WO2015146514A1 - Method for suppressing desialylation of sugar chain in preparation of labeled sugar chain sample - Google Patents

Method for suppressing desialylation of sugar chain in preparation of labeled sugar chain sample Download PDF

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WO2015146514A1
WO2015146514A1 PCT/JP2015/056324 JP2015056324W WO2015146514A1 WO 2015146514 A1 WO2015146514 A1 WO 2015146514A1 JP 2015056324 W JP2015056324 W JP 2015056324W WO 2015146514 A1 WO2015146514 A1 WO 2015146514A1
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sugar chain
polymer particles
sugar
acid
reaction
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PCT/JP2015/056324
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French (fr)
Japanese (ja)
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秀行 島岡
碧 阪口
雅哲 豊田
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住友ベークライト株式会社
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8836—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving saccharides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins

Definitions

  • the present invention relates to a method for suppressing the elimination of sialic acid from a sugar chain in the preparation of a labeled sugar chain sample, and a method for analyzing a sugar chain prepared by the method.
  • Biopolymers such as sugar chains, glycoproteins, glycopeptides, peptides, oligopeptides, proteins, nucleic acids, and lipids play an important role in biotechnology fields such as medicine, cell engineering, and organ engineering. Clarifying the control mechanism of biological reactions will lead to the development of the biotechnology field.
  • 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 extremely diverse and are deeply involved in various functions of naturally occurring organisms, such as cell-to-cell information transmission and protein functions and interactions. It is becoming clear.
  • Sugar chains often exist as complex carbohydrates bound to proteins and lipids in vivo.
  • biopolymers having sugar chains include proteoglycans on the cell wall of plant cells 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 sugar chains contained in these biopolymers control advanced and precise biological reactions while acting, assisting, amplifying, regulating, or inhibiting the functions of these biopolymers are gradually being clarified. If the relationship between these sugar chains and cell differentiation / proliferation, cell adhesion, immunity, and cell carcinogenesis is clarified, this sugar chain engineering is closely related to medicine, cell engineering, or organ engineering. We can expect new developments.
  • sugar chain In glycoprotein drugs, the sugar chain often plays an important role in the expression of biological activity. Therefore, the evaluation of sugar chains is extremely important as a quality control parameter for glycoprotein pharmaceuticals. Particularly for antibody drugs, it has been reported that the sugar chain structure affects antibody-dependent cytotoxic activity (ADCC activity), and the importance of sugar chain structure analysis is increasing.
  • ADCC activity antibody-dependent cytotoxic activity
  • sugar chains In order to analyze sugar chains using these various techniques, 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 these sugar chains takes time and man-hours, and it is difficult to prepare a large number of samples at once.
  • Patent Document 1 a sugar chain sample preparation method using polymer particles having a functional group for capturing a sugar chain such as a hydrazide group or an aminooxy group has been reported.
  • an object of the present invention is to suppress the elimination of sialic acid in a method for preparing a labeled sugar chain sample using polymer particles having a functional group for capturing a sugar chain.
  • the inventors of the present invention have prepared a method for preparing a labeled sugar chain sample using polymer particles having a functional group for capturing a sugar chain.
  • the labeled sugar can be obtained by reducing the reaction conditions in each step of the step of capturing the polymer chain, the step of releasing the sugar chain from the polymer particle capturing the sugar chain, and the step of labeling the released sugar chain. It has been found that the elimination of sialic acid during the preparation of the chain sample is effectively suppressed.
  • the present invention relates to a method for suppressing the elimination of sialic acid from a sugar chain in the preparation of a labeled sugar chain sample, and a method for analyzing a sugar chain prepared by the method. Is to provide.
  • a method for inhibiting detachment of sialic acid from a sugar chain in the preparation of a labeled sugar chain sample containing a sugar chain having sialic acid at the end (A) contacting a sample containing a sugar chain having sialic acid at a terminal with a polymer particle having a functional group for capturing the sugar chain, and capturing the sugar chain on the polymer particle; (B) a step of washing the polymer particles capturing the sugar chain, (C) releasing the sugar chain from the polymer particles having captured the sugar chain; (D) a step of labeling the released sugar chain, And the reaction of at least one of steps (a), (c) and (d) is performed at 0 to 60 ° C. for 10 seconds to 24 hours.
  • [6] The method according to [4], wherein the polymer particles have a crosslinked polymer structure represented by the following (formula 2).
  • a method for analyzing a sugar chain in a sample the step of preparing a labeled sugar chain by the method according to any one of [1] to [7], and analyzing the prepared labeled sugar chain
  • a method comprising the step of:
  • detachment of sialic acid can be effectively suppressed when preparing a labeled sugar chain sample using polymer particles having a functional group for capturing a sugar chain. It was.
  • the area of the peak derived from the neutral sugar chain (neutral)
  • the area of the peak derived from the sugar chain having one sialic acid (1NA) the area of the peak derived from the sugar chain having two sialic acids (2NA)
  • the area of the peak derived from the sugar chain having three sialic acids (3NA) were calculated, and a bar graph for each reaction condition It showed in.
  • the present invention provides a method for suppressing the elimination of sialic acid from a sugar chain in the preparation of a labeled sugar chain sample, and a method for analyzing a sugar chain in a sample.
  • a sample containing a sugar chain having sialic acid at the terminal is brought into contact with polymer particles having a functional group for capturing a sugar chain, and the sugar chain is captured by the polymer particle ( Step (a)).
  • sample containing sugar chains in the present invention can be prepared from a biological sample, for example.
  • biological samples include whole blood, serum, plasma, urine, saliva, cells, tissues, viruses, plant tissues, and the like.
  • purified or unpurified glycoprotein can be used.
  • the sample may be pretreated by a method such as degreasing, desalting, protein fractionation, and heat denaturation.
  • Sugar chains can be released from molecules containing sugar chains (for example, glycoproteins) contained in the biological sample using sugar chain releasing means.
  • sugar chain releasing means methods such as glycosidase treatment using N-glycosidase or O-glycosidase, hydrazine degradation, and ⁇ elimination by alkali treatment can be used.
  • a method using N-glycosidase is preferable.
  • protease treatment of biological samples may be performed using trypsin, chymotrypsin, etc., but in the protease inactivation treatment, it is necessary to expose the sample to a high temperature, and sialic acid may be eliminated.
  • the sugar chain is the only substance in the living body that has an aldehyde group.
  • a cyclic hemiacetal type and an acyclic aldehyde type exist in an equilibrium state in an aqueous solution or the like.
  • In vivo substances other than sugar chains such as proteins, nucleic acids, and lipids do not contain aldehyde groups. From this, it is possible to selectively capture only sugar chains by using polymer particles having hydrazide groups or aminooxy groups that react specifically with aldehyde groups to form stable bonds.
  • polymer particles solid particles or gel particles can be suitably used. If such polymer particles are used, the polymer particles can be easily collected by means such as centrifugation or filtration 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 filling the column and using it is particularly important from the viewpoint of continuous operation.
  • a filter plate for example, MultiScreen Solvinert Filter Plate manufactured by Millipore
  • a plurality of samples can be processed at the same time.
  • conventional purification means by column operation represented by gel filtration In comparison, the purification throughput is greatly improved.
  • magnetic beads are used as the polymer particles, the beads can be easily collected on the container wall surface by using magnetic force, so that the beads can be easily washed.
  • the shape of the polymer particles is not particularly limited, but a spherical shape or a similar shape is preferable.
  • the average particle size is preferably 0.05 to 1000 ⁇ m, more preferably 0.05 to 200 ⁇ m, still more preferably 0.1 to 200 ⁇ m, and most preferably 0.1 to 200 ⁇ m. 100 ⁇ m. If the average particle diameter is less than the lower limit, when the polymer particles are packed in a column and used, liquid permeability becomes poor, and it is necessary to apply a large pressure. Moreover, it becomes difficult to collect the polymer particles by centrifugation or filtration. When the average particle size exceeds the upper limit, the contact area between the polymer particles and the sample solution decreases, and the sugar chain capture efficiency decreases.
  • the “functional group for capturing sugar chains” in the polymer particles is preferably a hydrazide group or an aminooxy group. These functional groups can react with the aldehyde group of the sugar chain to capture the sugar chain through a hydrazide bond or an oxime bond. In the present invention, a hydrazide group is particularly preferred.
  • crosslinked polymer particles having a structure represented by the following (formula 1) are preferable.
  • R 1 and R 2 represent a hydrocarbon chain having 1 to 20 carbon atoms that may be interrupted by —O—, —S—, —NH—, —CO—, —CONH—, and R 3 , R 4 and R 5 each represent H, CH 3 or a hydrocarbon chain having 2 to 5 carbon atoms.
  • crosslinkable polymer particles crosslinkable polymer particles having a structure represented by the following (formula 2) are particularly preferable.
  • the amount of polymer particles used for capturing sugar chains is preferably 0.1 mg to 100 mg, more preferably 0.1 mg to 10 mg. Since the reaction time can be shortened by reducing the reaction system, the amount is more preferably 0.1 mg to 5 mg for a small amount of sample.
  • a mixed solvent of an acid and an organic solvent can be used.
  • the acid to be used is not particularly limited.
  • acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid and sulfuric acid are preferable, and acetic acid, formic acid, trifluoroacetic acid and phosphoric acid are more preferable. More preferably, acetic acid and trifluoroacetic acid are suitably used.
  • the organic solvent is not limited as long as it can dissolve sugar and the above-mentioned acid. However, since the trapping efficiency often increases when solidified during trapping, a relatively low boiling point organic solvent such as acetonitrile is preferably used. .
  • the mixing 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 most preferably 1:99 to 5:95. .
  • an aqueous system can be used in addition to the organic solvent system.
  • the pH of the reaction system is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6.
  • Various buffers can be used for pH adjustment.
  • the step (a) may be performed at 0 to 60 ° C., may be performed at 0 to 40 ° C., or may be performed at 20 to 40 ° C.
  • the reaction time in step (a) may be 10 seconds to 24 hours, 1 minute to 16 hours, 1 to 16 hours, or 5 to 16 hours. It may be 10 to 16 hours.
  • Step (a) may be performed, for example, at a reaction temperature of 0 to 60 ° C. and a reaction time of 10 seconds to 24 hours.
  • step (a) may be performed, for example, at a reaction temperature of 0 to 40 ° C. and a reaction time of 1 minute to 16 hours.
  • the reaction is preferably carried out in an open system to evaporate the solvent completely from the viewpoint of efficiently carrying out the reaction for releasing the sugar chain.
  • it is effective to apply heat or vacuum. This promotes drying and is expected to shorten the reaction time.
  • the amount of the solution used is preferably 1 ⁇ L to 1 mL, more preferably 1 ⁇ L to 500 ⁇ L.
  • the polymer particles capturing the sugar chains are then washed (step (b)).
  • Substances other than sugar chains (substances adsorbed non-specifically) among substances trapped by the polymer particles can be removed by washing the polymer particles that have captured the sugar chains.
  • Methods for removing substances other than sugar chains include washing with a guanidine aqueous solution, a chaotropic reagent capable of dissociating hydrophobic bonds, washing with a surfactant aqueous solution, pure water or a water-soluble buffer (for example, phosphorous).
  • a method of washing with an acid buffer, a Tris buffer, or the like can be used.
  • the temperature may be 0 to 60 ° C., 4 to 40 ° C., or 15 to 25 ° C.
  • the washing time may be 10 seconds to 2 hours, 10 seconds to 1 hour, or 10 seconds to 30 minutes.
  • Examples of the cleaning method include a method in which polymer particles are immersed in a cleaning solution and the replacement of the cleaning solution is repeated.
  • the polymer particles are put into a centrifuge tube or tube, a washing solution is added, and after shaking, the polymer particles are precipitated by centrifugation and washed by repeating the operation of removing the supernatant.
  • a washing solution for example, it can be washed by repeating the operation of putting polymer particles in a centrifuge tube, adding a washing solution, allowing the polymer particles to settle naturally or by forced centrifugation, and then removing the supernatant.
  • the washing operation is preferably performed 3 to 6 times. When magnetic beads are used, centrifugation is unnecessary and simple.
  • a filter tube which is a tube-like container and is equipped with a filter having a pore size which allows liquid permeation and does not allow polymer particles to pass through on the bottom surface.
  • 96-well multiwell plate has been developed as a solution dispensing device, a suction removal system, a plate transport system, and the like, and is optimal for high throughput.
  • the washing treatment may be performed continuously from the sugar chain capture reaction by passing a washing solution through the column.
  • substances other than the polymer particles that have captured the sugar chains may be removed by filtration or centrifugation.
  • the surplus functional group on the polymer particle can be capped using, for example, acetic anhydride.
  • the sugar chains are then released from the polymer particles that have captured the sugar chains (step (c)).
  • the content of water in the reaction solution for carrying out this step is preferably 0.1 to 99.9% by volume, more preferably 1 to 99% by volume, and even more preferably 5 to 5%. 98% by volume.
  • a mixed solvent of an acid, water and an organic solvent can also be used as a reaction solution for carrying out this step.
  • the salt concentration of the aqueous buffer is preferably 0.1 mM to 1 M, more preferably 0.1 mM to 500 mM, and even more preferably 1 mM to 100 mM.
  • the pH of the reaction solution is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6.
  • the acid to be used is not particularly limited.
  • acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid and sulfuric acid are preferable, and acetic acid, formic acid, trifluoroacetic acid and phosphoric acid are more preferable. More preferably, acetic acid and trifluoroacetic acid are suitably used.
  • the organic solvent is not limited as long as it can dissolve sugar and the above-mentioned acid. However, since the trapping efficiency often increases when solidified during trapping, a relatively low boiling point organic solvent such as acetonitrile is preferably used. .
  • step (c) is performed at a reaction temperature of 0 to 60 ° C.
  • Step (c) may be performed at 0 to 40 ° C. or may be performed at 20 to 40 ° C.
  • the reaction time in step (c) may be 10 seconds to 24 hours, 1 minute to 16 hours, 1 to 16 hours, or 5 to 16 hours. It may be 10 to 16 hours.
  • Step (c) may be performed, for example, at a reaction temperature of 0 to 60 ° C. and a reaction time of 10 seconds to 24 hours.
  • step (c) may be performed, for example, at a reaction temperature of 0 to 40 ° C. and a reaction time of 1 minute to 16 hours.
  • the reaction is preferably carried out in an open system to evaporate the solvent completely from the viewpoint of efficiently carrying out the reaction for releasing the sugar chain.
  • it is effective to apply heat or vacuum. This promotes drying and is expected to shorten the reaction time.
  • the amount of the solution used is preferably 1 ⁇ L to 1 mL, more preferably 1 ⁇ L to 500 ⁇ L.
  • the sugar chain can be liberated from weakly acidic to neutral, so that the conventional strong acidic treatment, for example, in the presence of a strong acid such as excision by 10% trifluoroacetic acid treatment.
  • a strong acid such as excision by 10% trifluoroacetic acid treatment.
  • sugar chain hydrolysis such as elimination of sialic acid residues.
  • the elimination of sialic acid can be further suppressed.
  • the released sugar chain is then labeled (step (d)).
  • a reductive amination method and an exchange reaction method can be suitably used.
  • the reductive amination method will be described.
  • a sugar chain is labeled with a compound having an amino group.
  • the results obtained when the pH is measured are preferably acidic to neutral conditions, preferably 2 to 9, more preferably 2 to 8, and even more preferably 2 to 7. is there.
  • the concentration of the amino compound is preferably 1 mM to 10M, and the concentration of the reducing agent is preferably 1 mM to 10M.
  • step (d) is performed at a reaction temperature of 0 to 60 ° C.
  • Step (d) may be performed at 0 to 40 ° C. or at 20 to 40 ° C.
  • the reaction time in step (d) may be 10 seconds to 24 hours, 1 minute to 16 hours, 1 to 16 hours, or 5 to 16 hours. It may be 10 to 16 hours.
  • Step (d) may be performed, for example, at a reaction temperature of 0 to 60 ° C. and a reaction time of 10 seconds to 24 hours.
  • step (d) may be performed, for example, at a reaction temperature of 0 to 40 ° C. and a reaction time of 1 minute to 16 hours.
  • the compound having an amino group preferably has ultraviolet-visible absorption characteristics or fluorescence characteristics, and specifically, is preferably at least one selected from the following group.
  • 8-aminopyrene-1,3,6-trisulfonate (8-Aminopyrene-1,3,6-trisulfonate), 8-aminonaphthalene-1,3,6-trisulfonate (8-Aminonaphthalene-1,3, 6-trisulfonate), 7-amino-1,3-naphthalenedisulfonic acid (7-amino-1,3-naphthalenedisulfonicacid), 2-amino9 (10H) -acridone (2-Amino9 (10H) -acridone), 5-Aminofluorescein, Dansylethylenediamine, 2-Aminopyridine, 7-A 7-Amino-4-methylcoumarin, 2-aminobenzamide, 2-aminobenzoic acid, 3-aminobenzoic acid, 7-Amino-1-naphthol, 3- (acetylamino) -6-aminoacridine (3- (Acetylamino)
  • the reaction conditions when the amino compound is 2-aminobenzamide (2-AB) are as follows.
  • a mixed solvent of an acid and an organic solvent or a mixed solvent of an acid, water and an organic solvent can be used.
  • the mixing ratio of the acid and the organic solvent is preferably 1:99 to 50:50, more preferably 5:95 to 50:50, and most preferably 10:90 to 50:50. It is.
  • the result obtained when the pH is measured regardless of the ratio of the organic solvent is preferably an acidic to neutral condition, preferably pH 2 to 9, More preferably, it is 2-8, and more preferably 2-7.
  • the concentration of the amino compound is 1 mM to 10M, preferably 10 mM to 10M, and more preferably 100 mM to 5M.
  • the concentration of the reducing agent is 1 mM to 10M, preferably 10 mM to 10M, more preferably 100 mM to 2M.
  • the reaction temperature may be 0 to 60 ° C., 0 to 40 ° C., or 20 to 40 ° C.
  • the reaction time may be 10 seconds to 24 hours, 1 minute to 16 hours, 1 to 16 hours, 5 to 16 hours, or 10 to 16 hours. There may be.
  • sodium cyanoborohydride for example, sodium cyanoborohydride, methylamine borane, dimethylamine borane, trimethylamine borane, picoline borane, pyridine borane and the like can be used, but sodium cyanoborohydride or picoline borane is used. Is preferable from the viewpoint of reactivity.
  • the resulting solution contains a labeled sugar chain and an unreacted amino compound and a reducing agent added in excess, it is preferable to perform a step of removing these excess reagents.
  • Any method of removal by silica column, removal by gel filtration, removal by ion exchange resin may be used, but the solvent used for preventing the elimination of sialic acid is preferably neutral.
  • sugar chain labeling by the exchange reaction method will be described.
  • a hydrazone-oxime exchange reaction when the functional group specifically reacting with the aldehyde group of the sugar chain is a hydrazide group
  • the sugar chain is cleaved from the polymer particle by an oxime exchange reaction (when the functional group that specifically reacts with the aldehyde group of the sugar chain is an aminooxy group) and simultaneously labeled with an aminooxy compound.
  • step (c)) the step of releasing the sugar chain from the polymer particles having captured the sugar chain (step (c)) and the step of labeling the released sugar chain (step (d)) are integrated. For this reason, it is not necessary to carry out the operation of separating the sugar chain from the polymer particles by the acid treatment described above.
  • the compound having an aminooxy group is preferably a substance selected from the following group or a salt thereof.
  • the compound having an aminooxy group preferably contains a moiety comprising at least one of an arginine residue, a tryptophan residue, a phenylalanine residue, a tyrosine residue, a cysteine residue, and a derivative thereof.
  • a portion can exert a sensitizing action when the sample is subjected to MALDI-TOF mass spectrometry, for example.
  • N-aminooxyacetyl-tryptophyll arginine methyl ester
  • N-aminooxyacetyl-tryptophyll arginine amide
  • formula 4 N-aminooxyacetyl-tryptophyll
  • a mixed solvent of an acid and an organic solvent or a mixed solvent of an acid, water and an organic solvent can be used.
  • the mixing 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, most preferably Is 1:99 to 5:95.
  • the pH obtained is preferably 2 to 7, more preferably 3 to 6, more preferably 3.5 to 5. 5 is most preferred.
  • the reaction solution can be adjusted to the above pH by adding an acetic acid / acetonitrile solution.
  • the reaction temperature during the exchange reaction may be 0 to 60 ° C., 0 to 40 ° C., or 20 to 40 ° C.
  • the reaction time may be 10 seconds to 24 hours, 1 minute to 16 hours, 1 to 16 hours, 5 to 16 hours, or 10 to 16 hours. There may be.
  • the amount of the solution used is preferably 1 ⁇ L to 1 mL, more preferably 1 ⁇ L to 500 ⁇ L. The smaller the reaction system is, the faster the solvent evaporates.
  • the amount of the solution to be used is more preferably 1 ⁇ L to 100 ⁇ L (for example, 1 ⁇ L to 50 ⁇ L).
  • At least one of the steps (a), (c) and (d) described above is performed at 0 to 60 ° C. for 10 seconds to 24 hours. From the viewpoint of suppressing elimination of sialic acid from the sugar chain, at least two steps among steps (a), (c) and (d) are carried out at 0 to 60 ° C. for 10 seconds to 24 hours. More preferably, the reaction in all steps (a), (c) and (d) is more preferably performed at 0 to 60 ° C. for 10 seconds to 24 hours. Furthermore, the step (b) is also preferably carried out at 0 to 60 ° C.
  • a labeled sugar chain prepared by the above method is analyzed.
  • the sugar chain solution recovered through the above method is used as it is, or after removing the excess labeled compound, chromatography (for example, HPLC), mass spectrometry (for example, MALDI-TOF MS), combination of chromatography and mass spectrometry
  • chromatography for example, HPLC
  • mass spectrometry for example, MALDI-TOF MS
  • electrophoresis for example, capillary electrophoresis
  • various databases for example, GlycoMod, Glycosite, SimGlycan (R), etc.
  • GlycoMod for example, Glycosite, SimGlycan (R), etc.
  • Example 1> Preparation of sugar chain sample 1 mg of bovine serum-derived Fetuin (manufactured by SIGMA, F3004) was dissolved in 50 ⁇ L of 100 mM ammonium bicarbonate (manufactured by Wako Pure Chemical Industries, Ltd., 017-02875), and then 120 mM DTT (dithiothreitol, manufactured by SIGMA, D9779) was added and reacted at 60 ° C. for 30 minutes. After completion of the reaction, 10 ⁇ L of 123 mM IAA (iodoacetamide, manufactured by Wako Pure Chemical Industries, Ltd., 093-02152) was added and reacted at room temperature for 1 hour in the dark.
  • 123 mM IAA iodoacetamide, manufactured by Wako Pure Chemical Industries, Ltd., 093-02152
  • protease treatment was carried out with 400 U trypsin (manufactured by SIGMA, T0303) to fragment the protein portion into peptides.
  • the reaction solution is treated at 90 ° C. for 5 minutes, then treated with 5 U glycosidase F (Roche, 1-365-193) to release sugar chains from the peptide, and pretreated biological sample (sugar sample) )
  • Capture sugar chains by sugar chain capture carrier Particles having a hydrazide group is a carrier for the sugar chain capturing 5mg (BlotGlyco (R)), the sugar chain solution (sugar samples disposable column containing the Sumitomo Bakelite Co., Ltd.)) 20 [mu] L and 180 ⁇ L of 2% acetic acid / Acetonitrile solution was added and reacted at 37 ° C. for 16 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried.
  • reaction solution 50 ⁇ L was collected and diluted 10-fold with acetonitrile, to adsorb the labeled sugar chain silica gel was added to a silica column (BlotGlyco (R) kit accessory). After washing the column with acetonitrile, the labeled sugar chain was recovered with 50 ⁇ L of ultrapure water.
  • the obtained labeled sugar chain was measured by HITACHI HPLC (FL). Measurement was performed using an amino column (Shodex Asahipak NH2P-50) at an excitation wavelength of 330 nm and a fluorescence wavelength of 420 nm.
  • Example 2 (Preparation of sugar chain sample) A sugar chain sample was prepared in the same manner as in Example 1.
  • Capture sugar chains by sugar chain capture carrier Particles having a hydrazide group is a carrier for the sugar chain capturing 5mg (BlotGlyco (R)), the sugar chain solution (sugar samples disposable column containing the Sumitomo Bakelite Co., Ltd.)) 20 [mu] L and 180 ⁇ L of 2% acetic acid / Acetonitrile solution was added and reacted at 25 ° C. for 16 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried.
  • Capture sugar chains by sugar chain capture carrier Particles having a hydrazide group is a carrier for the sugar chain capturing 5mg (BlotGlyco (R)), the sugar chain solution (sugar samples disposable column containing the Sumitomo Bakelite Co., Ltd.)) 20 [mu] L and 180 ⁇ L of 2% acetic acid / Acetonitrile solution was added 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 was completely evaporated and the particles were dried.
  • FIG. 1 shows a peak area derived from a neutral sugar chain, a peak area derived from a sugar chain having one sialic acid (1NA), and a sugar having two sialic acids.
  • Examples of peak areas derived from chains (2NA), peak areas derived from sugar chains having three sialic acids (3NA), and peak areas derived from sugar chains having four sialic acids (4NA) The comparison of the data of the comparative example is shown. In Example 1 and Example 2 where the reaction temperature was low, the area ratio of 3NA and 4NA was higher than that in the comparative example, and it was confirmed that elimination of sialic acid was suppressed.
  • the method for preparing a labeled sugar chain sample of the present invention is excellent in suppressing the elimination of sialic acid from a sugar chain, particularly in the preparation of a labeled sugar chain sample containing a sugar chain having a sialic acid at the terminal. ing.
  • the present invention is useful for, for example, diagnosis of diseases such as cancer by analysis including isomers of sialo-sugar chains, development of new biomarkers targeting sialo-sugar chains, drug screening and quality control. Therefore, the present invention can greatly contribute to the medical field, for example.

Abstract

This method for suppressing desialylation of a sugar chain in the preparation of a labeled sugar chain sample comprising a sugar chain including terminal sialic acid comprises: (a) a step for capturing sugar chains using polymer particles by bringing a sample containing sugar chains having terminal sialic acid into contact with polymer particles having functional groups for capturing sugar chains; (b) a step for washing the polymer particles that have captured the sugar chains; (c) a step for causing the sugar chains to separate from the polymer particles that have trapped the sugar chains; and (d) a step for labeling the separated sugar chains. The reaction of at least one of steps (a), (c) and (d) is performed under conditions of 0 to 60˚C for 10 seconds to 24 hours.

Description

標識された糖鎖試料の調製において糖鎖からのシアル酸の脱離を抑制する方法Method for suppressing elimination of sialic acid from a sugar chain in the preparation of a labeled sugar chain sample
　本発明は、標識された糖鎖試料の調製において糖鎖からのシアル酸の脱離を抑制する方法、および該方法により調製された糖鎖の分析方法に関する。 The present invention relates to a method for suppressing the elimination of sialic acid from a sugar chain in the preparation of a labeled sugar chain sample, and a method for analyzing a sugar chain prepared by the method.
　糖鎖、糖タンパク、糖ペプチド、ペプチド、オリゴペプチド、タンパク質、核酸、脂質などといった生体高分子は、医学、細胞工学、臓器工学などのバイオテクノロジー分野において重要な役割を担っており、これら物質による生体反応の制御機構を明らかにすることはバイオテクノロジー分野の発展に繋がることになる。 Biopolymers such as sugar chains, glycoproteins, glycopeptides, peptides, oligopeptides, proteins, nucleic acids, and lipids play an important role in biotechnology fields such as medicine, cell engineering, and organ engineering. Clarifying the control mechanism of biological reactions will lead to the development of the biotechnology field.
　糖鎖は、グルコース、ガラクトース、マンノース、フコース、キシロース、Ｎ－アセチルグルコサミン、Ｎ－アセチルガラクトサミン、シアル酸などの単糖およびこれらの誘導体がグリコシド結合によって鎖状に結合した分子の総称である。生体高分子の中でも、糖鎖は、非常に多様性に富んでおり、天然に存在する生物が有する様々な機能、例えば、細胞間情報伝達や、タンパク質の機能や相互作用の調整などに深く関わっていることが明らかになりつつある。 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. Among biopolymers, sugar chains are extremely diverse and are deeply involved in various functions of naturally occurring organisms, such as cell-to-cell information transmission and protein functions and interactions. It is becoming clear.
　糖鎖は生体内でタンパク質や脂質などに結合した複合糖質として存在することが多い。糖鎖を有する生体高分子としては、例えば、細胞の安定化に寄与する植物細胞の細胞壁のプロテオグリカン、細胞の分化、増殖、接着、移動などに影響を与える糖脂質、および細胞間相互作用や細胞認識に関与している糖タンパク質などが挙げられる。これらの生体高分子に含まれる糖鎖が、この生体高分子と互いに機能を代行、補助、増幅、調節、あるいは阻害しあいながら高度で精密な生体反応を制御する機構が次第に明らかにされつつある。このような糖鎖と細胞の分化増殖、細胞接着、免疫、および細胞の癌化との関係が明確にされれば、この糖鎖工学と、医学、細胞工学、あるいは臓器工学とを密接に関連させて新たな展開を図ることが期待できる。 Sugar chains often exist as complex carbohydrates bound to proteins and lipids in vivo. Examples of biopolymers having sugar chains include proteoglycans on the cell wall of plant cells 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 sugar chains contained in these biopolymers control advanced and precise biological reactions while acting, assisting, amplifying, regulating, or inhibiting the functions of these biopolymers are gradually being clarified. If the relationship between these sugar chains and cell differentiation / proliferation, cell adhesion, immunity, and cell carcinogenesis is clarified, this sugar chain engineering is closely related to medicine, cell engineering, or organ engineering. We can expect new developments.
　糖タンパク質医薬品では、その糖鎖が生物活性発現などに重要な役割を担っている場合が多い。したがって、糖タンパク質医薬品の品質管理のパラメーターとして、糖鎖の評価はきわめて重要である。特に抗体医薬品については、その糖鎖構造が抗体依存性細胞傷害活性（ＡＤＣＣ活性）を左右するとの報告がされており、糖鎖構造解析の重要性が高まっている。 In glycoprotein drugs, the sugar chain often plays an important role in the expression of biological activity. Therefore, the evaluation of sugar chains is extremely important as a quality control parameter for glycoprotein pharmaceuticals. Particularly for antibody drugs, it has been reported that the sugar chain structure affects antibody-dependent cytotoxic activity (ADCC activity), and the importance of sugar chain structure analysis is increasing.
　このため、近年、糖鎖構造を迅速、簡便、かつ精度高く解析する方法が求められるようになり、高速液体クロマトグラフィ（ＨＰＬＣ）、核磁気共鳴法、キャピラリー電気泳動法（ＣＥ法）、質量分析法、レクチンアレイ法などの多種多様の方法により糖鎖解析が行われている。 For this reason, in recent years, there has been a demand for a method for analyzing sugar chain structures quickly, simply, and with high accuracy. High-performance liquid chromatography (HPLC), nuclear magnetic resonance, capillary electrophoresis (CE), mass spectrometry Sugar chains are analyzed by a wide variety of methods such as the lectin array method.
　これら種々の手法を用いて糖鎖を解析するためには、あらかじめ生体試料中に含まれるタンパク質、ペプチド、脂質、核酸などと糖鎖を分離・精製することが必要である。これら糖鎖の精製や標識化は時間と工数がかかり、一度に多種多量の試料を調製するのは困難を要する。 In order to analyze sugar chains using these various techniques, 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 these sugar chains takes time and man-hours, and it is difficult to prepare a large number of samples at once.
　この問題を解決する技術として、ヒドラジド基やアミノオキシ基などの糖鎖を捕捉するための官能基を有するポリマー粒子を利用した糖鎖試料の調製方法が報告されている（特許文献１）。 As a technique for solving this problem, a sugar chain sample preparation method using polymer particles having a functional group for capturing a sugar chain such as a hydrazide group or an aminooxy group has been reported (Patent Document 1).
国際公開第２００８／０１８１７０号International Publication No. 2008/018170
　上記の糖鎖を捕捉するための官能基を有するポリマー粒子を利用した糖鎖試料の調製方法において、特に、末端にシアル酸を有する糖鎖（シアロ糖鎖）を含む標識された糖鎖試料を調製しようとする場合、糖鎖からのシアル酸の脱離が生じ易い。そこで、本発明は、糖鎖を捕捉するための官能基を有するポリマー粒子を利用した標識された糖鎖試料の調製方法において、シアル酸の脱離を抑制することを目的とする。 In the method for preparing a sugar chain sample using polymer particles having a functional group for capturing a sugar chain, a labeled sugar chain sample including a sugar chain having a sialic acid at the end (sialog sugar chain) is used. When preparing, sialic acid is easily detached from the sugar chain. Accordingly, an object of the present invention is to suppress the elimination of sialic acid in a method for preparing a labeled sugar chain sample using polymer particles having a functional group for capturing a sugar chain.
　本発明者らは、上記課題を解決すべく鋭意検討を行った結果、糖鎖を捕捉するための官能基を有するポリマー粒子を利用した標識された糖鎖試料の調製方法において、糖鎖を当該ポリマー粒子に捕捉する工程、糖鎖を捕捉したポリマー粒子から糖鎖を遊離させる工程、および遊離させた糖鎖を標識する工程の各工程における反応条件を低温化することなどにより、標識された糖鎖試料を調製する際のシアル酸の脱離が効果的に抑制されることを見出した。 As a result of intensive studies to solve the above problems, the inventors of the present invention have prepared a method for preparing a labeled sugar chain sample using polymer particles having a functional group for capturing a sugar chain. The labeled sugar can be obtained by reducing the reaction conditions in each step of the step of capturing the polymer chain, the step of releasing the sugar chain from the polymer particle capturing the sugar chain, and the step of labeling the released sugar chain. It has been found that the elimination of sialic acid during the preparation of the chain sample is effectively suppressed.
　すなわち、本発明は、標識された糖鎖試料の調製において糖鎖からのシアル酸の脱離を抑制する方法、および該方法により調製された糖鎖の分析方法に関し、より詳しくは、以下の発明を提供するものである。 That is, the present invention relates to a method for suppressing the elimination of sialic acid from a sugar chain in the preparation of a labeled sugar chain sample, and a method for analyzing a sugar chain prepared by the method. Is to provide.
［１］末端にシアル酸を有する糖鎖を含む、標識された糖鎖試料の調製において、糖鎖からのシアル酸の脱離を抑制する方法であって、
（ａ）末端にシアル酸を有する糖鎖を含む試料を、糖鎖を捕捉するための官能基を有するポリマー粒子に接触させ、当該糖鎖を当該ポリマー粒子に捕捉する工程、
（ｂ）糖鎖を捕捉したポリマー粒子を洗浄する工程、
（ｃ）糖鎖を捕捉したポリマー粒子から糖鎖を遊離させる工程、
（ｄ）遊離させた糖鎖を標識する工程、
を含み、工程（ａ）、（ｃ）および（ｄ）の少なくとも１の工程の反応を、０～６０℃で１０秒～２４時間の条件で行う方法。 [1] A method for inhibiting detachment of sialic acid from a sugar chain in the preparation of a labeled sugar chain sample containing a sugar chain having sialic acid at the end,
(A) contacting a sample containing a sugar chain having sialic acid at a terminal with a polymer particle having a functional group for capturing the sugar chain, and capturing the sugar chain on the polymer particle;
(B) a step of washing the polymer particles capturing the sugar chain,
(C) releasing the sugar chain from the polymer particles having captured the sugar chain;
(D) a step of labeling the released sugar chain,
And the reaction of at least one of steps (a), (c) and (d) is performed at 0 to 60 ° C. for 10 seconds to 24 hours.
［２］工程（ａ）、（ｃ）および（ｄ）の少なくとも１の工程の反応を、０～４０℃で１分～１６時間の条件で行う、［１］に記載の方法。 [2] The method according to [1], wherein the reaction in at least one of steps (a), (c) and (d) is performed at 0 to 40 ° C. for 1 minute to 16 hours.
［３］工程（ａ）、（ｃ）および（ｄ）の反応を、酸を含む溶媒中で行う、［１］または［２］に記載の方法。 [3] The method according to [1] or [2], wherein the reaction of steps (a), (c) and (d) is carried out in a solvent containing an acid.
［４］ポリマー粒子における糖鎖を捕捉するための官能基がヒドラジド基である、［１］～［３］のいずれかに記載の方法。 [4] The method according to any one of [1] to [3], wherein the functional group for capturing a sugar chain in the polymer particle is a hydrazide group.
［５］ポリマー粒子が下記（式１）で表される架橋型ポリマー構造を有するものである、［４］に記載の方法。 [5] The method according to [4], wherein the polymer particles have a crosslinked polymer structure represented by the following (formula 1).
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
（Ｒ１，Ｒ２は－Ｏ－，－Ｓ－，－ＮＨ－，－ＣＯ－，－ＣＯＮＨ－で中断されてもよい炭素数１～２０の炭化水素鎖を表し、Ｒ３，Ｒ４，Ｒ５はＨ，ＣＨ３または炭素数２～５の炭化水素鎖を表す。ｍ，ｎはモノマーユニット数を表し、ｍ：ｎ＝９９：１～７０：３０である。）
［６］ポリマー粒子が下記（式２）で表される架橋型ポリマー構造を有するものである、［４］に記載の方法。 (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—, and R 3 , R 4 , R 5 represents H, CH 3 or a hydrocarbon chain having 2 to 5 carbon atoms, m and n represent the number of monomer units, and m: n = 99: 1 to 70:30.
[6] The method according to [4], wherein the polymer particles have a crosslinked polymer structure represented by the following (formula 2).
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
（ｍ，ｎはモノマーユニット数を表し、ｍ：ｎ＝９９：１～７０：３０である。）
［７］糖鎖の標識が２－アミノベンズアミドにより行われる、［１］～［６］のいずれかに記載の方法。 (M and n represent the number of monomer units, and m: n = 99: 1 to 70:30)
[7] The method according to any one of [1] to [6], wherein the sugar chain is labeled with 2-aminobenzamide.
［８］試料中の糖鎖の分析方法であって、［１］～［７］のいずれかに記載の方法により標識された糖鎖を調製する工程、および調製した標識された糖鎖を分析する工程を含む方法。 [8] A method for analyzing a sugar chain in a sample, the step of preparing a labeled sugar chain by the method according to any one of [1] to [7], and analyzing the prepared labeled sugar chain A method comprising the step of:
　本発明により、糖鎖を捕捉するための官能基を有するポリマー粒子を利用して、標識された糖鎖試料を調製する際に、シアル酸の脱離を効果的に抑制することが可能となった。 According to the present invention, detachment of sialic acid can be effectively suppressed when preparing a labeled sugar chain sample using polymer particles having a functional group for capturing a sugar chain. It was.
ＢｌｏｔＧｌｙｃｏ（Ｒ）（住友ベークライト株式会社製）を利用して、ウシ血清由来Ｆｅｔｕｉｎから各反応条件（実施例１、２、比較例１）で標識された糖鎖試料を調製し、ＨＰＬＣに供した。ＨＰＬＣチャートから、中性糖鎖に由来するピークの面積（ｎｅｕｔｒａｌ）、シアル酸を１つ持つ糖鎖に由来するピークの面積（１ＮＡ）、シアル酸を２つ持つ糖鎖に由来するピークの面積（２ＮＡ）、シアル酸を３つ持つ糖鎖に由来するピークの面積（３ＮＡ）、およびシアル酸を４つ持つ糖鎖に由来するピークの面積（４ＮＡ）を算出し、各反応条件毎に棒グラフで示した。Using BlotGlyco (R) (manufactured by Sumitomo Bakelite Co., Ltd.), sugar chain samples labeled with each reaction condition (Examples 1 and 2 and Comparative Example 1) were prepared from bovine serum-derived Fetuin and subjected to HPLC. . From the HPLC chart, the area of the peak derived from the neutral sugar chain (neutral), the area of the peak derived from the sugar chain having one sialic acid (1NA), the area of the peak derived from the sugar chain having two sialic acids (2NA), the area of the peak derived from the sugar chain having three sialic acids (3NA), and the area of the peak derived from the sugar chain having four sialic acids (4NA) were calculated, and a bar graph for each reaction condition It showed in.
　本発明は、標識された糖鎖試料の調製において糖鎖からのシアル酸の脱離を抑制する方法、および試料中の糖鎖の分析方法を提供する。 The present invention provides a method for suppressing the elimination of sialic acid from a sugar chain in the preparation of a labeled sugar chain sample, and a method for analyzing a sugar chain in a sample.
　本発明の方法においては、まず、末端にシアル酸を有する糖鎖を含む試料を、糖鎖を捕捉するための官能基を有するポリマー粒子に接触させ、当該糖鎖を当該ポリマー粒子に捕捉する（工程（ａ））。 In the method of the present invention, first, a sample containing a sugar chain having sialic acid at the terminal is brought into contact with polymer particles having a functional group for capturing a sugar chain, and the sugar chain is captured by the polymer particle ( Step (a)).
　本発明における「糖鎖を含む試料」は、例えば、生体試料から調製することができる。生体試料としては、例えば全血、血清、血漿、尿、唾液、細胞、組織、ウイルス、植物組織などが挙げられる。また、本発明においては、精製された、あるいは未精製の糖タンパク質を用いることができる。試料は脱脂、脱塩、タンパク質分画、熱変性などの方法により前処理されていてもよい。 The “sample containing sugar chains” in the present invention can be prepared from a biological sample, for example. Examples of biological samples include whole blood, serum, plasma, urine, saliva, cells, tissues, viruses, plant tissues, and the like. Further, in the present invention, purified or unpurified glycoprotein can be used. The sample may be pretreated by a method such as degreasing, desalting, protein fractionation, and heat denaturation.
　糖鎖遊離手段を用いて上記生体試料に含まれる糖鎖を含む分子（例えば、糖タンパク質）から糖鎖を遊離させることができる。糖鎖遊離手段としては、Ｎ－グリコシダーゼあるいはＯ－グリコシダーゼを用いたグリコシダーゼ処理、ヒドラジン分解、アルカリ処理によるβ脱離などの方法を用いることができる。Ｎ型糖鎖の分析を行う場合は、Ｎ－グリコシダーゼを用いる方法が好ましい。なお、グリコシダーゼ処理に先立って、トリプシンやキモトリプシンなどを用いて生体試料のプロテアーゼ処理を行ってもよいが、プロテアーゼ失活処理においては試料を高温にさらす必要があり、シアル酸が脱離する可能性があるため、注意が必要である。生体試料のプロテアーゼ失活処理が必要な場合には、生体試料を６０℃を超える条件下に置く時間を最低限にすることが好ましい。より具体的には、生体試料を６０℃を超える条件下に置く時間を３０分以下にすることが好ましく、１０分以下にすることがより好ましく、５分以下にすることがさらに好ましい。 Sugar chains can be released from molecules containing sugar chains (for example, glycoproteins) contained in the biological sample using sugar chain releasing means. As the sugar chain releasing means, methods such as glycosidase treatment using N-glycosidase or O-glycosidase, hydrazine degradation, and β elimination by alkali treatment can be used. When analyzing an N-type sugar chain, a method using N-glycosidase is preferable. Prior to glycosidase treatment, protease treatment of biological samples may be performed using trypsin, chymotrypsin, etc., but in the protease inactivation treatment, it is necessary to expose the sample to a high temperature, and sialic acid may be eliminated. Because there is, attention is necessary. When protease inactivation treatment of a biological sample is necessary, it is preferable to minimize the time for which the biological sample is placed under conditions exceeding 60 ° C. More specifically, the time for placing the biological sample under conditions exceeding 60 ° C. is preferably 30 minutes or less, more preferably 10 minutes or less, and even more preferably 5 minutes or less.
　こうして調製した試料を、糖鎖を捕捉するための官能基を有するポリマー粒子に接触させることにより、糖鎖を当該ポリマー粒子に捕捉することができる。糖鎖は生体内物質のなかで唯一、アルデヒド基をもつ物質である。糖鎖は水溶液などの状態で環状のヘミアセタール型と、非環状型のアルデヒド型とが平衡で存在する。タンパク質や核酸、脂質など糖鎖以外の生体内物質にはアルデヒド基が含まれていない。このことから、アルデヒド基と特異的に反応して安定な結合を形成するヒドラジド基やアミノオキシ基を有するポリマー粒子を利用すれば、糖鎖のみを選択的に捕捉することが可能である。 By bringing the sample thus prepared into contact with polymer particles having a functional group for capturing sugar chains, the sugar chains can be captured by the polymer particles. The sugar chain is the only substance in the living body that has an aldehyde group. In the sugar chain, a cyclic hemiacetal type and an acyclic aldehyde type exist in an equilibrium state in an aqueous solution or the like. In vivo substances other than sugar chains such as proteins, nucleic acids, and lipids do not contain aldehyde groups. From this, it is possible to selectively capture only sugar chains by using polymer particles having hydrazide groups or aminooxy groups that react specifically with aldehyde groups to form stable bonds.
　「ポリマー粒子」としては、固体粒子やゲル粒子を好適に用いることができる。このようなポリマー粒子を用いれば、ポリマー粒子に糖鎖を捕捉させたのち、遠心分離やろ過などの手段によって、当該ポリマー粒子を容易に回収することができる。また、ポリマー粒子をカラムに充填して用いることも可能である。カラムに充填して用いる方法は、特に連続操作化の観点から重要となる。反応容器としてフィルタープレート（例えば、Ｍｉｌｌｉｐｏｒｅ社製のＭｕｌｔｉＳｃｒｅｅｎ　Ｓｏｌｖｉｎｅｒｔ　Ｆｉｌｔｅｒ　Ｐｌａｔｅ）を用いることにより、複数のサンプルを同時に処理することが可能となり、例えば、ゲルろ過に代表されるカラム操作による従来の精製手段と比較して、精製のスループットが大幅に向上される。また、当該ポリマー粒子として磁性体ビーズを用いれば、磁力を使って容器壁面などにビーズを集積できるため、ビーズの洗浄を容易に行うことができる。 As the “polymer particles”, solid particles or gel particles can be suitably used. If such polymer particles are used, the polymer particles can be easily collected by means such as centrifugation or filtration 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 filling the column and using it is particularly important from the viewpoint of continuous operation. By using a filter plate (for example, MultiScreen Solvinert Filter Plate manufactured by Millipore) as a reaction vessel, a plurality of samples can be processed at the same time. For example, conventional purification means by column operation represented by gel filtration In comparison, the purification throughput is greatly improved. Further, if magnetic beads are used as the polymer particles, the beads can be easily collected on the container wall surface by using magnetic force, so that the beads can be easily washed.
　ポリマー粒子の形状は特に限定しないが、球状またはそれに類する形状が好ましい。ポリマー粒子が球状の場合、平均粒径は好ましくは０．０５～１０００μｍであり、より好ましくは０．０５～２００μｍであり、さらに好ましくは０．１～２００μｍであり、最も好ましくは０．１～１００μｍである。平均粒径が下限値未満では、ポリマー粒子をカラムに充填して用いる際、通液性が悪くなるために大きな圧力を加える必要がある。また、ポリマー粒子を遠心分離やろ過で回収することも困難となる。平均粒径が上限値を超えると、ポリマー粒子と試料溶液の接触面積が少なくなり、糖鎖捕捉の効率が低下する。 The shape of the polymer particles is not particularly limited, but a spherical shape or a similar shape is preferable. When the polymer particles are spherical, the average particle size is preferably 0.05 to 1000 μm, more preferably 0.05 to 200 μm, still more preferably 0.1 to 200 μm, and most preferably 0.1 to 200 μm. 100 μm. If the average particle diameter is less than the lower limit, when the polymer particles are packed in a column and used, liquid permeability becomes poor, and it is necessary to apply a large pressure. Moreover, it becomes difficult to collect the polymer particles by centrifugation or filtration. When the average particle size exceeds the upper limit, the contact area between the polymer particles and the sample solution decreases, and the sugar chain capture efficiency decreases.
　ポリマー粒子における「糖鎖を捕捉するための官能基」としては、ヒドラジド基またはアミノオキシ基が好ましい。これら官能基は、糖鎖のアルデヒド基と反応して、ヒドラジド結合またはオキシム結合により糖鎖を捕捉することができる。本発明においては、ヒドラジド基が特に好ましい。 The “functional group for capturing sugar chains” in the polymer particles is preferably a hydrazide group or an aminooxy group. These functional groups can react with the aldehyde group of the sugar chain to capture the sugar chain through a hydrazide bond or an oxime bond. In the present invention, a hydrazide group is particularly preferred.
　ヒドラジド基を有するポリマー粒子としては、下記（式１）で表される構造を有する架橋型ポリマー粒子が好ましい。 As the polymer particles having a hydrazide group, crosslinked polymer particles having a structure represented by the following (formula 1) are preferable.
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
　（式１）中、Ｒ１，Ｒ２は－Ｏ－，－Ｓ－，－ＮＨ－，－ＣＯ－，－ＣＯＮＨ－で中断されてもよい炭素数１～２０の炭化水素鎖を表し、Ｒ３，Ｒ４，Ｒ５はＨ，ＣＨ３または炭素数２～５の炭化水素鎖を表す。ｍ，ｎはモノマーユニット数を表し、ｍ：ｎ＝９９：１～７０：３０である。 In the formula (1), R 1 and R 2 represent a hydrocarbon chain having 1 to 20 carbon atoms that may be interrupted by —O—, —S—, —NH—, —CO—, —CONH—, and R 3 , R 4 and R 5 each represent H, CH 3 or a hydrocarbon chain having 2 to 5 carbon atoms. m and n represent the number of monomer units, and m: n = 99: 1 to 70:30.
　架橋型ポリマー粒子としては、下記（式２）で表される構造を有する架橋型ポリマー粒子が特に好ましい。 As the crosslinkable polymer particles, crosslinkable polymer particles having a structure represented by the following (formula 2) are particularly preferable.
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006
（ｍ，ｎはモノマーユニット数を表し、ｍ：ｎ＝９９：１～７０：３０である。）
　上記の構造を有するポリマー粒子を調製する方法は、例えば、国際公開第２００８／０１８１７０号公報に記載されている。上記の構造を有するポリマー粒子の製品としては、ヒドラジド基含有ポリマー粒子である「ＢｌｏｔＧｌｙｃｏ（Ｒ）」（住友ベークライト株式会社製）を好適に用いることができる。 (M and n represent the number of monomer units, and m: n = 99: 1 to 70:30)
A method for preparing the polymer particles having the above structure is described in, for example, International Publication No. 2008/018170. As a product of polymer particles having the above structure, “BlotGlyco (R) ” (manufactured by Sumitomo Bakelite Co., Ltd.), which is a hydrazide group-containing polymer particle, can be suitably used.
　糖鎖を捕捉する際に用いるポリマー粒子の量は、好ましくは０．１ｍｇ～１００ｍｇであり、より好ましくは０．１ｍｇ～１０ｍｇである。反応系を小さくすることで、反応時間を短縮することが可能となるため、少量の試料に対しては、０．１ｍｇ～５ｍｇにするのがさらに好ましい。 The amount of polymer particles used for capturing sugar chains is preferably 0.1 mg to 100 mg, more preferably 0.1 mg to 10 mg. Since the reaction time can be shortened by reducing the reaction system, the amount is more preferably 0.1 mg to 5 mg for a small amount of sample.
　糖鎖を捕捉する際の溶媒としては、例えば、酸と有機溶媒の混合溶媒を用いることができる。使用する酸としては、特に制限されるものではないが、例えば、酢酸、ギ酸、トリフルオロ酢酸、塩酸、クエン酸、リン酸、硫酸が好ましく、より好ましくは酢酸、ギ酸、トリフルオロ酢酸、リン酸、さらに好ましくは酢酸、トリフルオロ酢酸が好適に用いられる。有機溶剤としては糖と前記の酸を溶解するものであれば制限はないが、捕捉時に乾固すると捕捉効率が上昇することが多いため、アセトニトリルなど比較的低沸点の有機溶媒が好適に用いられる。酸と有機溶媒の混合比率は、好ましくは０．１：９９．９～１０：９０、より好ましくは０．５：９９．５～５：９５、最も好ましくは１：９９～５：９５である。 As the solvent for capturing the sugar chain, for example, a mixed solvent of an acid and an organic solvent can be used. The acid to be used is not particularly limited. For example, acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid and sulfuric acid are preferable, and acetic acid, formic acid, trifluoroacetic acid and phosphoric acid are more preferable. More preferably, acetic acid and trifluoroacetic acid are suitably used. The organic solvent is not limited as long as it can dissolve sugar and the above-mentioned acid. However, since the trapping efficiency often increases when solidified during trapping, a relatively low boiling point organic solvent such as acetonitrile is preferably used. . The mixing 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 most preferably 1:99 to 5:95. .
　本発明における糖鎖捕捉時の反応系としては、上記有機溶媒系以外に、水系を用いることもできる。この場合、反応系のｐＨは、好ましくは２～９、より好ましくは２～７であり、さらに好ましくは２～６である。ｐＨ調整のためには、各種緩衝液を用いることができる。 As the reaction system at the time of sugar chain capture in the present invention, an aqueous system can be used in addition to the organic solvent system. In this case, the pH of the reaction system is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6. Various buffers can be used for pH adjustment.
　本発明においては、工程（ａ）を０～６０℃で行ってもよく、０～４０℃で行ってもよく、２０～４０℃で行ってもよい。また、工程（ａ）の反応時間は１０秒～２４時間であってもよく、１分～１６時間であってもよく、１～１６時間であってもよく、５～１６時間であってもよく、１０～１６時間であってもよい。工程（ａ）は、例えば、０～６０℃の反応温度、１０秒～２４時間の反応時間で行ってもよい。また、工程（ａ）は、例えば、０～４０℃の反応温度、１分～１６時間の反応時間で行ってもよい。 In the present invention, the step (a) may be performed at 0 to 60 ° C., may be performed at 0 to 40 ° C., or may be performed at 20 to 40 ° C. The reaction time in step (a) may be 10 seconds to 24 hours, 1 minute to 16 hours, 1 to 16 hours, or 5 to 16 hours. It may be 10 to 16 hours. Step (a) may be performed, for example, at a reaction temperature of 0 to 60 ° C. and a reaction time of 10 seconds to 24 hours. In addition, step (a) may be performed, for example, at a reaction temperature of 0 to 40 ° C. and a reaction time of 1 minute to 16 hours.
　反応は、糖鎖を遊離させる反応を効率よく行う観点から、開放系で行って溶媒を完全に蒸発させることが好ましい。蒸発を促すために、熱をかけるあるいは真空引きするのが有効である。これにより乾固が促進し、反応時間の短縮が期待される。用いる溶液量は、１μＬ～１ｍＬが好ましく、より好ましくは１μＬ～５００μＬである。反応系が小さいほど溶媒の蒸発が速いため、反応系を小さくすることによっても反応時間の短縮が期待される。よって、反応時間を短くする必要がある場合は、用いる溶液量を１μＬ～１００μＬ（例えば、１μＬ～５０μＬ）にするのがさらに好ましい。 The reaction is preferably carried out in an open system to evaporate the solvent completely from the viewpoint of efficiently carrying out the reaction for releasing the sugar chain. In order to promote evaporation, it is effective to apply heat or vacuum. This promotes drying and is expected to shorten the reaction time. The amount of the solution used is preferably 1 μL to 1 mL, more preferably 1 μL to 500 μL. The smaller the reaction system is, the faster the solvent evaporates. Therefore, shortening the reaction system is expected to shorten the reaction time. Therefore, when it is necessary to shorten the reaction time, the amount of the solution to be used is more preferably 1 μL to 100 μL (for example, 1 μL to 50 μL).
　本発明の方法においては、次いで、糖鎖を捕捉したポリマー粒子を洗浄する（工程（ｂ））。 In the method of the present invention, the polymer particles capturing the sugar chains are then washed (step (b)).
　糖鎖を捕捉したポリマー粒子の洗浄により、ポリマー粒子に捕捉された物質のうち糖鎖以外の物質（非特異的に吸着した物質）を除去することができる。 <Substances other than sugar chains (substances adsorbed non-specifically) among substances trapped by the polymer particles can be removed by washing the polymer particles that have captured the sugar chains.
　糖鎖以外の物質を除去する方法としては、疎水結合を解離する能力のあるカオトロピック試薬であるグアニジン水溶液で洗浄する方法、界面活性剤水溶液で洗浄する方法、純水や水溶性緩衝液（例えばリン酸緩衝液、トリス緩衝液など）で洗浄する方法を用いることができる。洗浄工程における洗浄条件としては、温度が０～６０℃であってもよく、４～４０℃であってもよく、１５～２５℃であってもよい。また、洗浄時間は１０秒から２時間であってもよく、１０秒～１時間であってもよく、１０秒～３０分であってもよい。洗浄方法としては、ポリマー粒子を、洗浄液に浸漬し、洗浄液の交換を繰り返す方法等が挙げられる。 Methods for removing substances other than sugar chains include washing with a guanidine aqueous solution, a chaotropic reagent capable of dissociating hydrophobic bonds, washing with a surfactant aqueous solution, pure water or a water-soluble buffer (for example, phosphorous). A method of washing with an acid buffer, a Tris buffer, or the like can be used. As cleaning conditions in the cleaning step, the temperature may be 0 to 60 ° C., 4 to 40 ° C., or 15 to 25 ° C. The washing time may be 10 seconds to 2 hours, 10 seconds to 1 hour, or 10 seconds to 30 minutes. Examples of the cleaning method include a method in which polymer particles are immersed in a cleaning solution and the replacement of the cleaning solution is repeated.
　具体的には、遠沈管やチューブにポリマー粒子を入れ、洗浄液を加え、振とうの後、遠心操作によりポリマー粒子を沈殿させて、上清を除去する操作を繰り返すことにより洗浄する。例えば、遠心チューブ内にポリマー粒子を入れ、洗浄液を加え、ポリマー粒子を自然沈降、または、遠心分離により強制的に沈降させた後、上清を除去する操作を繰り返すことで洗浄することができる。前記洗浄操作は３～６回行うことが好ましい。磁性ビーズを用いる場合には、遠心操作は不要であり、簡便である。 Specifically, the polymer particles are put into a centrifuge tube or tube, a washing solution is added, and after shaking, the polymer particles are precipitated by centrifugation and washed by repeating the operation of removing the supernatant. For example, it can be washed by repeating the operation of putting polymer particles in a centrifuge tube, adding a washing solution, allowing the polymer particles to settle naturally or by forced centrifugation, and then removing the supernatant. The washing operation is preferably performed 3 to 6 times. When magnetic beads are used, centrifugation is unnecessary and simple.
　また、チューブ状の容器であって、底面部に、液体透過可能で該ポリマー粒子が不透過な孔径を有するフィルターを装着するフィルターチューブを用いることも可能である。該フィルターチューブにポリマー粒子を入れて使用することで、洗浄に要した洗浄液を、フィルターを介して除去することが可能となり、前記の遠心操作後の上清除去の工程が必要なくなり、作業性の向上を図ることができる。 Further, it is also possible to use a filter tube which is a tube-like container and is equipped with a filter having a pore size which allows liquid permeation and does not allow polymer particles to pass through on the bottom surface. By using polymer particles in the filter tube, it becomes possible to remove the washing liquid required for washing through the filter, eliminating the need for the step of removing the supernatant after the centrifugation, and improving workability. Improvements can be made.
　また、６～３８４穴のマルチウェルプレートの底部が前記フィルターを装着したものが各種市販されており、これらのプレートを用いることでハイスループット化することが可能である。特に９６穴マルチウェルプレートは、溶液分注機器、吸引除去システム、およびプレートの搬送システムなどが開発されており、ハイスループット化に最適である。 In addition, various types of multi-well plates having 6 to 384 holes with the filter attached to the bottom are commercially available, and high throughput can be achieved by using these plates. In particular, a 96-well multiwell plate has been developed as a solution dispensing device, a suction removal system, a plate transport system, and the like, and is optimal for high throughput.
　ポリマー粒子を充填させたカラムを利用して糖鎖捕捉反応を行った場合には、洗浄処理は、カラムに洗浄溶液を通すことにより、糖鎖捕捉反応から連続的に処理してもよい。また、マルチプレートを用いた場合には、ろ過操作あるいは遠心操作により糖鎖を捕捉したポリマー粒子以外の物質を除去してもよい。 When the sugar chain capture reaction is performed using a column packed with polymer particles, the washing treatment may be performed continuously from the sugar chain capture reaction by passing a washing solution through the column. When a multiplate is used, substances other than the polymer particles that have captured the sugar chains may be removed by filtration or centrifugation.
　なお、ポリマー粒子上の余剰官能基は、例えば、無水酢酸などを利用してキャッピングすることができる。 The surplus functional group on the polymer particle can be capped using, for example, acetic anhydride.
　本発明の方法においては、次いで、糖鎖を捕捉したポリマー粒子から糖鎖を遊離させる（工程（ｃ））。 In the method of the present invention, the sugar chains are then released from the polymer particles that have captured the sugar chains (step (c)).
　ポリマー粒子から糖鎖を切り離すためには、酸処理を行うことが好ましい。本反応には水が必要であり、本工程を実施する反応溶液中の水の含有率は好ましくは０．１～９９．９容量％、より好ましくは１～９９容量％、さらに好ましくは５～９８容量％である。また、本工程を実施する反応溶液として、酸と水と有機溶媒の混合溶媒を用いることもできる。さらに、本工程を実施する反応溶液として、水性緩衝液を使用してもよい。水性緩衝液の塩濃度は好ましくは０．１ｍＭ～１Ｍ、より好ましくは０．１ｍＭ～５００ｍＭ、さらに好ましくは１ｍＭ～１００ｍＭである。反応溶液のｐＨは、好ましくは２～９、より好ましくは２～７であり、さらに好ましくは２～６である。使用する酸としては、特に制限されるものではないが、例えば、酢酸、ギ酸、トリフルオロ酢酸、塩酸、クエン酸、リン酸、硫酸が好ましく、より好ましくは酢酸、ギ酸、トリフルオロ酢酸、リン酸、さらに好ましくは酢酸、トリフルオロ酢酸が好適に用いられる。有機溶剤としては糖と前記の酸を溶解するものであれば制限はないが、捕捉時に乾固すると捕捉効率が上昇することが多いため、アセトニトリルなど比較的低沸点の有機溶媒が好適に用いられる。 In order to detach sugar chains from polymer particles, it is preferable to perform acid treatment. Water is required for this reaction, and the content of water in the reaction solution for carrying out this step is preferably 0.1 to 99.9% by volume, more preferably 1 to 99% by volume, and even more preferably 5 to 5%. 98% by volume. Moreover, a mixed solvent of an acid, water and an organic solvent can also be used as a reaction solution for carrying out this step. Furthermore, you may use an aqueous buffer as a reaction solution which implements this process. The salt concentration of the aqueous buffer is preferably 0.1 mM to 1 M, more preferably 0.1 mM to 500 mM, and even more preferably 1 mM to 100 mM. The pH of the reaction solution is preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6. The acid to be used is not particularly limited. For example, acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid and sulfuric acid are preferable, and acetic acid, formic acid, trifluoroacetic acid and phosphoric acid are more preferable. More preferably, acetic acid and trifluoroacetic acid are suitably used. The organic solvent is not limited as long as it can dissolve sugar and the above-mentioned acid. However, since the trapping efficiency often increases when solidified during trapping, a relatively low boiling point organic solvent such as acetonitrile is preferably used. .
　本発明においては、工程（ｃ）を、０～６０℃の反応温度で行う。工程（ｃ）は、０～４０℃で行ってもよく、２０～４０℃で行ってもよい。また、工程（ｃ）の反応時間は１０秒～２４時間であってもよく、１分～１６時間であってもよく、１～１６時間であってもよく、５～１６時間であってもよく、１０～１６時間であってもよい。工程（ｃ）は、例えば、０～６０℃の反応温度、１０秒～２４時間の反応時間で行ってもよい。また、工程（ｃ）は、例えば、０～４０℃の反応温度、１分～１６時間の反応時間で行ってもよい。 In the present invention, step (c) is performed at a reaction temperature of 0 to 60 ° C. Step (c) may be performed at 0 to 40 ° C. or may be performed at 20 to 40 ° C. The reaction time in step (c) may be 10 seconds to 24 hours, 1 minute to 16 hours, 1 to 16 hours, or 5 to 16 hours. It may be 10 to 16 hours. Step (c) may be performed, for example, at a reaction temperature of 0 to 60 ° C. and a reaction time of 10 seconds to 24 hours. In addition, step (c) may be performed, for example, at a reaction temperature of 0 to 40 ° C. and a reaction time of 1 minute to 16 hours.
　反応は、糖鎖を遊離させる反応を効率よく行う観点から、開放系で行って溶媒を完全に蒸発させることが好ましい。蒸発を促すために、熱をかけるあるいは真空引きするのが有効である。これにより乾固が促進し、反応時間の短縮が期待される。用いる溶液量は、１μＬ～１ｍＬが好ましく、より好ましくは１μＬ～５００μＬである。反応系が小さいほど溶媒の蒸発が速いため、反応系を小さくすることによっても反応時間の短縮が期待される。よって、反応時間を短くする必要がある場合は、用いる溶液量を１μＬ～１００μＬ（例えば、１μＬ～５０μＬ）にするのがさらに好ましい。 The reaction is preferably carried out in an open system to evaporate the solvent completely from the viewpoint of efficiently carrying out the reaction for releasing the sugar chain. In order to promote evaporation, it is effective to apply heat or vacuum. This promotes drying and is expected to shorten the reaction time. The amount of the solution used is preferably 1 μL to 1 mL, more preferably 1 μL to 500 μL. The smaller the reaction system is, the faster the solvent evaporates. Therefore, shortening the reaction system is expected to shorten the reaction time. Therefore, when it is necessary to shorten the reaction time, the amount of the solution to be used is more preferably 1 μL to 100 μL (for example, 1 μL to 50 μL).
　本発明においては、弱酸性から中性付近で、糖鎖の遊離反応を行うことができるため、従来の強酸性処理、例えば、１０％トリフルオロ酢酸処理による切出しのような強酸の存在下での切出し反応に比べて、シアル酸残基の脱離など糖鎖の加水分解などを引き起こすことを抑制することが可能となる。また、低温で反応することにより、よりシアル酸の脱離を抑制することができる。 In the present invention, the sugar chain can be liberated from weakly acidic to neutral, so that the conventional strong acidic treatment, for example, in the presence of a strong acid such as excision by 10% trifluoroacetic acid treatment. Compared to the excision reaction, it is possible to suppress the occurrence of sugar chain hydrolysis such as elimination of sialic acid residues. Moreover, by reacting at a low temperature, the elimination of sialic acid can be further suppressed.
　本発明の方法においては、次いで、遊離させた糖鎖を標識する（工程（ｄ））。 In the method of the present invention, the released sugar chain is then labeled (step (d)).
　標識化の方法としては、還元的アミノ化法と交換反応法を好適に用いることができる。まず、還元的アミノ化法について説明する。還元的アミノ化法においては、アミノ基を有する化合物により、糖鎖を標識化する。反応系においては、ｐＨ測定した際に得られた結果が、酸性から中性の条件であるのが好ましく、好ましくはｐＨ２～９、より好ましくは２～８であり、さらに好ましくは２～７である。アミノ化合物の濃度は、１ｍＭ～１０Ｍであるのが好ましく、還元剤の濃度は、１ｍＭ～１０Ｍであるのが好ましい。 As the labeling method, a reductive amination method and an exchange reaction method can be suitably used. First, the reductive amination method will be described. In the reductive amination method, a sugar chain is labeled with a compound having an amino group. In the reaction system, the results obtained when the pH is measured are preferably acidic to neutral conditions, preferably 2 to 9, more preferably 2 to 8, and even more preferably 2 to 7. is there. The concentration of the amino compound is preferably 1 mM to 10M, and the concentration of the reducing agent is preferably 1 mM to 10M.
　本発明においては、工程（ｄ）を、０～６０℃の反応温度で行う。工程（ｄ）は、０～４０℃で行ってもよく、２０～４０℃で行ってもよい。また、工程（ｄ）の反応時間は１０秒～２４時間であってもよく、１分～１６時間であってもよく、１～１６時間であってもよく、５～１６時間であってもよく、１０～１６時間であってもよい。工程（ｄ）は、例えば、０～６０℃の反応温度、１０秒～２４時間の反応時間で行ってもよい。また、工程（ｄ）は、例えば、０～４０℃の反応温度、１分～１６時間の反応時間で行ってもよい。 In the present invention, step (d) is performed at a reaction temperature of 0 to 60 ° C. Step (d) may be performed at 0 to 40 ° C. or at 20 to 40 ° C. The reaction time in step (d) may be 10 seconds to 24 hours, 1 minute to 16 hours, 1 to 16 hours, or 5 to 16 hours. It may be 10 to 16 hours. Step (d) may be performed, for example, at a reaction temperature of 0 to 60 ° C. and a reaction time of 10 seconds to 24 hours. In addition, step (d) may be performed, for example, at a reaction temperature of 0 to 40 ° C. and a reaction time of 1 minute to 16 hours.
　ここで、アミノ基を有する化合物は、紫外可視吸収特性または蛍光特性を有することが好ましく、具体的には下記の群から選ばれる少なくとも１つであることが好ましい。 Here, the compound having an amino group preferably has ultraviolet-visible absorption characteristics or fluorescence characteristics, and specifically, is preferably at least one selected from the following group.
　８－アミノピレン－１，３，６－トリスルフォネート（８－Ａｍｉｎｏｐｙｒｅｎｅ－１，３，６－ｔｒｉｓｕｌｆｏｎａｔｅ），８－アミノナフタレン－１，３，６－トリスルフォネート（８－Ａｍｉｎｏｎａｐｈｔｈａｌｅｎｅ－１，３，６－ｔｒｉｓｕｌｐｈｏｎａｔｅ），７－アミノ－１，３－ナフタレンジスルフォニックアシド（７－ａｍｉｎｏ－１，３－ｎａｐｈｔａｌｅｎｅｄｉｓｕｌｆｏｎｉｃａｃｉｄ），２－アミノ９（１０Ｈ）－アクリドン（２－Ａｍｉｎｏ９（１０Ｈ）－ａｃｒｉｄｏｎｅ），５－アミノフルオロセイン（５－Ａｍｉｎｏｆｌｕｏｒｅｓｃｅｉｎ），ダンシルエチレンジアミン（Ｄａｎｓｙｌｅｔｈｙｌｅｎｅｄｉａｍｉｎｅ），２－アミノピリジン（２－Ａｍｉｎｏｐｙｒｉｄｉｎｅ），７－アミノ－４－メチルクマリン（７－Ａｍｉｎｏ－４－ｍｅｔｈｙｌｃｏｕｍａｒｉｎｅ），２－アミノベンズアミド（２－Ａｍｉｎｏｂｅｎｚａｍｉｄｅ），２－アミノベンゾイックアシド（２－Ａｍｉｎｏｂｅｎｚｏｉｃａｃｉｄ），３－アミノベンゾイックアシド（３－Ａｍｉｎｏｂｅｎｚｏｉｃａｃｉｄ），７－アミノ－１－ナフトール（７－Ａｍｉｎｏ－１－ｎａｐｈｔｈｏｌ），３－（アセチルアミノ）－６－アミノアクリジン（３－（Ａｃｅｔｙｌａｍｉｎｏ）－６－ａｍｉｎｏａｃｒｉｄｉｎｅ），２－アミノ－６－シアノエチルピリジン（２－Ａｍｉｎｏ－６－ｃｙａｎｏｅｔｈｙｌｐｙｒｉｄｉｎｅ），エチルｐ－アミノベンゾエート（Ｅｔｈｙｌｐ－ａｍｉｎｏｂｅｎｚｏａｔｅ），ｐ－アミノベンゾニトリル（ｐ－Ａｍｉｎｏｂｅｎｚｏｎｉｔｒｉｌｅ），および７－アミノナフタレン－１，３－ジスルフォニックアシド（７－ａｍｉｎｏｎａｐｈｔｈａｌｅｎｅ－１，３－ｄｉｓｕｌｆｏｎｉｃａｃｉｄ）。 8-aminopyrene-1,3,6-trisulfonate (8-Aminopyrene-1,3,6-trisulfonate), 8-aminonaphthalene-1,3,6-trisulfonate (8-Aminonaphthalene-1,3, 6-trisulfonate), 7-amino-1,3-naphthalenedisulfonic acid (7-amino-1,3-naphthalenedisulfonicacid), 2-amino9 (10H) -acridone (2-Amino9 (10H) -acridone), 5-Aminofluorescein, Dansylethylenediamine, 2-Aminopyridine, 7-A 7-Amino-4-methylcoumarin, 2-aminobenzamide, 2-aminobenzoic acid, 3-aminobenzoic acid, 7-Amino-1-naphthol, 3- (acetylamino) -6-aminoacridine (3- (Acetylamino) -6-aminoacididine), 2-amino-6-cyanoethylpyridine (2 -Amino-6-cyanoethylpyridine, ethyl p-aminobenzoate, p-aminobenzonitrile (p-Am) nobenzonitrile), and 7-aminonaphthalene-1,3-Soo Gandolfo nick A Sid (7-aminonaphthalene-1,3-disulfonicacid).
　特に、アミノ化合物が２－アミノベンズアミド（２－ａｍｉｎｏｂｅｎｚａｍｉｄｅ；２ＡＢ）の場合の反応条件は以下の通りである。反応系においては、酸と有機溶媒の混合溶媒あるいは酸と水と有機溶媒の混合溶媒を用いることができる。酸と有機溶媒の混合溶媒の場合、酸と有機溶媒の混合比率は、好ましくは１：９９～５０：５０、より好ましくは５：９５～５０：５０、最も好ましくは１０：９０～５０：５０である。また、酸と水と有機溶媒の混合溶媒の場合、有機溶媒の比率に関わらずｐＨ測定した際に得られた結果が、酸性から中性の条件であるのが好ましく、好ましくはｐＨ２～９、より好ましくは２～８であり、さらに好ましくは２～７である。アミノ化合物の濃度は１ｍＭ～１０Ｍ、好ましくは１０ｍＭ～１０Ｍで、さらに好ましくは１００ｍＭ～５Ｍである。還元剤の濃度は、１ｍＭ～１０Ｍ、好ましくは１０ｍＭ～１０Ｍ、さらに好ましくは１００ｍＭ～２Ｍである。反応温度は０～６０℃であってもよく、０～４０℃であってもよく、２０～４０℃であってもよい。反応時間は１０秒～２４時間であってもよく、１分～１６時間であってもよく、１～１６時間であってもよく、５～１６時間であってもよく、１０～１６時間であってもよい。 In particular, the reaction conditions when the amino compound is 2-aminobenzamide (2-AB) are as follows. In the reaction system, a mixed solvent of an acid and an organic solvent or a mixed solvent of an acid, water and an organic solvent can be used. In the case of a mixed solvent of an acid and an organic solvent, the mixing ratio of the acid and the organic solvent is preferably 1:99 to 50:50, more preferably 5:95 to 50:50, and most preferably 10:90 to 50:50. It is. In the case of a mixed solvent of an acid, water and an organic solvent, the result obtained when the pH is measured regardless of the ratio of the organic solvent is preferably an acidic to neutral condition, preferably pH 2 to 9, More preferably, it is 2-8, and more preferably 2-7. The concentration of the amino compound is 1 mM to 10M, preferably 10 mM to 10M, and more preferably 100 mM to 5M. The concentration of the reducing agent is 1 mM to 10M, preferably 10 mM to 10M, more preferably 100 mM to 2M. The reaction temperature may be 0 to 60 ° C., 0 to 40 ° C., or 20 to 40 ° C. The reaction time may be 10 seconds to 24 hours, 1 minute to 16 hours, 1 to 16 hours, 5 to 16 hours, or 10 to 16 hours. There may be.
　また、還元剤は、例えば、シアノ水素化ホウ素ナトリウム、メチルアミンボラン、ジメチルアミンボラン、トリメチルアミンボラン、ピコリンボラン、ピリジンボランなどが使用可能であるが、シアノ水素化ホウ素ナトリウムあるいはピコリンボランを使用するのが反応性の面から考えて好ましい。 As the reducing agent, for example, sodium cyanoborohydride, methylamine borane, dimethylamine borane, trimethylamine borane, picoline borane, pyridine borane and the like can be used, but sodium cyanoborohydride or picoline borane is used. Is preferable from the viewpoint of reactivity.
　この工程の後、得られる溶液は標識された糖鎖と過剰量加えた未反応アミノ化合物、還元剤が存在するため、これら余剰試薬を除去する工程を行うのが好ましい。シリカカラムによる除去、ゲル濾過による除去、イオン交換樹脂による除去、いずれの方法を用いても良いが、シアル酸の脱離を防ぐために使用する溶媒は中性であることが好ましい。 After this step, since the resulting solution contains a labeled sugar chain and an unreacted amino compound and a reducing agent added in excess, it is preferable to perform a step of removing these excess reagents. Any method of removal by silica column, removal by gel filtration, removal by ion exchange resin may be used, but the solvent used for preventing the elimination of sialic acid is preferably neutral.
　次に、交換反応法による糖鎖の標識化について説明する。交換反応法においては、洗浄操作後、アミノオキシ基を有する化合物を作用させることにより、ヒドラゾン－オキシム交換反応（糖鎖のアルデヒド基と特異的に反応する官能基がヒドラジド基の場合）またはオキシム－オキシム交換反応（糖鎖のアルデヒド基と特異的に反応する官能基がアミノオキシ基の場合）によって糖鎖がポリマー粒子から切り離され、同時にアミノオキシ化合物によって標識化される。よって、本標識方法の場合、糖鎖を捕捉したポリマー粒子から糖鎖を遊離させる工程（工程（ｃ））と遊離させた糖鎖を標識する工程（工程（ｄ））とは一体となる。このため、上記した酸処理などによって糖鎖をポリマー粒子から切り離す操作を別途実施する必要はない。 Next, sugar chain labeling by the exchange reaction method will be described. In the exchange reaction method, a hydrazone-oxime exchange reaction (when the functional group specifically reacting with the aldehyde group of the sugar chain is a hydrazide group) or oxime--by reacting with a compound having an aminooxy group after the washing operation The sugar chain is cleaved from the polymer particle by an oxime exchange reaction (when the functional group that specifically reacts with the aldehyde group of the sugar chain is an aminooxy group) and simultaneously labeled with an aminooxy compound. Therefore, in the present labeling method, the step of releasing the sugar chain from the polymer particles having captured the sugar chain (step (c)) and the step of labeling the released sugar chain (step (d)) are integrated. For this reason, it is not necessary to carry out the operation of separating the sugar chain from the polymer particles by the acid treatment described above.
　アミノオキシ基を有する化合物としては、下記の群から選ばれた物質またはその塩であることが好ましい。
　Ｏ－ベンジルヒドロキシルアミン（Ｏ－ｂｅｎｚｙｌｈｙｄｒｏｘｙｌａｍｉｎｅ），Ｏ－フェニルヒドロキシルアミン（Ｏ－ｐｈｅｎｙｌｈｙｄｒｏｘｙｌａｍｉｎｅ），Ｏ－（２，３，４，５，６－ペンタフルオロベンジル）ヒドロキシルアミン（Ｏ－（２，３，４，５，６－ｐｅｎｔａｆｌｕｏｒｏｂｅｎｚｙｌ）ｈｙｄｒｏｘｙｌａｍｉｎｅ），Ｏ－（４－ニトロベンジル）ヒドロキシルアミン（Ｏ－（４－ｎｉｔｒｏｂｅｎｚｙｌ）ｈｙｄｒｏｘｙｌａｍｉｎｅ），２－アミノオキシピリジン（２－ａｍｉｎｏｏｘｙｐｙｒｉｄｉｎｅ），２－アミノオキシメチルピリジン（２－ａｍｉｎｏｏｘｙｍｅｔｈｙｌｐｙｒｉｄｉｎｅ），４－［（アミノオキシアセチル）アミノ］ベンゾイックアシドメチルエステル（４－［（ａｍｉｎｏｏｘｙａｃｅｔｙｌ）ａｍｉｎｏ］ｂｅｎｚｏｉｃ　ａｃｉｄ　ｍｅｔｈｙｌ　ｅｓｔｅｒ），４－［（アミノオキシアセチル）アミノ］ベンゾイックアシドエチルエステル（４－［（ａｍｉｎｏｏｘｙａｃｅｔｙｌ）ａｍｉｎｏ］ｂｅｎｚｏｉｃ　ａｃｉｄ　ｅｔｈｙｌ　ｅｓｔｅｒ），および４－［（アミノオキシアセチル）アミノ］ベンゾイックアシドｎ－ブチルエステル（４－［（ａｍｉｎｏｏｘｙａｃｅｔｙｌ）ａｍｉｎｏ］ｂｅｎｚｏｉｃ　ａｃｉｄ　ｎ－ｂｕｔｙｌｅｓｔｅｒ），Ｎ－アミノオキシアセチル－トリプトフィル（アルギニンメチルエステル）（Ｎ－ａｍｉｎｏｏｘｙａｃｅｔｙｌ－ｔｒｙｐｔｏｐｈｙｌ（ａｒｇｉｎｉｎｅ　ｍｅｔｈｙｌ　ｅｓｔｅｒ）），Ｎ－アミノオキシアセチル－トリプトフィル（アルギニンアミド）（Ｎ－Ａｍｉｎｏｏｘｙａｃｅｔｙｌ－ｔｒｙｐｔｏｐｈｙｌ(ａｒｇｉｎｉｎｅ　ａｍｉｄｅ)） The compound having an aminooxy group is preferably a substance selected from the following group or a salt thereof.
O-benzylhydroxylamine, O-phenylhydroxylamine, O- (2,3,4,5,6-pentafluorobenzyl) hydroxylamine (O- (2,3,4) , 5,6-pentafluorobenzyl) hydroxylamine), O- (4-nitrobenzyl) hydroxylamine (O- (4-nitrobenzyl) hydroxylamine), 2-aminooxypyridine, 2-aminooxymethylpyridine (2) -Aminomethoxypyridine), 4-[(aminooxyacetyl) amino] benzoic acid methyl ester (4-[(amin oxyacetyl) amino] benzoic acid methyl ester), 4-[(aminooxyacetyl) amino] benzoic acid ethyl ester (4-[(aminooxyacetyl) amino] benzoic acid ethyl ester), and 4-[(aminooxyacetyl) amino Benzoic acid n-butyl ester (4-[(aminooxyacetyl) amino) benzoic acid n-butylester), N-aminooxyacetyl-tryptophyll (arginine methyl ester) -Aminooxyacetyl-tryptophyll (arginine amide (N-Aminooxyacetyl-tryptophyl (arginine amide))
　アミノオキシ基を有する化合物は、アルギニン残基、トリプトファン残基、フェニルアラニン残基、チロシン残基、システイン残基およびこれら誘導体の少なくとも一つからなる部分を含むことが好ましい。このような部分は、例えば、試料をＭＡＬＤＩ－ＴＯＦ質量分析に供した場合に、増感作用を発揮することができる。特に、下記（式３）で表されるＮ－アミノオキシアセチル－トリプトフィル（アルギニンメチルエステル）、または下記（式４）で表されるＮ－アミノオキシアセチル－トリプトフィル（アルギニンアミド）が好ましい。 The compound having an aminooxy group preferably contains a moiety comprising at least one of an arginine residue, a tryptophan residue, a phenylalanine residue, a tyrosine residue, a cysteine residue, and a derivative thereof. Such a portion can exert a sensitizing action when the sample is subjected to MALDI-TOF mass spectrometry, for example. In particular, N-aminooxyacetyl-tryptophyll (arginine methyl ester) represented by the following (formula 3) or N-aminooxyacetyl-tryptophyll (arginine amide) represented by the following (formula 4) is preferable.
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000008
　交換反応時の反応系においては、酸と有機溶媒の混合溶媒あるいは酸と水と有機溶媒の混合溶媒を用いることができる。酸と有機溶媒の混合溶媒の場合、酸と有機溶媒の混合比率は、好ましくは０．１：９９．９～１０：９０、より好ましくは０．５：９９．５～５：９５、最も好ましくは１：９９～５：９５である。また、酸と水と有機溶媒の混合溶媒の場合、有機溶媒の比率に関わらずｐＨ測定した際に得られた結果がｐＨ２～７が好ましく、ｐＨ３～６がより好ましく、ｐＨ３．５～５．５が最も好ましい。酢酸／アセトニトリル溶液を加えることにより、反応液を上記のｐＨに調整することができる。交換反応時の反応温度は０～６０℃であってもよく、０～４０℃であってもよく、２０～４０℃であってもよい。反応時間は１０秒～２４時間であってもよく、１分～１６時間であってもよく、１～１６時間であってもよく、５～１６時間であってもよく、１０～１６時間であってもよい。交換反応を効率よく行う観点から、工程（ｄ）を開放系で行って溶媒を完全に蒸発させることが好ましい。蒸発を促すために、熱をかけるあるいは真空引きするのが有効である。これにより乾固が促進し、反応時間の短縮が期待される。用いる溶液量は、１μＬ～１ｍＬが好ましく、より好ましくは１μＬ～５００μＬである。反応系が小さいほど溶媒の蒸発が速いため、反応系を小さくすることによっても反応時間の短縮が期待される。よって、反応時間を短くする必要がある場合は、用いる溶液量を１μＬ～１００μＬ（例えば、１μＬ～５０μＬ）にするのがさらに好ましい。 In the reaction system during the exchange reaction, a mixed solvent of an acid and an organic solvent or a mixed solvent of an acid, water and an organic solvent can be used. In the case of a mixed solvent of an acid and an organic solvent, the mixing 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, most preferably Is 1:99 to 5:95. In the case of a mixed solvent of acid, water, and organic solvent, the pH obtained is preferably 2 to 7, more preferably 3 to 6, more preferably 3.5 to 5. 5 is most preferred. The reaction solution can be adjusted to the above pH by adding an acetic acid / acetonitrile solution. The reaction temperature during the exchange reaction may be 0 to 60 ° C., 0 to 40 ° C., or 20 to 40 ° C. The reaction time may be 10 seconds to 24 hours, 1 minute to 16 hours, 1 to 16 hours, 5 to 16 hours, or 10 to 16 hours. There may be. From the viewpoint of efficiently performing the exchange reaction, it is preferable to perform the step (d) in an open system to completely evaporate the solvent. In order to promote evaporation, it is effective to apply heat or vacuum. This promotes drying and is expected to shorten the reaction time. The amount of the solution used is preferably 1 μL to 1 mL, more preferably 1 μL to 500 μL. The smaller the reaction system is, the faster the solvent evaporates. Therefore, shortening the reaction system is expected to shorten the reaction time. Therefore, when it is necessary to shorten the reaction time, the amount of the solution to be used is more preferably 1 μL to 100 μL (for example, 1 μL to 50 μL).
　上述した工程（ａ）、（ｃ）および（ｄ）の少なくとも１つの工程は、０～６０℃で１０秒～２４時間の条件で行う。糖鎖からのシアル酸の脱離を抑制する観点からは、工程（ａ）、（ｃ）および（ｄ）のうち、少なくとも２つの工程を、０～６０℃で１０秒～２４時間の条件で行うことがより好ましく、工程（ａ）、（ｃ）および（ｄ）全ての工程の反応を、０～６０℃で１０秒～２４時間の条件で行うことがさらに好ましい。さらに、工程（ｂ）についても０～６０℃の条件で行うことが好ましい。 At least one of the steps (a), (c) and (d) described above is performed at 0 to 60 ° C. for 10 seconds to 24 hours. From the viewpoint of suppressing elimination of sialic acid from the sugar chain, at least two steps among steps (a), (c) and (d) are carried out at 0 to 60 ° C. for 10 seconds to 24 hours. More preferably, the reaction in all steps (a), (c) and (d) is more preferably performed at 0 to 60 ° C. for 10 seconds to 24 hours. Furthermore, the step (b) is also preferably carried out at 0 to 60 ° C.
　本発明における試料中の糖鎖の分析方法においては、上記方法により調製した標識された糖鎖を分析する。上記の方法を経て回収した糖鎖溶液はそのまま、あるいは、過剰に含まれる標識化合物を除去したのち、クロマトグラフィ（例えば、ＨＰＬＣ）、質量分析（例えば、ＭＡＬＤＩ－ＴＯＦ　ＭＳ）、クロマトグラフィと質量分析の組み合わせ（例えば、ＬＣ－ＭＳ）、電気泳動（例えば、キャピラリー電気泳動）などの公知の方法により、糖鎖の分析（例えば、糖鎖の構造や量の分析）に供することができる。糖鎖の分析においては、必要に応じて、各種データベース（例えば、ＧｌｙｃｏＭｏｄ、Ｇｌｙｃｏｓｕｉｔｅ、ＳｉｍＧｌｙｃａｎ（Ｒ）など）を参照することができる。 In the method for analyzing a sugar chain in a sample in the present invention, a labeled sugar chain prepared by the above method is analyzed. The sugar chain solution recovered through the above method is used as it is, or after removing the excess labeled compound, chromatography (for example, HPLC), mass spectrometry (for example, MALDI-TOF MS), combination of chromatography and mass spectrometry It can be used for analysis of sugar chains (for example, analysis of structure and amount of sugar chains) by known methods such as (for example, LC-MS) and electrophoresis (for example, capillary electrophoresis). In the analysis of sugar chains, various databases (for example, GlycoMod, Glycosite, SimGlycan (R), etc.) can be referred to as necessary.
　以下、実施例および比較例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples and comparative examples, but the present invention is not limited to the following examples.
＜実施例１＞
（糖鎖サンプルの調製）
　ウシ血清由来Ｆｅｔｕｉｎ（ＳＩＧＭＡ社製、Ｆ３００４）１ｍｇを１００ｍＭ重炭酸アンモニウム（和光純薬工業株式会社製、０１７－０２８７５）５０μＬに溶解させた後、１２０ｍＭ　ＤＴＴ（ジチオスレイトール、ＳＩＧＭＡ社製、Ｄ９７７９）を５μＬ加え、６０℃で３０分間反応させた。反応終了後、１２３ｍＭ　ＩＡＡ（ヨードアセトアミド、和光純薬工業株式会社製、０９３－０２１５２）１０μＬを加えて遮光下、室温で１時間反応させた。続いて４００Ｕのトリプシン（ＳＩＧＭＡ社製、Ｔ０３０３）によってプロテアーゼ処理をし、タンパク質部分をペプチド断片化した。反応溶液を９０℃で５分処理した後、５ＵのグリコシダーゼＦ（Ｒｏｃｈｅ社製、１－３６５－１９３）による処理を行って糖鎖をペプチドから遊離させ、予備処理済の生体試料（糖鎖サンプル）を得た。 <Example 1>
(Preparation of sugar chain sample)
1 mg of bovine serum-derived Fetuin (manufactured by SIGMA, F3004) was dissolved in 50 μL of 100 mM ammonium bicarbonate (manufactured by Wako Pure Chemical Industries, Ltd., 017-02875), and then 120 mM DTT (dithiothreitol, manufactured by SIGMA, D9779) Was added and reacted at 60 ° C. for 30 minutes. After completion of the reaction, 10 μL of 123 mM IAA (iodoacetamide, manufactured by Wako Pure Chemical Industries, Ltd., 093-02152) was added and reacted at room temperature for 1 hour in the dark. Subsequently, protease treatment was carried out with 400 U trypsin (manufactured by SIGMA, T0303) to fragment the protein portion into peptides. The reaction solution is treated at 90 ° C. for 5 minutes, then treated with 5 U glycosidase F (Roche, 1-365-193) to release sugar chains from the peptide, and pretreated biological sample (sugar sample) )
（糖鎖捕捉担体による糖鎖の捕捉）
　糖鎖捕捉用の担体であるヒドラジド基を有する粒子５ｍｇ（ＢｌｏｔＧｌｙｃｏ（Ｒ））、住友ベークライト株式会社製）が入ったディスポカラムに上記糖鎖溶液（糖鎖サンプル）２０μＬおよび１８０μＬの２％酢酸／アセトニトリル溶液を加え、３７℃で１６時間反応させた。反応は開放系で行い、溶媒が完全に蒸発し粒子が乾固した状態であることを目視で確認した。 (Capture sugar chains by sugar chain capture carrier)
Particles having a hydrazide group is a carrier for the sugar chain capturing 5mg (BlotGlyco (R)), the sugar chain solution (sugar samples disposable column containing the Sumitomo Bakelite Co., Ltd.)) 20 [mu] L and 180μL of 2% acetic acid / Acetonitrile solution was added and reacted at 37 ° C. for 16 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried.
（洗浄およびキャッピング）
　グアニジン溶液、水、メタノール、トリエチルアミノ溶液にて粒子を洗浄後、１０％無水酢酸／メタノールを添加し、室温で３０分間反応させ、未反応のヒドラジド基をキャッピングした。キャッピング後、メタノール、塩酸水溶液、水にて粒子を洗浄した。 (Cleaning and capping)
After washing the particles with guanidine solution, water, methanol and triethylamino solution, 10% acetic anhydride / methanol was added and reacted at room temperature for 30 minutes to cap unreacted hydrazide groups. After capping, the particles were washed with methanol, aqueous hydrochloric acid and water.
（糖鎖の遊離）
　続いて、粒子の入ったディスポカラムに超純水２０μＬおよび２％酢酸／アセトニトリル溶液１８０μＬを加え、３７℃で１６時間反応させた。反応は開放系で行い、溶媒が完全に蒸発し粒子が乾固した状態であることを目視で確認した。 (Free sugar chain)
Subsequently, 20 μL of ultrapure water and 180 μL of a 2% acetic acid / acetonitrile solution were added to the disposable column containing the particles, and reacted at 37 ° C. for 16 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried.
（糖鎖の標識）
　２－アミノベンズアミド（２－ＡＢ、和光純薬工業株式会社製、５７４－９２４４１）による標識を行った。粒子の入ったディスポカラムに、２－ＡＢおよびシアノ水素化ホウ素ナトリウムの終濃度がそれぞれ０．７Ｍ、１Ｍになるように３０％酢酸／ジメチルスルホシキド（ＤＭＳＯ）混合溶媒に溶解させて調製した溶液１００μＬを添加し、３７℃で１６時間反応させた。 (Sugar chain labeling)
Labeling with 2-aminobenzamide (2-AB, manufactured by Wako Pure Chemical Industries, Ltd., 574-92441) was performed. It was prepared by dissolving in a 30% acetic acid / dimethylsulfoxide (DMSO) mixed solvent in a disposable column containing particles so that the final concentrations of 2-AB and sodium cyanoborohydride were 0.7M and 1M, respectively. 100 μL of the solution was added and reacted at 37 ° C. for 16 hours.
（余剰２ＡＢの除去）
　反応溶液５０μＬを回収し、アセトニトリルで１０倍に希釈した後、シリカカラム（ＢｌｏｔＧｌｙｃｏ（Ｒ）キット付属品）に添加してシリカゲルに標識糖鎖を吸着させた。アセトニトリルにてカラムを洗浄後、超純水５０μＬにて標識糖鎖を回収した。 (Removal of surplus 2AB)
The reaction solution 50μL was collected and diluted 10-fold with acetonitrile, to adsorb the labeled sugar chain silica gel was added to a silica column (BlotGlyco (R) kit accessory). After washing the column with acetonitrile, the labeled sugar chain was recovered with 50 μL of ultrapure water.
（標識化糖鎖の検出）
　得られた標識糖鎖をＨＩＴＡＣＨＩ　ＨＰＬＣ（ＦＬ）にて測定した。アミノカラム（Ｓｈｏｄｅｘ　Ａｓａｈｉｐａｋ　ＮＨ２Ｐ－５０）を用いて励起波長３３０ｎｍ、蛍光波長４２０ｎｍにて測定した。 (Detection of labeled sugar chain)
The obtained labeled sugar chain was measured by HITACHI HPLC (FL). Measurement was performed using an amino column (Shodex Asahipak NH2P-50) at an excitation wavelength of 330 nm and a fluorescence wavelength of 420 nm.
＜実施例２＞
（糖鎖サンプルの調製）
　実施例１と同様の方法により、糖鎖サンプルを調製した。 <Example 2>
(Preparation of sugar chain sample)
A sugar chain sample was prepared in the same manner as in Example 1.
（糖鎖捕捉担体による糖鎖の捕捉）
　糖鎖捕捉用の担体であるヒドラジド基を有する粒子５ｍｇ（ＢｌｏｔＧｌｙｃｏ（Ｒ））、住友ベークライト株式会社製）が入ったディスポカラムに上記糖鎖溶液（糖鎖サンプル）２０μＬおよび１８０μＬの２％酢酸／アセトニトリル溶液を加え、２５℃で１６時間反応させた。反応は開放系で行い、溶媒が完全に蒸発し粒子が乾固した状態であることを目視で確認した。 (Capture sugar chains by sugar chain capture carrier)
Particles having a hydrazide group is a carrier for the sugar chain capturing 5mg (BlotGlyco (R)), the sugar chain solution (sugar samples disposable column containing the Sumitomo Bakelite Co., Ltd.)) 20 [mu] L and 180μL of 2% acetic acid / Acetonitrile solution was added and reacted at 25 ° C. for 16 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried.
（洗浄およびキャッピング）
　グアニジン溶液、水、メタノール、トリエチルアミノ溶液にて粒子を洗浄後、１０％無水酢酸／メタノールを添加し、室温で３０分間反応させ、未反応のヒドラジド基をキャッピングした。キャッピング後、メタノール、塩酸水溶液、水にて粒子を洗浄した。 (Cleaning and capping)
After washing the particles with guanidine solution, water, methanol and triethylamino solution, 10% acetic anhydride / methanol was added and reacted at room temperature for 30 minutes to cap unreacted hydrazide groups. After capping, the particles were washed with methanol, aqueous hydrochloric acid and water.
（糖鎖の遊離）
　続いて、粒子の入ったディスポカラムに超純水２０μＬおよび２％酢酸／アセトニトリル溶液１８０μＬを加え、２５℃で１６時間反応させた。反応は開放系で行い、溶媒が完全に蒸発し粒子が乾固した状態であることを目視で確認した。 (Free sugar chain)
Subsequently, 20 μL of ultrapure water and 180 μL of a 2% acetic acid / acetonitrile solution were added to the disposable column containing the particles and reacted at 25 ° C. for 16 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried.
（糖鎖の標識）
　２－アミノベンズアミド（２－ＡＢ、和光純薬工業株式会社製、５７４－９２４４１）による標識を行った。粒子の入ったディスポカラムに、２－ＡＢおよびシアノ水素化ホウ素ナトリウムの終濃度がそれぞれ０．７Ｍ、１Ｍになるように３０％酢酸／ジメチルスルホシキド（ＤＭＳＯ）混合溶媒に溶解させて調製した溶液１００μＬを添加し、２５℃で１６時間反応させた。 (Sugar chain labeling)
Labeling with 2-aminobenzamide (2-AB, manufactured by Wako Pure Chemical Industries, Ltd., 574-92441) was performed. It was prepared by dissolving in a 30% acetic acid / dimethylsulfoxide (DMSO) mixed solvent in a disposable column containing particles so that the final concentrations of 2-AB and sodium cyanoborohydride were 0.7M and 1M, respectively. 100 μL of the solution was added and reacted at 25 ° C. for 16 hours.
（余剰２ＡＢの除去）
　実施例１と同様の方法により、余剰２ＡＢを除去した。 (Removal of surplus 2AB)
Excess 2AB was removed by the same method as in Example 1.
（標識化糖鎖の検出）
　実施例１と同様の方法により、標識化糖鎖を検出した。 (Detection of labeled sugar chain)
A labeled sugar chain was detected by the same method as in Example 1.
＜比較例１＞
（糖鎖サンプルの調製）
　実施例１と同様の方法により、糖鎖サンプルを調製した。 <Comparative Example 1>
(Preparation of sugar chain sample)
A sugar chain sample was prepared in the same manner as in Example 1.
（糖鎖捕捉担体による糖鎖の捕捉）
　糖鎖捕捉用の担体であるヒドラジド基を有する粒子５ｍｇ（ＢｌｏｔＧｌｙｃｏ（Ｒ））、住友ベークライト株式会社製）が入ったディスポカラムに上記糖鎖溶液（糖鎖サンプル）２０μＬおよび１８０μＬの２％酢酸／アセトニトリル溶液を加え、８０℃で１時間反応させた。反応は開放系で行い、溶媒が完全に蒸発し粒子が乾固した状態であることを目視で確認した。 (Capture sugar chains by sugar chain capture carrier)
Particles having a hydrazide group is a carrier for the sugar chain capturing 5mg (BlotGlyco (R)), the sugar chain solution (sugar samples disposable column containing the Sumitomo Bakelite Co., Ltd.)) 20 [mu] L and 180μL of 2% acetic acid / Acetonitrile solution was added 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 was completely evaporated and the particles were dried.
（洗浄およびキャッピング）
　グアニジン溶液、水、メタノール、トリエチルアミノ溶液にて粒子を洗浄後、１０％無水酢酸／メタノールを添加し、室温で３０分間反応させ、未反応のヒドラジド基をキャッピングした。キャッピング後、メタノール、塩酸水溶液、水にて粒子を洗浄した。 (Cleaning and capping)
After washing the particles with guanidine solution, water, methanol and triethylamino solution, 10% acetic anhydride / methanol was added and reacted at room temperature for 30 minutes to cap unreacted hydrazide groups. After capping, the particles were washed with methanol, aqueous hydrochloric acid and water.
（糖鎖の遊離）
　続いて、粒子の入ったディスポカラムに超純水２０μＬおよび２％酢酸／アセトニトリル溶液１８０μＬを加え、７０℃で１．５時間反応させた。反応は開放系で行い、溶媒が完全に蒸発し粒子が乾固した状態であることを目視で確認した。 (Free sugar chain)
Subsequently, 20 μL of ultrapure water and 180 μL of a 2% acetic acid / acetonitrile solution were added to the disposable column containing the particles, and reacted at 70 ° C. for 1.5 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the particles were dried.
（糖鎖の標識）
　２－アミノベンズアミド（２－ＡＢ、和光純薬工業株式会社製、５７４－９２４４１）による標識を行った。粒子の入ったディスポカラムに、２－ＡＢおよびシアノ水素化ホウ素ナトリウムの終濃度がそれぞれ０．７Ｍ、１Ｍになるように３０％酢酸／ジメチルスルホシキド（ＤＭＳＯ）混合溶媒に溶解させて調製した溶液１００μＬを添加し、６０℃で２時間反応させた。 (Sugar chain labeling)
Labeling with 2-aminobenzamide (2-AB, manufactured by Wako Pure Chemical Industries, Ltd., 574-92441) was performed. It was prepared by dissolving in a 30% acetic acid / dimethylsulfoxide (DMSO) mixed solvent in a disposable column containing particles so that the final concentrations of 2-AB and sodium cyanoborohydride were 0.7M and 1M, respectively. 100 μL of the solution was added and reacted at 60 ° C. for 2 hours.
（余剰２ＡＢの除去）
　実施例１と同様の方法により、余剰２ＡＢを除去した。 (Removal of surplus 2AB)
Excess 2AB was removed by the same method as in Example 1.
（標識化糖鎖の検出）
　実施例１と同様の方法により、標識化糖鎖を検出した。 (Detection of labeled sugar chain)
A labeled sugar chain was detected by the same method as in Example 1.
　図１に、上記ＨＰＬＣチャートから読み取ったピークのうち、中性糖鎖に由来するピークの面積、シアル酸を１つ持つ糖鎖に由来するピークの面積（１ＮＡ）、シアル酸を２つ持つ糖鎖に由来するピークの面積（２ＮＡ）、シアル酸を３つ持つ糖鎖に由来するピークの面積（３ＮＡ）、シアル酸を４つ持つ糖鎖に由来するピークの面積（４ＮＡ）について実施例と比較例のデータを比較したものを示した。反応温度が低い、実施例１や実施例２では、比較例と比べて３ＮＡや４ＮＡの面積比率が高くなり、シアル酸の脱離が抑制される事が確認された。 FIG. 1 shows a peak area derived from a neutral sugar chain, a peak area derived from a sugar chain having one sialic acid (1NA), and a sugar having two sialic acids. Examples of peak areas derived from chains (2NA), peak areas derived from sugar chains having three sialic acids (3NA), and peak areas derived from sugar chains having four sialic acids (4NA) The comparison of the data of the comparative example is shown. In Example 1 and Example 2 where the reaction temperature was low, the area ratio of 3NA and 4NA was higher than that in the comparative example, and it was confirmed that elimination of sialic acid was suppressed.
　本発明の標識された糖鎖試料の調製方法は、特に、末端にシアル酸を有する糖鎖を含む、標識化された糖鎖試料の調製において、糖鎖からのシアル酸の脱離抑制に優れている。本発明は、例えば、シアロ糖鎖の異性体まで含めた解析による癌などの疾患の診断や、シアロ糖鎖を標的とした新たなバイオマーカーの開発、医薬品のスクリーニングや品質管理に有用である。従って、本発明は、例えば、医療分野において大きく貢献しうるものである。 The method for preparing a labeled sugar chain sample of the present invention is excellent in suppressing the elimination of sialic acid from a sugar chain, particularly in the preparation of a labeled sugar chain sample containing a sugar chain having a sialic acid at the terminal. ing. The present invention is useful for, for example, diagnosis of diseases such as cancer by analysis including isomers of sialo-sugar chains, development of new biomarkers targeting sialo-sugar chains, drug screening and quality control. Therefore, the present invention can greatly contribute to the medical field, for example.

Claims (8)

  1. 　末端にシアル酸を有する糖鎖を含む、標識された糖鎖試料の調製において、糖鎖からのシアル酸の脱離を抑制する方法であって、
    （ａ）末端にシアル酸を有する糖鎖を含む試料を、糖鎖を捕捉するための官能基を有するポリマー粒子に接触させ、当該糖鎖を当該ポリマー粒子に捕捉する工程、
    （ｂ）糖鎖を捕捉したポリマー粒子を洗浄する工程、
    （ｃ）糖鎖を捕捉したポリマー粒子から糖鎖を遊離させる工程、
    （ｄ）遊離させた糖鎖を標識する工程、
    　を含み、工程（ａ）、（ｃ）および（ｄ）の少なくとも１の工程の反応を、０～６０℃で１０秒～２４時間の条件で行う方法。     In the preparation of a labeled sugar chain sample containing a sugar chain having a sialic acid at the end, a method for suppressing the elimination of sialic acid from the sugar chain,
    (A) contacting a sample containing a sugar chain having sialic acid at a terminal with a polymer particle having a functional group for capturing the sugar chain, and capturing the sugar chain on the polymer particle;
    (B) a step of washing the polymer particles capturing the sugar chain,
    (C) releasing the sugar chain from the polymer particles having captured the sugar chain;
    (D) a step of labeling the released sugar chain,
    And the reaction of at least one of steps (a), (c) and (d) is performed at 0 to 60 ° C. for 10 seconds to 24 hours.
  2. 　工程（ａ）、（ｃ）および（ｄ）の少なくとも１の工程の反応を、０～４０℃で１分～１６時間の条件で行う、請求項１に記載の方法。 The method according to claim 1, wherein the reaction in at least one of steps (a), (c) and (d) is carried out at 0 to 40 ° C for 1 minute to 16 hours.
  3. 　工程（ａ）、（ｃ）および（ｄ）の反応を、酸を含む溶媒中で行う、請求項１または２に記載の方法。 The method according to claim 1 or 2, wherein the reactions of steps (a), (c) and (d) are carried out in a solvent containing an acid.
  4. 　ポリマー粒子における糖鎖を捕捉するための官能基がヒドラジド基である、請求項１～３のいずれか一項に記載の方法。 The method according to any one of claims 1 to 3, wherein the functional group for capturing a sugar chain in the polymer particle is a hydrazide group.
  5. 　ポリマー粒子が下記（式１）で表される架橋型ポリマー構造を有するものである、請求項４に記載の方法。
    Figure JPOXMLDOC01-appb-C000001
     （Ｒ１，Ｒ２は－Ｏ－，－Ｓ－，－ＮＨ－，－ＣＯ－，－ＣＯＮＨ－で中断されてもよい炭素数１～２０の炭化水素鎖を表し、Ｒ３，Ｒ４，Ｒ５はＨ，ＣＨ３または炭素数２～５の炭化水素鎖を表す。ｍ，ｎはモノマーユニット数を表し、ｍ：ｎ＝９９：１～７０：３０である。）     The method according to claim 4, wherein the polymer particles have a crosslinked polymer structure represented by the following (formula 1).
    Figure JPOXMLDOC01-appb-C000001
     (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—, and R 3 , R 4 , R 5 represents H, CH 3 or a hydrocarbon chain having 2 to 5 carbon atoms, m and n represent the number of monomer units, and m: n = 99: 1 to 70:30.
  6. 　ポリマー粒子が下記（式２）で表される架橋型ポリマー構造を有するものである、請求項４に記載の方法。
    Figure JPOXMLDOC01-appb-C000002
     （ｍ，ｎはモノマーユニット数を表し、ｍ：ｎ＝９９：１～７０：３０である。）     The method according to claim 4, wherein the polymer particles have a crosslinked polymer structure represented by the following (formula 2).
    Figure JPOXMLDOC01-appb-C000002
     (M and n represent the number of monomer units, and m: n = 99: 1 to 70:30)
  7. 　糖鎖の標識が２－アミノベンズアミドにより行われる、請求項１～６のいずれか一項に記載の方法。 The method according to any one of claims 1 to 6, wherein the sugar chain is labeled with 2-aminobenzamide.
  8. 　試料中の糖鎖の分析方法であって、請求項１～７のいずれかに記載の方法により標識された糖鎖を調製する工程、および調製した標識された糖鎖を分析する工程を含む方法。 A method for analyzing a sugar chain in a sample, comprising the steps of preparing a labeled sugar chain by the method according to any one of claims 1 to 7, and analyzing the prepared labeled sugar chain .
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WO2008018170A1 (en) * 2006-08-09 2008-02-14 Sumitomo Bakelite Co., Ltd. Sugar chain-capturing substance and use thereof
JP2011072970A (en) * 2009-10-01 2011-04-14 Sumitomo Bakelite Co Ltd Tablet containing sugar chain trapping material and use thereof
US8198063B1 (en) * 2006-09-12 2012-06-12 Pro Zyme, Inc Rapid deglycosylation of glycoproteins
JP2012201653A (en) * 2011-03-28 2012-10-22 Sumitomo Bakelite Co Ltd Sugar chain library, method for forming the same, and sugar chain array immobilizing the same

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* Cited by examiner, † Cited by third party
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JP2006038674A (en) * 2004-07-28 2006-02-09 Kazuaki Kakehi Analysis method of glycoprotein sugar chain, and manufacturing method of unlabeled sugar chain
WO2008018170A1 (en) * 2006-08-09 2008-02-14 Sumitomo Bakelite Co., Ltd. Sugar chain-capturing substance and use thereof
US8198063B1 (en) * 2006-09-12 2012-06-12 Pro Zyme, Inc Rapid deglycosylation of glycoproteins
JP2011072970A (en) * 2009-10-01 2011-04-14 Sumitomo Bakelite Co Ltd Tablet containing sugar chain trapping material and use thereof
JP2012201653A (en) * 2011-03-28 2012-10-22 Sumitomo Bakelite Co Ltd Sugar chain library, method for forming the same, and sugar chain array immobilizing the same

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