JP6064541B2 - Sugar chain purification method - Google Patents

Description

The present invention relates to a method for efficiently purifying and labeling a sugar chain contained in a biological sample.

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

Sugar chains are glucose, galactose, mannose, fucose, xylose,
N-acetylglucosamine, N-acetylgalactosamine, sialic acid and other monosaccharides and their derivatives are generic names for molecules linked in a chain by glycosidic bonds.
For example, biopolymers having sugar chains include plant cell wall proteoglycans that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc., and cell-cell interactions and cells. Examples include glycoproteins involved in recognition. The mechanisms by which 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. Furthermore, if the relationship between such sugar chains and cell differentiation / proliferation, cell adhesion, immunity, and cell carcinogenesis is clarified, this sugar chain engineering and medicine, cell engineering, or organ engineering are closely related. We can expect new developments related to

In 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 drugs, and it has been reported that the sugar chain structure affects antibody-dependent cytotoxic activity (ADCC activity), especially for antibody drugs. Therefore, the importance of sugar chain structure analysis is increasing.

Therefore, in recent years, there has been a demand for a method for analyzing a sugar chain structure quickly, simply, and with high accuracy. High-performance liquid chromatography (HPLC), nuclear magnetic resonance, capillary electrophoresis (CE), mass spectrometry, lectin Sugar chains are analyzed by various methods such as an array method.

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. MALDI-TOF-MS is widely used because it is simple and excellent in high throughput. However, since sugar chains have poor ionization efficiency compared to other contaminants such as peptides, purification with high accuracy is required to obtain high sensitivity. However, 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.
As a technique for solving the above-described problems, for example, there is a sample preparation method realized using a specific sugar chain-capturing substance described in Patent Document 1.

International Publication No. 2008/018170

Realize a method for efficiently and reliably purifying only sugar chains from biological samples with much contamination.

The present invention is as follows.
(1) A method for purifying a sugar chain contained in a biological sample, wherein (a) a step of capturing a sugar chain in a sugar chain-capturing substance that is a substance that specifically captures a sugar chain from a biological sample; And (c) a step of releasing the sugar chain from the sugar chain-trapping substance, wherein the steps (a), (b), and (c) are performed in the same reaction vessel. In step (b), the sugar chain-trapping substance in which the sugar chain has been captured in step (a) is washed with a water-soluble cleaning solution to which a surfactant is added. A method for purifying a sugar chain, which is a linear alkyl ester salt of sulfate.
(2) The method for purifying a sugar chain according to (1), wherein the concentration of the sulfate linear alkyl ester salt is 0.05 to 5% by weight.
(3) The method for purifying a sugar chain according to (1) or (2), wherein the sulfate linear alkyl ester salt is sodium dodecyl sulfate.
(4) The sugar chain purification according to any one of (1) to (3), wherein in the step (a), the sugar chain trapping substance is a carrier having a functional group that specifically reacts with an aldehyde group of the sugar chain. Method.
(5) The method for purifying a sugar chain according to (4), wherein the functional group is a hydrazide group or an aminooxy group.
(6) The method for purifying a sugar chain according to (5), wherein the sugar chain-trapping substance has a crosslinked polymer structure represented by the following (formula 1).

(R 1, R 2 are hydrocarbon chains having 1 to 20 carbon atoms which may be interrupted by —O—, —S—, —NH—, —CO—, —CONH—, R 3, R 4, R 5 are H, CH 3, Or a hydrocarbon chain having 2 to 5 carbon atoms, m and n represent the number of monomer units.)
(7) The method for purifying a sugar chain according to (6), wherein the sugar chain-trapping substance has a crosslinked polymer structure represented by the following (formula 2).

(M and n indicate the number of monomer units.)
(8) The method for purifying a sugar chain according to any one of (1) to (7), wherein in the step (c), the sugar chain-trapping substance is subjected to an acid treatment.
(9) The method for purifying a sugar chain according to any one of (1) to (8), wherein the steps (a) and (c) have a step of evaporating the reaction solvent.

It has become possible to provide a method for efficiently and reliably purifying only sugar chains from an unpurified biological sample containing a large amount of contaminants.

The MALDI-TOF-MS measurement result of an Example and a comparative example.

The present invention is a method for labeling a sugar chain contained in a biological sample,
(A) a step of capturing a sugar chain in a sugar chain-trapping substance that is a substance that specifically captures a sugar chain from a biological sample;
(B) a step of washing the sugar chain-trapping substance that has captured the sugar chain (c) a step of releasing the sugar chain from the sugar chain-trapping substance;
In this method, the steps (a), (b), and (c) are continuously carried out in the same reaction vessel.

In the step (a), the substance that specifically captures the sugar chain preferably has a functional group that reacts with the aldehyde group of the sugar chain. The functional group is more preferably a hydrazide group or an oxylamino group.
As such a sugar chain-trapping substance, a cross-linked polymer having a structure represented by the following (formula 1) or (formula 2) is preferably used as a carrier.

(R 1 and R 2 are hydrocarbon chains having 1 to 20 carbon atoms which may be interrupted by —O—, —S—, —NH—, —CO—, —CON H—, R 3, R 4 and R 5 are H, CH 3 Or a hydrocarbon chain having 2 to 5 carbon atoms, m and n represent the number of monomer units.)
R 1 represents a hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted by —O—, —S—, —NH—, —CO— or —CONH—, and examples thereof include the following. . In the formula, a, b, and d represent an integer of 1 to 5, and c represents an integer of 1 to 10.

(M and n indicate the number of monomer units.)
Other commercially available hydrazide group-containing crosslinked particles such as Affigel Hz (BIO-RAD, 153-6047), CarboLink (TM) Coupling Gel (PIERCE, 20391), UltraLink (R) Hydrazide Gel (PIERCE, 53149) and the like may be used.

The washing step (b) is a step of washing the sugar chain-trapping substance in which the sugar chain has been captured in the step (a) and removing biological substances other than the captured sugar chain.
Examples of methods for removing biological substances other than sugar chains include washing with an aqueous solution of guanidine, which is a chaotropic reagent capable of dissociating hydrophobic bonds, or simply with pure water or a water-soluble buffer. The inventor has found that it is effective to use a surfactant, particularly a sulfuric acid linear alkyl ester sodium salt alone, not in combination with a chaotropic reagent or the like.

Examples of the linear alkyl ester salts of sulfuric acid include sodium alkylsulfate, sulfuric acid alkyl ester sodium salt, alkylsulfate sodium salt, alcohol sulfuric ester sodium salt, sodium dodecyl sulfate, sodium lauryl sulfate, and the like. From the standpoint of ease, sodium dodecyl sulfate (sodium dodecyl sulfate, abbreviated as SDS) is preferably used.

When sodium dodecyl sulfate is used as the cleaning agent, it is used as an aqueous solution. As the solution, pure water and a buffer solution can be preferably used.
As a buffer solution used here, a phosphate buffer solution, a Tris buffer solution, etc. can be used, for example. As a density | concentration of sodium dodecyl sulfate aqueous solution, Preferably it is 0.05 to 5 weight%, More preferably, it is 0.1 to 1 weight%.
As washing conditions in the washing step, the temperature is 4 to 40 ° C., and the washing time is 10 seconds to 30 minutes.

As a washing method, in the case of beads, the beads can be washed by immersing them in a washing solution and repeating the exchange of the washing solution.

Specifically, the beads are put into a centrifuge tube or a tube, a washing solution is added, and after shaking, washing is performed by repeating the operation of precipitating the beads by centrifugation and removing the supernatant.
For example, it can be washed by repeating the operation of putting beads in a centrifuge tube, adding a washing solution, and allowing the beads to settle naturally or by centrifugal separation and then removing the supernatant. The washing operation is preferably performed 3 to 6 times.
In the case of a plate, the washing liquid can be simply washed by dispensing and sucking and removing in each well. Moreover, you may use the centrifuge which can centrifuge a plate as needed.

Moreover, it is also possible to use a filter tube which is a tube-shaped container and is equipped with a filter having a pore size that allows liquid permeation and does not allow permeation of the beads to the bottom surface portion. By using beads in the filter tube, it is possible to remove the washing solution required for washing through the filter, eliminating the need for the supernatant removal step after the centrifugal operation, and improving workability. Can be achieved.

Various types of multi-well plates having 6 to 384 holes with the filter attached are commercially available, and high throughput can be achieved by using these plates. In particular, a 96-well multi-well 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.

In the case where the sugar chain capture reaction is performed in a continuous manner, this 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 sugar chain-trapping substance may be removed by filtration or centrifugation.

In the step (c), a sugar chain is released from the sugar chain-trapping substance in which the sugar chain has been trapped, that is, a reaction for cutting out the sugar chain from the sugar chain-trapping substance is performed. In this step, it is preferable to subject the sugar chain-trapping substance to an acid treatment with a mixed solvent of an acid and an organic solvent or a mixed solvent of an acid, water and an organic solvent. In the case of a mixed solvent of acid, water and organic solvent, the water content is preferably 0.1% to 90%, more preferably 0.1% to 80%, and even more preferably 0.1% to 50%. . An aqueous buffer may be contained instead of water. The concentration of the buffer is preferably 0.1 mM to 1M, 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 2-9, more preferably 2-7, and even more preferably 2-6. The acid used is preferably, for example, acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid, sulfuric acid, more preferably acetic acid, formic acid, trifluoroacetic acid, phosphoric acid, and more preferably acetic acid, trifluoroacetic acid. . The reaction temperature is preferably 4 to 90 ° C, preferably 25 to 90 ° C, more preferably 40 to 90 ° C. The reaction time is 10 minutes to 24 hours, preferably 10 minutes to 8 hours, more preferably 10 minutes to 3 hours. The reaction is preferably performed in an open system to evaporate the solvent completely from the viewpoint of efficiently carrying out the reaction for releasing the sugar chain.

Since the sugar chain release reaction can be carried out from weakly acidic to neutral, compared to conventional strong acid treatment, for example, excision reaction in the presence of strong acid such as excision by 10% trifluoroacetic acid treatment, It is possible to suppress the occurrence of sugar chain hydrolysis such as elimination of acid residues.

The next step is a step of releasing the sugar chain from the sugar chain-trapping carrier.
There are a reductive amination method and an exchange reaction method as a method for releasing a sugar chain from a sugar chain-trapping carrier.
The reductive amination method is a method of inducing a reductive amination reaction by adding an excess of a substance having an amino group, for example, and releasing a sugar chain from a sugar chain-trapping carrier.
In addition, the exchange reaction method is a method of releasing a sugar chain from a hyzolazone bond using an oxime exchange reaction using an oxime reagent.

First, the reductive amination method will be described in detail.
The reductive amination method is a reaction in which a sugar chain is released from a sugar chain-trapping substance in which the sugar chain has been trapped, that is, a sugar chain is excised from the sugar chain-trapping substance. In this step, it is preferable to subject the sugar chain-trapping substance to an acid treatment with a mixed solvent of an acid and an organic solvent or a mixed solvent of an acid, water and an organic solvent. In the case of a mixed solvent of acid, water and organic solvent, the water content is preferably 0.1% to 90%, more preferably 0.1% to 80%, and even more preferably 0.1% to 50%. . An aqueous buffer may be contained instead of water. The concentration of the buffer is preferably 0.1 mM to 1M, 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 2-9, more preferably 2-7, and even more preferably 2-6. The acid used is preferably, for example, acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid, sulfuric acid, more preferably acetic acid, formic acid, trifluoroacetic acid, phosphoric acid, and more preferably acetic acid, trifluoroacetic acid. . The reaction temperature is preferably 4 to 90 ° C, preferably 25 to 90 ° C, more preferably 40 to 90 ° C. The reaction time is 10 minutes to 24 hours, preferably 10 minutes to 8 hours, more preferably 10 minutes to 3 hours. The reaction is preferably performed in an open system to evaporate the solvent completely from the viewpoint of efficiently carrying out the reaction for releasing the sugar chain.

Since the sugar chain release reaction can be carried out from weakly acidic to neutral, compared to the conventional cleavage reaction in the presence of a strong acid such as cleavage by 10% trifluoroacetic acid treatment, It is possible to suppress the occurrence of sugar chain hydrolysis such as elimination of sialic acid residues.

It is also possible to label the free sugar chain obtained in the above step.
The labeling method is preferably a reaction in which an amino group-containing compound is labeled with any amino compound using, for example, a reductive amination reaction. In the reaction system, the pH is preferably from acidic to neutral, preferably from 2 to 9, more preferably from 2 to 8, and even more preferably from 2 to 7. The reaction temperature is preferably 4 to 90 ° C, preferably 25 to 90 ° C, more preferably 40 to 90 ° C. The concentration of the amino compound is 1 m
It is preferable that it is M-10M, and it is preferable that the density | concentration of a reducing agent is 1 mM-10M. The reaction time is 10 minutes to 24 hours, preferably 10 minutes to 8 hours, more preferably 10 minutes to 3 hours.

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-Aminopyrene-1,3,6-trisulfonate, 8-Aminophathalene-1,3,6-trisulphonate, 7-amino-1,3-naphthalenedisulfonicacid, 2-Amino9 (10H) -acidonediene, 5-Aminofluoresceine, Dynefluorescence -Aminopyridine, 7-Amino-4-methylcoumarin, 2-Aminobenzomide, 2-Aminobenzoicacid, 3-Aminobenzoicacid, 7-Amio-1-naphthol, 3- (Acetylamino) -6-aminoacrino6-caminoaminocrine6-caminoamino ethylpyridine, Ethylp-amino
benzoate, p-Aminobenzonitrile, and 7-aminophenathalene-1,3-disulphonicacid.

In particular, when the amino compound is 2-aminobenzamide, the pH is preferably 2 to 9, more preferably 2 to 8, and further preferably 2 to 7 under acidic to neutral conditions. Regarding the reaction temperature, it is 4-90 degreeC, Preferably it is 30-90 degreeC, More preferably, it is 40-80 degreeC. The concentration of the amino compound is 1 mM to 10M, preferably 10 mM to 10M, and more preferably 100 mM to 1M. The concentration of the reducing agent is 1 mM to 10M, preferably 10 mM to 10M, more preferably 100 mM to 2M. The reaction time is 10
Minutes to 24 hours, preferably 10 minutes to 8 hours, and more preferably 1 hour to 3 hours.
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 it is reactive to use sodium cyanoborohydride. It is preferable from the viewpoint.

After the previous sugar chain separation step, the resulting solution contains a labeled sugar chain, an unreacted amino compound and a reducing agent added in excess, and therefore, a step of removing these excess reagents is preferably performed. Any method of removal by silica column, removal by gel filtration, removal by ion exchange resin may be used, but the solvent used to prevent sialic acid from leaving is preferably neutral.

The other exchange reaction uses a reaction in which a sugar chain is separated from a polymer particle by a hydrazone-oxime exchange reaction and simultaneously labeled with an aminooxy compound by allowing a compound having an aminooxy group to act after the washing operation. To release sugar chains.

The compound having an aminooxy group is preferably a substance selected from the following or a salt thereof.
O-phenylhydroxylamine; O- (2,3,4,5,6-pentafluorobenzoyl) hydroxylamine; O- (4-nitrobenzoyl) hydrolamine;
2-aminoxypyridine; 2-aminooxymethylpyr
idin; 4-[(aminooxyethyl) amino] benzoic a
cid methyl ester; 4-[(aminooxyethyl) amino] benzoic acid ethyl ester; 4-[(aminooxyethyl) amino] benzoic acid n-butylester.

The compound having an aminooxy group preferably contains a moiety consisting of at least one of an arginine residue, a tryptophan residue, a phenylalanine residue, a tyrosine residue, a cysteine residue and a derivative thereof. In particular, those having a structure represented by the following (formula 3) are preferable.

  The pH of the reaction solution during the exchange reaction is preferably pH 2 to 7, more preferably pH 3 to 6, and most preferably pH 3.5 to 5.5. The reaction solution can be adjusted to the above pH by adding an acetic acid / acetonitrile solution. The temperature during the exchange reaction is preferably 50 to 95 ° C, more preferably 60 to 90 ° C, and most preferably 70 to 90 ° C. During the exchange reaction, by opening the reaction vessel and performing a heating operation, the reaction proceeds while evaporating the solvent, and finally, the exchange reaction can be efficiently performed by drying.

The collected labeled sugar chain solution can be analyzed as it is or after removing the excessively contained aminooxy compound, by an analytical means such as mass spectrometry or HPLC. The sugar chain purification method has been described above. Next, the specific method will be described with reference to examples.

Hereinafter, the present invention will be described by way of examples, but is not limited thereto.

<Example>
(Adjustment of sugar chain sample)
1 mg of bovine serum-derived IgG (SIGMA, I5506) was dissolved in 50 μL of 100 mM ammonium bicarbonate (Wako Pure Chemicals, 017-02875), and then 5 μL of 120 mM DTT (dithiothreitol, SIGMA, D9779) was added at 60 ° C. The reaction was allowed for 30 minutes. After completion of the reaction, 10 μL of 123 mM IAA (iodoacetamide, Wako Pure Chemical, 093-02152) was added and reacted at room temperature for 1 hour under light shielding. Subsequently, protease treatment was performed with 400 U of trypsin (SIGMA, T0303) to fragment the protein portion into peptides. The reaction solution was treated at 90 ° C. for 5 minutes, and then treated with 5 U of glycosidase F (Roche, 1-365-193) to release sugar chains from the peptide to obtain a pretreated biological sample.

(Sugar chain purification by sugar chain capture carrier)
The sugar chain solution 20 μL and 180 μL of 2% acetic acid / acetonitrile in a disposable column containing 5 mg of particles having a hydrazide group (BlotGlyco®), manufactured by Sumitomo Bakelite Co., Ltd., BS-45603) as a carrier for capturing sugar chains The 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. After washing with a 0.5% SDS aqueous solution, the particles were further washed with water and a triethylamine solution. Subsequently, 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 water.

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 to release sugar chains from the sugar chain-trapping carrier. 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.

(Collecting sugar chains)
50 μL of water was added to the dried particles, and only the solution was recovered. The same operation was further performed twice to obtain a total sugar chain solution of 150 μL.

(Sugar chain detection)
The resulting glycan solution was analyzed with a matrix-assisted laser ionization-time-of-flight mass spectrometer (MALDI-TOF-MS) (MALDI-TOF-MS, Bruker's 'autoflex III'). . 2,5-Dihydroxybenzoic acid was used for the matrix. The measurement results are shown in FIG.

<Comparative example>
(Adjustment of sugar chain sample)
Implemented in the same way as the example.

(Sugar chain purification by sugar chain capture carrier)
The same as in the Examples, except that the cleaning solution used for cleaning the particles was changed from a 0.5% SDS aqueous solution to a guanidine hydrochloride aqueous solution.

(Collecting sugar chains)
Implemented in the same way as the example.

(Sugar chain detection)
Implemented in the same way as the example. The measurement results are shown in the upper part of FIG.

(effect)
Contaminant peaks that could not be removed by washing with an aqueous guanidine hydrochloride solution can be eliminated by washing with SDS.

Claims (8)

A method for purifying sugar chains contained in a biological sample,
(A) a step of capturing a sugar chain in a sugar chain-trapping substance that is a substance that specifically captures a sugar chain from a biological sample;
(B) a step of washing the sugar chain-trapping substance that has captured the sugar chain (c) a step of releasing the sugar chain from the sugar chain-trapping substance;
In the step (b), the steps (a), (b), and (c) are continuously performed in the same reaction vessel. In the step (b), the sugar chain-trapping substance in which the sugar chain is captured in the step (a) Is washed with a water-soluble cleaning solution to which a surfactant is added,
The surfactant, Ri sulfate linear alkyl ester salts der, wherein a sulfate linear alkyl ester salt, sodium dodecyl sulfate, and a sugar chain purification method characterized by not in combination with chaotropic agents, and the like. The method for purifying a sugar chain according to claim 1, wherein the concentration of the sulfuric acid linear alkyl ester salt is 0.05 to 5% by weight. The method for purifying a sugar chain according to claim 1 or 2 , wherein in the step (a), the sugar chain-trapping substance is a carrier having a functional group that specifically reacts with an aldehyde group of the sugar chain. The sugar chain purification method according to claim 3 , wherein the functional group is a hydrazide group or an aminooxy group. The method for purifying a sugar chain according to claim 4, wherein the sugar chain-trapping substance has a crosslinked polymer structure represented by the following (formula 1).

(R 1, R 2 are hydrocarbon chains having 1 to 20 carbon atoms which may be interrupted by —O—, —S—, —NH—, —CO—, —CONH—, R 3, R 4, R 5 are H, CH 3, Or a hydrocarbon chain having 2 to 5 carbon atoms, m and n represent the number of monomer units.) 6. The method for purifying a sugar chain according to claim 5, wherein the sugar chain-trapping substance has a crosslinked polymer structure represented by the following (formula 2).

(M and n indicate the number of monomer units.) The method for purifying a sugar chain according to any one of claims 1 to 6, further comprising a step of performing an acid treatment on the sugar chain-trapping substance in the step (c). The method for purifying a sugar chain according to any one of claims 1 to 7, further comprising a step of evaporating the reaction solvent in the steps (a) and (c).