the above problems, the present inventors have achieved high throughput by using a plate in which a plurality of columns filled with monolithic silica having a macro diameter of 1 to 20 μm are arranged in the flow path. Found that is possible. That is, according to this plate, even when a plurality of sugar chain samples are processed simultaneously, the present inventors can effectively remove excess labeling reagent while maintaining a high sugar chain recovery rate. In addition, the inventors have found that it is possible to perform a rapid treatment by passing a sugar chain sample through the monolithic silica portion of the column by suction (vacuum) or the like, and the present invention has been completed.

  More specifically, the present invention provides the following inventions.

(1) A method for preparing a sugar chain sample,
(Step 1) By injecting a sample solution containing labeled sugar chains into a plate in which a plurality of columns filled with a monolithic silica having a macro diameter of 1 to 20 μm in the flow path is arranged, sugar is added to the monolithic silica. Adsorbing chain components,
(Step 2) A step of washing the monolithic silica with a washing liquid,
(Step 3) A step of contacting the eluate with monolithic silica to elute the adsorbed sugar chain component
A method comprising the steps of:

  (2) The preparation method according to (1), wherein in step 1, the sample solution is passed through the column by suction, natural drop, pressurization, or centrifugation.

  (3) The preparation method according to (1) or (2), wherein in step 3, the eluate is a solution containing 20% or more of water.

  (4) A sugar chain sample prepared by the method according to any one of (1) to (3).

  (5) A plate in which a plurality of columns filled with monolithic silica having a macro diameter of 1 to 20 μm in a flow path is used for carrying out the method according to any one of (1) to (3).

  (6) A method for analyzing a sugar chain, comprising analyzing the sugar chain sample according to (4) by HPLC, mass spectrometry, LC-MS, or capillary electrophoresis.

  According to the method for preparing a sugar chain sample of the present invention using a plate in which a plurality of columns filled with monolithic silica having a macro diameter of 1 to 20 μm in the flow path are arranged, it becomes possible to increase the throughput. That is, according to the present invention, even when a plurality of sugar chain samples are processed at the same time, unreacted labeling reagent in the sample solution containing labeled sugar chains can be reduced, and sugar chain analysis can be made highly efficient. It becomes possible. In addition, the sample solution can be passed through the monolithic silica portion of the column by suction or the like, and rapid processing is also possible. Furthermore, it is possible to suppress variations in the amount of sugar chain recovered.

FIG. 1 is a diagram showing the HPLC chart obtained in Example 1. FIG. 2 is a graph showing the results of comparing the total amount of area values of the sugar chain-derived peaks in HPLC obtained in Examples 1 and 2 and Comparative Example 1.

(Glycan sample)
As the sugar chain sample used in the present invention, for example, those prepared from biological samples such as whole blood, serum, plasma, urine, saliva, cells, tissues, viruses, plant tissues, and the like can be used. In addition, purified or prepared from unpurified glycoprotein can be used. A pure sugar chain purified separately may be used as a sample. The sugar chain is released from the glycoprotein contained in the biological sample using a sugar chain releasing means. As means for releasing the sugar chain, methods such as glycosidase treatment using N-glycosidase or O-glycosidase, hydrazine degradation, and β elimination by alkali treatment can be used. When N-type sugar chains are analyzed, a method using N-glycosidase is preferable. Prior to glycosidase treatment, protease treatment may be performed using trypsin, chymotrypsin, or the like.

(Glycan purification)
Prior to labeling the sugar chain, it is preferable to remove impurities other than the sugar chain (eg, protein, peptide, nucleic acid, lipid, etc.). As a method for removing impurities, for example, gel filtration purification, ultrafiltration, dialysis, solid phase extraction using polarity, purification using an ion exchange resin, precipitation using a difference in solubility, and the like can be used. Purification using a solid phase carrier that selectively binds to a sugar chain (for example, BlotGlyco BS-45603 manufactured by Sumitomo Bakelite Co., Ltd., see Non-patent Document 6) can also be used. The sugar chain may be labeled without removing impurities.

(Labeling of sugar chains)
A sample containing a sugar chain is dried as necessary, and then labeled with a labeling reagent to be labeled. The labeling reagent is preferably a substance containing a functional group such as an amino group, a hydrazide group, or an aminooxy group that is reactive with the reducing end of the sugar chain.

  The substance having an amino group is preferably selected from the group of compounds listed below, but is not limited thereto.

2-aminopyridine; 2-aminobenzamide; 2-aminoanthranilic acid; 7-amino-1-naphthol; 3- (acetylamino) -6-aminoacridine; 9-aminopyrene-1,4,6-trisulfonic acid; 8-aminonaphtalene-1, 3,6-trisulfonic acid; 7-amino-1,3-naphtalenedisulfonic acid: 2-amino-9 (10H) -acridone; 5-aminofluorescein; Dansylethylenediamine; 7-amino-4-methylcoumarine; benzylamine.
Among these compounds, 2-aminopyridine (PA), 2-aminobenzamide (2AB), 2-aminoanthranilic acid (2AA), and 3- (acetylamino) -6-aminoacridine (AA-Ac) are used for HPLC It is frequently used as a label for analysis, and is particularly preferable.

  Further, 8-aminonaphtalene-1,3,6-trisulfonic acid (APTS) is frequently used when sugar chains are analyzed by capillary electrophoresis, and is particularly preferable.

  The substance having a hydrazide group is preferably selected from the group of compounds listed below, but is not limited thereto.

2-hydroxybenzhydrazide; 2-hydrazinobenzoic acid; benzylhydrazine; 5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (Dansylhydrazine); 2-hydrazinopyridine; 9-fluorenylmethyl carbazate (Fmoc hydrazine); 4,4-difluoro-5,7-dimethyl-4- bora-3a, 4a-diaza-s-indacene-3-propionoc acid, hydrazide; 2- (6,8-difluoro-7-hydroxy-4-methylcoumarin) acetohydrazide; 7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH ); Phenylhydrazine; 1-Naphthaleneacethydrazide; phenylacetic hydrazide.
The substance having an aminooxy group is preferably selected from the group of compounds listed below, but is not limited thereto.

N-aminooxyacetyl (tryptophyl) arginine methyl ester; O-benzylhydroxylamine; O-phenylhydroxylamine; O- (2,3,4,5,6-pentafluorobenzyl) hydroxylamine; O- (4-nitrobenzyl) hydroxylamine; 2-aminooxypyridine; 4-[(aminooxyacetyl) amino] benzoic acid methyl ester; 4-[(aminooxyacetyl) amino] benzoic acid ethyl ester; 4-[(aminooxyacetyl) amino] benzoic acid n-butyl ester; aminooxy-biotin.
Among these compounds, N-aminooxyacetyl (tryptophyl) arginine methyl ester (aoWR) is particularly preferable because it has an effect of improving sensitivity during MALDI-TOF MS measurement.

  In the method of the present invention, for example, it is preferable to add 10 equivalents or more of a labeling reagent to the sugar chain and perform the heating reaction in the presence of a reducing agent, but the conditions are limited to these labeling reactions. It is not a thing. When labeling is performed with a hydrazide group-containing compound or an aminooxy group-containing compound, a reducing agent is not necessarily added.

(Purification of labeled sugar chain)
Since the labeled sugar chain solution prepared by the above method contains an unreacted labeling reagent, it is difficult to directly perform the analysis. For this reason, it is necessary to remove the unreacted labeling reagent in advance before the measurement. For removal of the reagent, in general, a device in which a column or cartridge is filled with gel particles or silica gel particles is often used. In order to improve the separation performance of these columns (cartridges), a method of either increasing the packing density of the solid phase or decreasing the number average diameter of the packing is usually used. However, in the former, the packing density does not increase as expected due to variations in the shape and diameter of the packing material, and in the latter, the pressure load on the column and the apparatus increases, so that the processing speed tends to be limited. In a small-scale column (cartridge), the dead volume of the frit (bead blocking member) necessary to hold the packing tends to cause a reduction in separation performance and recovery rate.

  In the present invention, as a means for avoiding these problems, a column packed with monolithic silica in the flow path is used. Monolithic silica is silica that forms a filter-like porous continuous body having a three-dimensional network structure. Monolithic silica is a monolithic silica with two types of pores (mesopores and macropores). In this specification, the diameter of micrometer-sized pores is the macro diameter, and the diameter of nm-sized pores is This is called meso diameter. Monolithic silica has advantages such as better liquid permeability, no frit and less dead volume compared to conventional particulate silica.

  When processing a plurality of sample solutions at the same time (in order to increase the throughput), it is preferable to use a plate (multiwell plate) in which a plurality of these columns are arranged.

  In the present invention, monolithic silica having a pore diameter (macro diameter) of 1 to 20 μm can be used as monolithic silica suitable for high throughput. Therefore, the present invention also provides a plate in which a plurality of columns filled with monolithic silica having a macro diameter of 1 to 20 μm are packed in the flow path.

  The pore diameter (macro diameter) of monolithic silica in the column is more preferably 2 to 10 μm, still more preferably 3 to 8 μm, and most preferably 5 to 7 μm. When the pore diameter (macro diameter) of the monolithic silica is smaller than the most preferable range, the sample solution tends to be poorly passed. When the pore diameter is large, the recovery efficiency of the sugar chain sample is reduced, or the sugar chain is reduced. There is a tendency for the variation in the amount of collected to increase.

  As for the properties of monolithic silica, normal phase mode or HILIC (Hydrophilic interaction chromatography) mode is preferred.

  Monolithic silica is used by injecting a sample solution containing a labeled sugar chain into a column filled with monolithic silica in the flow path, and then using a method such as suction, natural drop, pressurization, or centrifugation for the sample solution. After passing through the monolithic silica part, it is washed with a washing solution, and an eluate is added to elute sugar chain components. Suction is preferred from the viewpoint that many sample solutions can be processed quickly.

  The sample solution applied to monolithic silica preferably contains 90% by volume or more of an organic solvent, and more preferably contains 95% by volume or more of an organic solvent. As the organic solvent, acetonitrile, methanol, ethanol, 2-propanol, hexane, ethyl acetate, methylene chloride, tetrahydrofuran and the like can be used, and acetonitrile is most preferable.

  After applying the sugar chain sample, the monolithic silica is washed with a washing solution. The cleaning liquid preferably contains 90% by volume or more of an organic solvent and 10% by volume or less of water, and more preferably contains 95% by volume or more of an organic solvent and 5% by volume or less of water. As the organic solvent, acetonitrile, methanol, ethanol, 2-propanol, hexane, ethyl acetate, methylene chloride, tetrahydrofuran and the like can be used, and acetonitrile is most preferable. It is also preferable to sequentially wash with cleaning liquids having different compositions (for example, 100% acetonitrile and acetonitrile / water (95: 5, v / v) mixed solvent).

  After the washing operation, the eluate is brought into contact with monolithic silica, and the adsorbed labeled sugar chain is eluted. The eluate preferably contains 20% by volume or more of water, more preferably contains 50% by volume or more of water, and most preferably pure water. In addition, it is preferable to dry the monolithic silica after the washing operation and before the elution operation because the solvent used in the washing operation can be effectively removed. By subjecting the monolithic silica to a suction treatment for about 30 seconds, the monolithic silica can be dried easily and quickly.

  The recovered sugar chain solution can be concentrated, dried, or diluted as necessary to be used for various measurements.

(Sugar chain analysis using MALDI-TOF MS)
The obtained labeled sugar chain can be analyzed by mass spectrometry represented by MALDI-TOF MS. In particular, when the sugar chain is labeled with N-aminooxyacetyl-tryptophanyl (arginine methyl ester), high-sensitivity analysis can be performed using MALDI-TOF MS.

(Glycan analysis using HPLC)
The resulting labeled sugar chain can be analyzed using HPLC. In particular, when the sugar chain is PA, 2AB, 2AA, or AA-Ac, it can be analyzed particularly preferably.

(Other sugar chain analysis)
The labeled sugar chain obtained can be subjected to various analyzes such as HPLC-MS and capillary electrophoresis.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

(Preparation of sugar chain sample)
IgG (derived from fetal bovine serum) was used as a model glycoprotein. 1 mg of IgG was placed in a container and dissolved in 50 μL of 100 mM ammonium bicarbonate solution. After adding 5 μL of 120 mM dithiothreitol solution and incubating at 60 ° C. for 30 minutes, 10 μL of 123 mM iodoacetamide solution was added and allowed to stand at room temperature for 1 hour. Furthermore, trypsin 400 units were added and allowed to stand at 37 ° C. for 16 hours. After inactivating trypsin by treating at 90 ° C. for 5 minutes, 1 unit of N-glycosidase F (Roche Diagnostics) was added and incubated at 37 ° C. for 16 hours to release sugar chains. Pure water was added to adjust the liquid volume to 100 μL.

(Glycan purification)
Sugar chain purification was performed using polymer beads having sugar chain-trapping properties. 20 μL of the IgG sugar chain solution obtained above (equivalent to IgG 200 μg) was added to 5 mg of hydrazide group-containing polymer beads (BS-45603, manufactured by Sumitomo Bakelite Co., Ltd.), and 180 μL of acetonitrile containing 2% acetic acid was added. The mixture was reacted at 80 ° C. for 1 hour and dried. After washing the polymer beads with 2M guanidine hydrochloride solution, water, methanol, and 1% triethylamine solution, 10% acetic anhydride / methanol solution was added and reacted at room temperature for 30 minutes to cap the hydrazide group. After capping, the polymer beads were washed with methanol and water. Sugar beads were released from the beads by adding 20 μL of pure water and 180 μL of acetonitrile containing 2% acetic acid to the beads, and heating and drying at 80 ° C. for 1 hour.

(Labeling of sugar chains)
Labeling of sugar chains was performed by the following method.

  2-aminobenzamide was dissolved in 30% (v / v) acetic acid / DMSO mixed solvent at a concentration of 350 mM. Further, sodium cyanoborohydride as a reducing agent was dissolved in the above solution at a concentration of 1M. 50 μL of this solution was added to the above beads and reacted at 80 ° C. for 2 hours to label the sugar chain with 2AB. The solution was recovered to obtain a 2AB-labeled sugar chain solution (containing unreacted 2AB).

(Purification of labeled sugar chain)
In order to remove the unreacted labeling reagent, solid phase extraction was performed by the following method.

[Example 1]
Unreacted 2AB was removed from the sample solution labeled with 2AB using a 96-well cleanup plate. In each well of the plate, a disk-shaped monolithic silica filter having a pore diameter (macro diameter) of 6.5 μm is fixed to the bottom of the well having a capacity of about 1 mL.

  The 2AB-labeled sugar chain solution obtained above was diluted with acetonitrile to prepare an acetonitrile content of 95% (v / v). The entire amount of this sample solution was injected into the wells of a 96-well cleanup plate, and then quickly aspirated (vacuum) to adsorb the labeled sugar chain to monolithic silica. The column was washed successively with acetonitrile and acetonitrile / water (95: 5, v / v) and then sucked (vacuum) for 30 seconds to dry the column. Thereafter, 100 μL of pure water was added to the column to elute the adsorbed components and collect them.

[Example 2]
Unreacted 2AB was removed from the sample solution labeled with 2AB using the 96-well cleanup plate used in Example 1.

  The 2AB-labeled sugar chain solution obtained above was diluted with acetonitrile to prepare an acetonitrile content of 95% (v / v). The whole amount of this sample solution was poured into the wells of a 96-well cleanup plate and allowed to fall naturally for 10 minutes. The remaining solution was vacuumed and the labeled sugar chain was adsorbed on monolithic silica. The column was washed successively with acetonitrile and acetonitrile / water (95: 5, v / v) and then sucked (vacuum) for 30 seconds to dry the column. Thereafter, 100 μL of pure water was added to the column to elute the adsorbed components and collect them.

[Comparative Example 1]
Unreacted 2AB was removed from the sample solution labeled with 2AB using monolithic silica. “MonoFas spin column” manufactured by GL Sciences Inc. was used as monolithic silica. In this column, a disk-shaped monolithic silica filter with a pore size of 15 μm is fixed to the bottom of a tube with a capacity of about 1 mL. The sample solution is injected into the tube and centrifuged to make the sample solution fine. It is designed to pass through the hole.

  The 2AB-labeled sugar chain solution obtained above was diluted with acetonitrile to prepare an acetonitrile content of 95% (v / v). The entire amount of this sample solution was injected into a monolithic silica spin column, and centrifuged through a table centrifuge to pass through to adsorb the labeled sugar chain. After sequentially washing the column with acetonitrile and acetonitrile / water (95: 5, v / v), 50 μL of pure water was added to elute and collect the adsorbed components.

(HPLC measurement)
The sugar chain solutions obtained by the methods of the above Examples and Comparative Examples were measured by HPLC. The HPLC apparatus was a Waters HPLC system, 2695 Alliance separations module, 2475 fluorescence detector, and the analytical column was Shodex Asahipak NH2P-50 4E manufactured by Showa Denko. The analysis was performed in gradient mode, and the mobile phase was (A) 2% (v / v) acetic acid / acetonitrile solution, (B) 3% (v / v) acetic acid, 5% (v / v) triethylamine / water solution. In 90 minutes, the ratio of (A) was changed from 70% to 5%. The column temperature was kept at 40 ° C. Detection is performed by fluorescence detection. In the case of Example 1 and Comparative Example (2AB sugar chain), detection is performed at an excitation wavelength of 330 nm and a fluorescence wavelength of 420 nm. In Example 2 (PA sugar chain), the excitation wavelength is 320 nm and the fluorescence is detected. Detection was performed at a wavelength of 400 nm. The injection amount of the sample solution was adjusted to be 1/20 of the total sample solution, that is, 10 μg in terms of IgG in both Examples and Comparative Examples.

  FIG. 1 shows the HPLC chart obtained in Example 1. The horizontal axis represents retention time (minutes), and the vertical axis represents fluorescence intensity. In the figure, (a) is a peak derived from an unreacted labeling reagent, (b) is a peak derived from a neutral sugar chain, (c) is a peak derived from a sugar chain containing one sialic acid, and (d) is a sialic acid. 2 shows a peak derived from a sugar chain containing two. FIG. 2 shows a graph comparing the total amount of area values of the peaks derived from the sugar chains of HPLC obtained in Examples 1 and 2 and Comparative Example 1.

  It was found that the recovered amount was almost the same in both Examples and Comparative Examples.

  For Examples 1 and 2, the same experiment was performed using a disk-shaped monolithic silica filter having a pore diameter (macro diameter) of 3 μm, 7 μm, 10 μm, and 15 μm. As a result, when a disc-shaped monolithic silica filter having a through-pore diameter (macro diameter) of 7 μm was used, the same results as in Examples 1 and 2 were obtained, but the through-pore diameter (macro diameter) was 10 μm or 15 μm. When a disc-shaped monolithic silica filter is used, when the sample solution is passed through the monolithic silica filter by suction, the amount of sugar chain recovered is lower than when the monolithic silica filter is allowed to pass by natural fall. Wells were created.

  On the other hand, when a disc-shaped monolithic silica filter having a pore diameter (macro diameter) smaller than 5 μm was used, the passage of the sugar chain sample through the filter deteriorated.

  When the method of the present invention is used, it is possible to efficiently perform the unreacted reagent removal step performed after the sugar chain labeling reaction even when the throughput is increased. Therefore, the present invention can be used not only for glycan research but also for the recovery of glycans from natural products and the production of glycan reference materials, and for medical treatment such as searching for disease markers by glycan analysis and diagnosis by glycan analysis. It can be applied to other fields.

Claims (6)

A method for preparing a sugar chain sample,
(Step 1) By injecting a sample solution containing labeled sugar chains into a plate in which a plurality of columns filled with a monolithic silica having a macro diameter of 1 to 20 μm in the flow path is arranged, sugar is added to the monolithic silica. Adsorbing chain components,
(Step 2) A step of washing the monolithic silica with a washing liquid,
(Step 3) A step of contacting the eluate with monolithic silica to elute the adsorbed sugar chain component
A method comprising the steps of: The preparation method according to claim 1, wherein in step 1, the sample solution is passed through the column by suction, natural drop, pressurization, or centrifugation. The preparation method according to claim 1 or 2, wherein in step 3, the eluate is a solution containing 20% or more of water. A sugar chain sample prepared by the method according to claim 1.   A plate in which a plurality of columns filled with monolithic silica having a macro diameter of 1 to 20 μm in a flow path are used for carrying out the method according to claim 1. A method for analyzing a sugar chain, comprising analyzing the sugar chain sample according to claim 4 by HPLC, mass spectrometry, LC-MS, or capillary electrophoresis.