JP2017067677A - Method for preparing purified substance of label sugar - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve a variation in the recovery rate according to the type of a sugar chain in purification of a sugar chain label body.SOLUTION: The method for preparing purified substance of a label sugar includes an applying step, a cleaning step, and an elution step. In the applying step, a sample containing a label sugar is applied on a solid phase of silica gel. The label sugar has a marker group indicating a polarity or a positive charge. In the cleaning step, the solid phase is cleaned with a cleaning solution. In the elution step, the label sugar is eluted with at least an acid solution. The elution step may include a first elution step for elusion with water or an aqueous solution with a pH higher than that of the acid solution and a second elution step for elusion with the acid solution.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a technique for purifying sugar. More specifically, the present invention relates to a technique for purifying a labeled sugar with a good recovery rate.

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 living organisms are deeply involved in cell-to-cell information transmission, protein function and coordination of interactions.

  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. Glycoproteins involved in recognition can be mentioned, but the mechanism by which these high-molecular sugar chains control advanced and precise biological reactions while acting, assisting, amplifying, regulating, or inhibiting each other's functions gradually. It is being revealed. Furthermore, if the relationship between such sugar chains and cell differentiation / proliferation, cell adhesion, immunity, and cell carcinogenesis is clarified, this sugar chain engineering and medicine, cell engineering, or organ engineering are closely related. We can expect new developments related to

  In order to detect a disease early and to maintain a high quality of life (QOL), a biomarker that can prevent the development of the disease and diagnose the transition is 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 a sugar chain is analyzed by HPLC or HPLC-MS, fluorescent labeling such as 2-aminobenzamide derivatization (2AB conversion) and 2-aminopyridine derivatization (PA conversion) is generally used (Non-patent Document 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. In order to easily remove such an excessive amount of the labeling reagent, a step of adsorbing the sugar chain component to the monolithic silica by bringing the sample solution containing the labeled sugar chain into contact with the monolithic silica; There is known a sugar chain sample preparation method including a step of washing with a washing liquid and a step of bringing the eluate into contact with monolithic silica and eluting the adsorbed sugar chain component (Patent Document 1). This method is applied to general labeled sugar chains such as 2AB and PA, and an eluent is water or an aqueous solution of an organic solvent selected from acetonitrile and alcohol.

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)

  The method of Patent Document 1 can easily remove an excessive amount of labeling reagent, but there is room for improvement in the variation in recovery rate depending on the type of sugar chain.

As a result of intensive studies, the inventor achieves the object of the present invention described above by using an acidic solution in the elution step from a solid phase when a sugar having a polar or positively charged labeling group is purified on a solid phase. As a result, the present invention has been completed.
The present invention includes the following inventions.

(1)
The method for preparing a purified product of the labeled sugar of the present invention includes an applying step, a washing step, and an elution step. In the applying step, a sample containing a labeled sugar is applied to a solid phase of silica gel. The labeled sugar has a labeling group that exhibits a polar or positive charge. In the washing step, the solid phase is washed with a washing solution. In the elution step, the labeled sugar is eluted with at least an acidic solution.

  Thereby, the recovery rate of the labeled sugar from the solid phase can be improved. In addition, an apply process, a washing | cleaning process, and an elution process are performed in this order. In addition, the sample containing the labeled sugar may be a sample known to contain at least a neutral sugar label, or a sample that only contains at least a neutral sugar label. May be.

(2)
In the method for preparing a purified product of labeled sugar of (1) above, the elution step includes a first elution step of elution with water or an aqueous solution having a pH higher than that of the acidic solution, a second elution step of elution with an acidic solution, May be included.

  As a result, a purified product of labeled sugar can be obtained with a good recovery rate regardless of the type of sugar that is the source of the labeled sugar contained in the sample. Therefore, whether the sample to be applied to the solid phase contains only a neutral sugar label as a labeled sugar, or a sample containing an acidic sugar together with a neutral sugar label, it is satisfactory. A purified product of labeled sugar can be obtained in a recovery rate.

(3)
In the method for preparing the purified product of the labeled sugar of (1) or (2) above, the acidic component in the acidic solution may be an organic acid.

  This facilitates purification under conditions that are mild to the labeled sugar.

(4)
In the method for preparing a purified product of labeled sugars of (1) to (3) above, the concentration of the acidic component in the acidic solution may be 0.0005% by volume or more.

  As a result, a purified product of the labeled sugar can be obtained with a better recovery rate.

(5)
In the method for preparing a purified product of the labeled sugar of (3) or (4) above, the organic acid may be acetic acid.

  Thus, the preparation method of the present invention is excellent in versatility. Furthermore, since acetic acid itself is volatile, it is convenient when removing the solvent, such as when the purified product is concentrated or dried.

(6)
In the method for preparing a purified product of a labeled sugar of (1) to (5) above, the labeled sugar may be a pyridylaminated sugar.

  Thus, the preparation method of the present invention is excellent in versatility.

The HPLC chart of the PA-ized sugar chain purified in Example 1 to Example 5 is shown. The HPLC chart of the PA-ized sugar chain refine | purified in the comparative example 1 and Example 6 is shown. The HPLC chart of the PA-ized sugar chain refine | purified in the comparative example 2 and Example 7 is shown. As a reference, an HPLC chart of a mixture (non-separated) of a PA oligomer of a glucose oligomer (neutral sugar chain) and a PA analogue of bovine fetuin (acidic sugar chain) is shown. The graph of the recovery rate of the PA-form of a neutral sugar chain calculated | required from the area value of the neutral sugar chain origin peak about the PA-ized sugar chain refine | purified in the comparative example 3 and Example 8 to Example 10 is shown. The graph of the recovery rate of the PA-form of a neutral sugar chain calculated | required from the area value of the peak derived from an acidic sugar chain about the PA-ized sugar chain refine | purified in Comparative Example 3 and Example 8 to Example 10 is shown. The enlarged view of the principal part of the HPLC chart of FIG. 4 is shown. The graph of the ratio of the area of each peak with respect to the sum of the areas of all the peaks calculated | required from the HPLC chart obtained in Example 10 (n = 3) is shown.

[1. Apply process]
[1-1. Sample containing labeled sugar]
The sample to be subjected to the method of the present invention contains a labeled sugar to be purified together with impurities to be separated and removed in a solvent. The sample may be a labeling reaction product obtained by performing a sugar labeling reaction using a labeling reagent selected according to the analysis technique in order to enable analysis of the sugar.

  When the sugar subjected to the labeling reaction is obtained by a sugar release treatment from a sugar complex (for example, glycoprotein), the sugar may be purified (in this case, the sugar release treatment). The product may be purified once and then subjected to a labeling reaction) and not purified (in this case, the sugar release treatment and the labeling reaction are performed in one pot).

  Examples of the impurities to be separated and removed include unreacted excess labeling reagent, by-products, and salts. In addition, depending on the background of obtaining the labeled sugar, components other than the above that can be added or can be generated in the background (for example, sugar chain-releasing enzyme, components other than the sugar of the above sugar complex, other proteins, peptides, nucleic acids, etc. And biomolecules such as lipids) may be included as contaminants.

[1-2. Labeling group]
The labeling group possessed by the labeling sugar is a derivative group of a sugar labeling reagent. The labeling group has a polar or positive charge.
The labeling group preferably contains a basic group. The basic group is a functional group having the property that the ionic rate increases under a pH environment lower than its pKa. For example, a functional group containing a basic nitrogen atom may be mentioned, specifically, an amino group, an amidino group, a guanidino group, a hydrazino group, a mono- or di-substituted amino group, a mono-, di- or tri-substituted amidino. Groups, mono-, di-, tri- or tetra-substituted guanidino groups, mono-, di- or tri-substituted hydrazino groups, and groups of these quaternary salts. Among these, an amino group (primary amino group), a mono-substituted amino group (secondary amino group), a di-substituted amino group (tertiary amino group), and a quaternary salt group thereof (quaternary). Ammonium group) is preferable.

  Examples of labeling reagents that give labeling groups include aromatic compounds having amino groups. In this case, in the labeling reagent, the amino group reacts with the reducing end of the sugar by reductive amination and is derivatized to the labeling group. An aromatic compound having an amino group has ultraviolet-visible absorption characteristics or fluorescence characteristics, and thus improves the sugar detection sensitivity in UV detection or fluorescence detection. Specifically, 2-aminobenzamide, 2-Aminopyridine, 7-amino-1-naphthol, 3- (acetylamino) -6-aminoacidine, 2-amino-9 (10H) -acidone 7-amino-4-methylcoumineline, , 2-aminobenzohydrazide; 2-hydrazinobenzoic acid; benzhydrydrazine; 2-hydrazinopyridine, 9-fluorenylmethyl-di-a-ne-a-ro-5,7-a4-di-4 a id, hydrazide; 2- (6,8-difluoro-7-hydroxy-4-methylcoumarin) acetohydrazide, phenylhydrazine; include phenylacetic hydrazide; 1-Naphthaleneacethydrazide. Among these, 2-Aminopyridine is preferable from the viewpoint of versatility and mildness of separation conditions. Moreover, as long as the function as a labeling reagent is maintained, these derivatives are also mentioned.

  Other examples of the labeling reagent that provides the labeling group include compounds having a sugar reactive group such as an oxylamino group or a hydrazide group. The compound having an oxylamino group or a hydrazide group can contain amino acid residues such as arginine residue, tryptophan residue, phenylalanine residue, tyrosine residue, cysteine residue, lysine residue and the like. When an arginine residue is contained, ionization at the time of measurement by a mass spectrometer is promoted, which is preferable in terms of improving the sugar detection sensitivity in mass spectrometry. When a tryptophan residue is included, the residue is fluorescent and hydrophobic, which is preferable in terms of improving the separation and improving the fluorescence detection sensitivity during reverse-phase HPLC detection of the labeled sugar chain. When it contains a phenylalanine residue and / or a tyrosine residue, it is preferable in that it is suitable for detection by UV absorption of a labeled sugar chain. When a cysteine residue is contained, labeling can be performed with a labeling reagent such as ICAT reagent (ABI, USA) targeting the -SH group of the residue. When a lysine residue is included, labeling with a labeling reagent such as iTRAQ reagent (Applied Biosystems, USA) or ExacTag reagent (Perkin, USA) can be performed targeting the amino group of the residue. When a tryptophan residue is included, labeling can be performed with an NBS reagent (Shimadzu Corporation, Japan) targeting the indole group of the residue.

  Still another example of a labeling reagent that provides a labeling group includes, for example, a compound having a basic nitrogen-containing group and a sugar-reactive group. The labeling reagent in this case is derivatized into a positively charged labeling group by modifying the sugar via a sugar reactive group and further quaternizing the basic nitrogen-containing group. As a result, a positive charge can be stably present in the labeled sugar, which is preferable in terms of improving the sugar detection sensitivity in mass spectrometry.

[1-3. sugar]
In the present invention, the sugar includes both monosaccharides and sugar chains. The molecular weight of the sugar (before labeling) may be, for example, 150 or more and 6000 or less, preferably 300 or more and 3000 or less.
The sugar that is the origin of the labeled sugar contained in the sample may be unknown whether the structure, or whether it is a neutral sugar chain, or may be known. This is useful when the sugar contains at least a neutral sugar label. A neutral sugar is a sugar that does not contain an acidic group such as a sulfate group or a carboxyl group.

  The labeled sugar contained in the sample may further contain an acidic sugar label. In this case, the sample contains a neutral sugar chain label and an acidic sugar label. An acidic sugar is a sugar containing an acidic group such as a sulfate group or a carboxyl group.

[1-4. Silica gel solid phase]
The solid phase used in the present invention is silica gel. Silica gel has silanol groups on the surface. The present invention does not specifically exclude the case where a modification group is attached to a part of the surface silanol groups, but the more unmodified silanol groups (Si—OH) are more preferable. Thereby, the labeled sugar to be purified can be preferably captured.

  Examples having a preferred amount of unmodified silanol groups include, for example, 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and even more preferably 95% of the entire surface silanol groups. % Or more, most preferably 100% may be unmodified silanol groups.

The shape of the solid phase is not particularly limited, and may be, for example, a bead type or a monolith type.
In the case of the bead type, the particle diameter may be, for example, 1 μm to 100 μm, preferably 1 μm to 50 μm, more preferably 1 μm to 10 μm, for example 5 μm. It is preferable from the viewpoint of liquid permeability that the particle diameter is not less than the above lower limit value, and it is preferable that it is not more than the above upper limit value from the viewpoint of preventing a decrease in the theoretical number.

  The monolith type silica gel is a bulk of silica gel having micrometer-sized three-dimensional network pores (macropores) and nanometer-size pores (mesopores). The diameter of the macropores may be, for example, 1 μm to 100 μm, preferably 1 μm to 50 μm, more preferably 1 μm to 30 μm, and still more preferably 1 μm to 20 μm. It is preferable from the viewpoint of liquid permeability that the macropores are equal to or higher than the lower limit value, and it is preferable to be equal to or lower than the upper limit value from the viewpoint of preventing a decrease in the theoretical number. The mesopore diameter may be, for example, 1 nm to 100 nm, preferably 1 nm to 70 nm. Thereby, the labeled sugar to be purified can be preferably captured.

The volume of the monolith silica gel (including the volume of macropores and mesopores), for example 0.01 cm ³ or more 1 cm ³ or less, preferably may be at 0.1 cm ³ or less 0.01 cm ³ or more. With this volume, it becomes easy to obtain an eluent after elution at a concentration suitable for HPLC analysis.

  The silica gel solid phase is used in a suitable container. The container is not particularly limited as long as it is capable of separating (fluidizing) a liquid while holding a solid phase. For example, a column, each well of a multiwell plate, each well of a filter plate, a microtube, etc. Is mentioned.

[1-5. solvent]
The solvent in the sample preferably contains an organic solvent as a main component. The amount of the organic solvent in the solvent may be, for example, 90% by volume or more, preferably 95% by volume or more. Thereby, the labeled sugar can be dissolved well. Examples of the organic solvent include acetonitrile, methanol, ethanol, 2-propanol, hexane, ethyl acetate, methylene chloride, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, hexamethylphosphoramide, etc. Among these, polar and aprotic Acetonitrile is preferred in that it has a low surface tension and low viscosity.

[1-6. Apply]
The above sample is applied to a solid phase. As a result, the labeled sugar is adsorbed on the solid phase. The labeled sugar is reversibly captured by the silanol group of the silica gel solid phase via the polarity or positive charge of the labeling group.

[2. Cleaning process]
[2-1. Cleaning fluid]
After applying the sample, the solid phase is washed with a washing solution. The cleaning liquid is, for example, 90% by volume to 100% by volume organic solvent and 0% by volume to 10% by volume water, preferably 95% by volume to 100% by volume organic solvent and 0% by volume to 5% by volume. May contain water. Examples of the organic solvent include acetonitrile, methanol, ethanol, 2-propanol, hexane, ethyl acetate, methylene chloride, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, hexamethylphosphoramide, and the like. In this respect, acetonitrile is preferable.

[2-2. Washing]
By passing the washing solution, contaminants are eluted while adsorbing the labeled sugar to the solid phase. Examples of the liquid passing method include natural dropping, suction, pressurization, and centrifugation (the same applies to the elution step described later). In cleaning, cleaning may be performed in stages using cleaning liquids having different compositions. For example, after passing 100% acetonitrile, acetonitrile containing water can also be passed.

[3. Elution process]
[3-1. Eluate]
After washing the solid phase, the eluate is passed through to elute the labeled sugar from the solid phase into the eluent. As an eluent, at least an acidic solution is used. The acidic solution only needs to contain at least an acidic component in water. Specifically, it may be an aqueous solution containing substantially only an acidic component as a solute, or may be a buffer solution prepared acidic including an acidic component and its conjugate base.

  The acidic component is preferably volatile. Volatility means the property that the boiling point at 1 atm is 50 ° C. or higher and 260 ° C. or lower, preferably 150 ° C. or lower (the same applies hereinafter). As a result, the labeled sugar in the eluent can be easily concentrated or dried, and the possibility that the acidic component remains and adversely affects the analysis can be prevented.

  The pKa of the acidic solution is preferably weakly acidic, for example, 3.5 to 5.0, preferably 4.0 to 5.0. It is preferable that the pKa of the acidic solution is not less than the above lower limit value because the elution conditions are mild. It is preferable that the pKa of the acidic solution is not more than the above upper limit in that the purification efficiency of the labeled sugar is good. For example, even when both a neutral sugar label and an acidic sugar label are included as labeled sugars, both are eluted well.

  Such an acidic component exhibiting weak acidity is preferably an organic acid, and more preferably a carboxylic acid. Examples include formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, lauric acid, and cyclohexanecarboxylic acid. Among these, acetic acid is most preferable in consideration of mildness of elution conditions, volatility, and availability.

  In the present invention, the acidic solution is preferably weakly acidic as described above. However, depending on the type of labeling group of the labeled sugar (for example, when the polarity is relatively small), the above weakly acidic solution is used for the purpose of increasing elution efficiency. It is not excluded that the acidity is stronger. Examples of acidic components in such cases include inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid; fluorine-containing carboxylic acids such as trifluoroacetic acid; and organic sulfonic acids.

  The concentration of the acidic component in the acidic solution may be, for example, 0.0005% by volume or more, preferably 0.002% by volume. Thereby, the labeled sugar can be obtained with a higher recovery rate. The upper limit value within the concentration range is not particularly limited, but the acidic solution as the eluent used in the present invention is excellent in elution capability, so that the elution capability with respect to the use amount of the eluate can be obtained more efficiently, for example 0.2. Volume%, preferably 0.15 volume%, more preferably 0.1 volume% is sufficient. The above concentration is preferable when the acidic component is a monovalent acid.

  The flow rate of the acidic solution is, for example, the apparent volume of the carrier (in the case of granular type silica gel, including the volume of voids between packed particles, and in the case of monolith type silica gel, the volume of macropores and mesopores is also included. For example) may be 0.1 times or more, preferably 0.2 times or more. Alternatively, when the support is monolithic silica type silica gel, it may be, for example, 0.1 times or more, preferably 1 or more times the total volume of macropores and mesopores. For example, it may be 10 μl or more, preferably 15 μl or more, more preferably 20 μl or more. Thereby, the labeled sugar can be obtained with a higher recovery rate.

  The upper limit value within the range of the flow rate is not particularly limited, but may be, for example, 10 times, preferably 1 time, the apparent volume of the carrier. Alternatively, when the support is monolithic silica type silica gel, it may be, for example, 10 times, preferably 1 time the total volume of macropores and mesopores. For example, it may be 30 μl, preferably 25 μl. Thereby, the elution ability with respect to the usage-amount of an eluate can be obtained more efficiently.

[3-2. First elution step]
In the elution step, water or an aqueous solution having a pH higher than that of the acidic solution can be passed as an eluent before elution with the acidic solution. When the labeled sugar includes a neutral sugar label as well as a neutral sugar label, both the neutral sugar label and the acidic sugar label are adsorbed on the solid phase. By passing water or an aqueous solution having a pH higher than that of the acidic solution, the acidic sugar label can be eluted first.

  The water or aqueous solution used as the eluent in the first elution step is not particularly limited as long as the pH is higher than that of the acidic solution.

  For example, it is preferable that the pH of water or an aqueous solution used as an eluent in the first elution step and the pH of the acidic solution have a difference of 1.5 or more, preferably 2.0 or more. Thus, the neutral sugar label and the acidic sugar label can be well eluted and separated. The upper limit value within the range of the pH difference is not particularly limited, but may be, for example, 5.0, preferably 4.0. This makes it possible to suppress the decomposition of silica.

  Alternatively, the pH of the water or aqueous solution used in the first elution step may be 5.8 or more and 8.6 or less, preferably 6.0 or more and 7.5 or less. It is preferable that the pH is equal to or higher than the lower limit in terms of being able to satisfactorily dissolve and separate the labeled body of neutral sugar and the labeled body of acidic sugar. Is preferable in that it can be suppressed.

  The solute that may be contained in the aqueous solution as the eluent is not particularly limited, and examples thereof include an acidic component as long as carbon dioxide, a surfactant, an organic solvent, a basic component, and a pH condition are satisfied. In addition, when an organic solvent is included, water is the main component, and is clearly distinguished from the above-described cleaning liquid in which the organic solvent is predominant.

  The first elution step is preferably performed under non-heating conditions. Specifically, it can be performed in a room temperature environment of 4 degrees or more and 40 degrees or less. In this case, it is preferable that the aqueous solution passing therethrough is closer to acidity in that the elimination of acidic sugar residues is favorably suppressed.

  The flow rate of the eluate in the first elution step is, for example, the apparent volume of the carrier (including the volume of voids between particles in the packed state in the case of granular type silica gel, and macropores in the case of monolith type silica gel). Including the volume of the mesopores), for example, 0.1 times or more, preferably 0.2 times or more. Alternatively, when the carrier is monolithic silica type silica gel, the total volume of macropores and mesopores may be, for example, 0.1 times or more, preferably 1 time or more. For example, it may be 10 μl or more, preferably 15 μl or more, more preferably 20 μl or more. Thereby, the labeled sugar can be obtained with a higher recovery rate.

  The upper limit value within the range of the flow rate is not particularly limited, but may be, for example, 10 times, preferably 1 time, the apparent volume of the carrier. Alternatively, when the support is monolithic silica type silica gel, it may be, for example, 10 times, preferably 1 time the total volume of macropores and mesopores. For example, it may be 30 μl, preferably 25 μl. Thereby, the elution ability with respect to the usage-amount of an eluate can be obtained more efficiently.

  If it is known that the labeled sugar does not contain an acidic sugar label, the first elution step may not be performed.

[3-3. Second elution step]
When the first elution step is performed, the acidic solution is passed as described above after the first elution step (second elution step). Thereby, the labeled body of neutral sugar is eluted. By using the acidic solution described above as the eluent, the neutral sugar label can be eluted with a good recovery rate.

[3-4. Eluent]
The eluent eluted from the solid phase in the elution step contains purified labeled sugar, and can be appropriately subjected to treatments such as concentration, drying, and dilution before being subjected to the analysis step. When the first elution step and the second elution step are performed, the eluent obtained in each step is individually collected for the purpose of separating the neutral sugar label and acidic sugar label. Alternatively, the eluent obtained in each step may be recovered in a mixed state for the purpose of analyzing the labeled body of neutral sugar and the labeled body of acidic sugar in a mixed state. Good.

  In addition, when the substance which comprises the elution liquid used at the elution process contains other components other than water and an acidic component, it is preferable that not only an acidic component but the said other component is also volatile. As a result, the labeled sugar in the eluent can be easily concentrated or dried, and the possibility that the other components remain and adversely affect the analysis can be prevented.

[4. analysis]
Labeled sugars prepared by the present invention can be used for chromatography (eg, liquid chromatography, high performance liquid chromatography or HPAE-PAD chromatography), mass spectrometry (eg, MALDI-TOF MS), combinations thereof (LC-MS, HPLC-MS ), Electrophoresis (for example, capillary electrophoresis) and the like, and can be analyzed qualitatively and / or quantitatively. In the analysis of sugar chains, various databases (for example, GlycoMod, Glycosuite, SimGlycan (registered trademark), etc.) can be used.

  Hereinafter, although an example is given and the present invention is explained still in detail, the present invention is not limited to these examples.

[Example 1]
(Purification of labeled sugar chain)
In order to purify the labeled sugar chain, solid phase extraction was performed by the following method.
A column holding monolithic silica was used as the solid phase. In this column, a disc-shaped monolithic silica filter having a through-pore diameter (macro diameter) of 6-12 μm and a meso diameter of 10-60 nm is fixed to the bottom of a tube having a capacity of about 1 mL. This column is structured such that the sample solution is passed through the pores of monolithic silica by injecting the sample solution into a tube and centrifuging it.

  10 pmol of PA-labeled sugar chain (Takara Bio Inc.) was diluted with acetonitrile to prepare an acetonitrile content of 95% (v / v). The whole amount of this sample solution was applied to a monolithic silica spin column, and the labeled sugar chain was adsorbed by passing it through a table centrifuge and allowing it to pass through. After sequentially washing the column with acetonitrile and acetonitrile / water (95: 5, v / v) as a washing solution, 50 μL of acetic acid water containing 0.0005 volume% acetic acid in pure water was added as an eluent. The adsorbed components were eluted and the eluent was collected. 1 μL of the collected eluent was subjected to LC.

(HPLC measurement)
The obtained eluent was subjected to HPLC measurement under the conditions shown in Table 1 below.

A liquid and B liquid of Table 1 are liquids which respectively comprise a mobile phase, These A liquid and B liquid were mixed, and the polarity of the mobile phase was adjusted. In Table 1, the description “B: a% (T ₁ min) → B: b% (T ₂ min)” means that the concentration of the B solution is changed from a% in (T ₂ −T ₁ ) min. It means that it was changed to b%. However, T ₁ , T ₂ , a, and b each represent a real number. In Table 1,% represents a percentage based on volume.

[Example 2]
The same operation as in Example 1 was performed except that the acetic acid concentration in the acetic acid water as the eluent was changed to 0.002% by volume.

[Example 3]
The same operation as in Example 1 was performed except that the concentration of acetic acid in acetic acid water as an eluent was changed to 0.01% by volume.

[Example 4]
The same operation as in Example 1 was performed except that the concentration of acetic acid in acetic acid water as an eluent was changed to 0.05% by volume.

[Example 5]
The same operation as in Example 1 was performed except that the concentration of acetic acid in acetic acid water as an eluent was changed to 0.2% by volume.

[Elution efficacy evaluation-1]
FIG. 1 shows an HPLC chart of the PA sugar chain purified in Example 1 to Example 5. FIG. 1 shows, for comparison, an HPLC chart of an unpurified PA sugar chain stock solution and a water HPLC chart. The horizontal axis represents the retention time (minutes), and the vertical axis represents the fluorescence intensity (mV) (hereinafter the same in all HPLC charts).

  As shown in FIG. 1, it was confirmed that the PA sugar chain was eluted with a good recovery rate in Examples 1 to 5. In addition, it was confirmed from Examples 2 to 5 and in particular from Example 4 to Example 5 that the PA sugar chain was eluted with a better recovery rate.

[Example 6]
Instead of the labeled sugar chain preparation, a labeled sugar chain sample mixed with an excess reagent obtained by actually labeling the sugar chain is used, and the acetic acid concentration in the acetic acid water as the eluent is 0.1% by volume. The same operation as Example 1 was performed except having changed into.

(Sugar chain labeling)
Labeling of sugar chains was performed as follows.
276 mg of 2-aminopyridine was dissolved in 100 μL of acetic acid (PA solution). 200 mg of dimethylamine-borane was dissolved in an acetic acid aqueous solution (mixed with 50 μL of pure water and 80 μL of acetic acid) (reducing agent solution).
10 μg of glucose oligomer and 50 μL of PA solution were mixed and reacted at 80 ° C. for 60 minutes. Thereafter, 110 μL of a reducing agent solution was added and reacted at 80 ° C. for 35 minutes. This gave a labeling reaction product.

[Comparative Example 1]
The same operation as in Example 6 was performed except that the eluent was changed to pure water.

[Elution efficacy evaluation-2]
FIG. 2 shows an HPLC chart of the PA sugar chain purified in Example 6 and Comparative Example 1.
As shown in FIG. 2, it was confirmed that the elution rate of PA sugar chain was greatly improved by Example 6.

[Comparative Example 2]
The same operation as in Example 6 was performed except that the sugar chain to be labeled was changed to a human IgG sugar chain and the eluate was changed to pure water.

[Example 7]
The same operation as in Example 6 was performed except that the sugar chain to be labeled was changed to a human IgG sugar chain and the acetic acid concentration in the acetic acid water as the eluent was changed to 0.1% by volume.

[Elution effect evaluation-3]
FIG. 3 shows an HPLC chart of the PA sugar chain purified in Comparative Example 2 and Example 7.
As shown in FIG. 3, it was confirmed that the elution rate of PA sugar chain was greatly improved by Example 7.

[Comparative Example 3]
Same as Example 6 except that the sugar chain to be labeled is changed to a mixture of glucose oligomer (neutral sugar chain) and bovine fetuin (acidic sugar chain), and the eluate is changed to 50 μL of pure water. The operation was performed.

[Example 8]
The sugar chain to be labeled is changed to a mixture of a glucose oligomer (neutral sugar chain) and bovine fetuin (acidic sugar chain), and in elution from monolithic silica, 10 μL of 0.1 μl of pure water is first eluted. The same operation as in Example 6 was performed, except that elution was performed with 1% by volume of acetic acid water.

[Example 9]
In the elution, the same operation as in Example 8 was performed except that the elution was performed with 20 μL of pure water and then with 20 μL of 0.1 volume% acetic acid water.

[Example 10]
In the elution, the same operation as in Example 8 was performed except that the elution was performed with 25 μL of pure water and then with 25 μL of 0.1 volume% acetic acid water.

[Elution efficiency evaluation-4]
For reference, FIG. 4 shows an HPLC chart of a mixture (non-separated) of a PA oligomer of a glucose oligomer (neutral sugar chain) and a PA analogue of bovine fetuin (acidic sugar chain).

  In the HPLC charts of PA sugar chains purified in Comparative Example 3 and Examples 8 to 10, the peak corresponding to the shorter retention time (derived from the neutral sugar chain) among the peaks indicated by arrows in FIG. From the area value, the recovery rate of the neutralized sugar PA was obtained and is shown in a graph in FIG. The vertical axis represents the recovery rate.

  In the HPLC charts of PA sugar chains purified in Comparative Example 3 and Examples 8 to 10, the peak area corresponding to the longer retention time (derived from acidic sugar chains) among the peaks indicated by arrows in FIG. From the value, the recovery rate of the PA product of the acidic sugar chain was determined and shown in the graph of FIG. 6 as in FIG.

  As shown in FIG. 5, it was confirmed that the recovery rate of the neutral sugar chain label was improved by Examples 8 to 10. Further, as shown in FIG. 5 and FIG. 6, the recovery rate of the neutral sugar chain label was further improved by Example 9 and Example 10, and the acidic sugar chain could be eluted with a good recovery rate. It was confirmed.

  For reference, an enlarged view of the main part of the HPLC chart of FIG. 4 is shown for reference, and each peak is numbered. Peaks numbered 1 to 7 are derived from neutral sugar chains, and peaks numbered 8 to 11 are peaks derived from acidic sugar chains (sialog sugar chains).

  Example 10 was performed 3 times using monolithic silicas of different lots, and HPLC charts were obtained respectively. In each of the three HPLC charts, the area of the peak corresponding to the peaks of numbers 1 to 11 in FIG. 7 was determined, and the ratio of the area of each peak to the sum of all the peak areas was determined. The ratio of the area of each peak obtained from each of the three HPLC charts was averaged and shown in a graph in FIG. In addition, in FIG. 8, the area ratio (b) of each peak obtained from the HPLC chart of the stock solution of FIG. 4 and the area ratio (c) of each peak obtained from the water elution HPLC chart of Comparative Example 3 are also shown. It was.

  As shown in FIG. 8, the area ratio (a) of each peak obtained in this example is within ± 10% of the peak area ratio (b) of the stock solution. It was shown that the peak derived from was improved to about twice the peak area ratio (c) in the case of water elution.

  The present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the spirit of the present invention.

Claims (6)

An applying step of applying a sample containing a labeled sugar having a polar or positive charge labeling group to a solid phase of silica gel;
A washing step of washing the solid phase with a washing solution;
An elution step of eluting the labeled sugar with at least an acidic solution from the solid phase;
A method for preparing a purified product of a labeled sugar. The elution step comprises
A first elution step of elution with water or an aqueous solution having a pH higher than that of the acidic solution;
A second elution step for elution with the acidic solution;
A method for preparing a purified product of a labeled sugar according to claim 1.   The method for preparing a purified product of a labeled sugar according to claim 1 or 2, wherein the acidic component in the acidic solution is an organic acid.   The method for preparing a purified product of labeled sugar according to any one of claims 1 to 3, wherein the concentration of the acidic component in the acidic solution is 0.0005 vol% or more.   The method for preparing a purified product of a labeled sugar according to claim 3 or 4, wherein the organic acid is acetic acid. The method for preparing a purified product of a labeled sugar according to any one of claims 1 to 5, wherein the labeled sugar is a pyridylaminated sugar.