WO2009133696A1 - 糖鎖標識方法 - Google Patents
糖鎖標識方法 Download PDFInfo
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- WO2009133696A1 WO2009133696A1 PCT/JP2009/001927 JP2009001927W WO2009133696A1 WO 2009133696 A1 WO2009133696 A1 WO 2009133696A1 JP 2009001927 W JP2009001927 W JP 2009001927W WO 2009133696 A1 WO2009133696 A1 WO 2009133696A1
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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
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- 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
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
- Y10T436/143333—Saccharide [e.g., DNA, etc.]
Definitions
- 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.
- sugar chains are very diverse and are substances involved in various functions of naturally occurring organisms.
- Sugar chains often exist as complex carbohydrates bound to proteins, lipids, and the like in vivo, and are one of the important components in vivo. It is becoming clear that sugar chains in living organisms are deeply involved in cell-to-cell information transmission, protein functions, and coordination of interactions.
- a sugar chain is a general term for molecules in which monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid, and derivatives thereof are linked in a chain by glycosidic bonds. is there.
- 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.
- this sugar chain engineering and medicine, cell engineering, or organ engineering are closely related. We can expect new developments related to
- 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 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.
- ADCC activity antibody-dependent cytotoxic activity
- An object of the present invention is to provide a method for simply performing a step of purifying and labeling a sugar chain from a biological sample.
- the present invention is as follows. (1) 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; (C) releasing sugar chains from the sugar chain-trapping substance; (D) a step of labeling the released sugar chain, A method for labeling a sugar chain, wherein the steps (a), (c), and (d) are continuously performed in the same reaction vessel.
- 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; (D) a step of labeling the released sugar chain, A method for labeling a sugar chain, wherein the steps (a), (b), (c), and (d) are continuously carried out in the same reaction vessel.
- the sugar chain-trapping substance is a carrier having a functional group that specifically reacts with an aldehyde group of the sugar chain.
- the functional group is a hydrazide group or an aminooxy group.
- R 1 and 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 Represents H, CH 3 , or a hydrocarbon chain having 2 to 5 carbon atoms, and m and n represent the number of monomer units.
- (6) The sugar chain labeling method according to (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 compound having an amino group is 8-Aminopyrene-1,3,6-trisulfonate, 8-Aminophathalene-1,3,6-trisulphonate, 7-amino-1,3-naphthalenedisulfonic acid, 2-Amino9 (10H) -acridoneceine, 5-Aminofluorescent 2-Aminopyridine, 7-Amino-4-methylcoumarin, 2-Aminobenzomide, 2-Aminobenzoic acid, 3-Aminobenzoic acid, 7-Amino-1-naphthol, 3- (Acetylamino) -6-amino6-amino-amino6-amino-amino-amino-6-amino-amino-6-amino-amino-amino-amino-amino-amin
- purification and labeling of a target compound such as a sugar chain contained in a biological sample can be performed in the same reaction vessel, and the target compound can be labeled easily.
- labeling with an amino compound generally used for analysis such as HPLC or CE can be easily performed.
- FIG. 6 is a chart showing the results of measuring the 2-AB-labeled bovine fetuin sugar chain obtained in Experimental Example 1 by high performance liquid chromatography.
- FIG. 6 is a chart showing the results of measuring the 2-AB-labeled bovine serum-derived IgG sugar chain obtained in Experimental Example 2 by high performance liquid chromatography.
- FIG. 6 is a chart showing the results of measuring the 2-AB-labeled bovine fetuin sugar chain obtained in Experimental Example 3 by high performance liquid chromatography.
- 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; (D) a step of labeling the released sugar chain, In this method, the steps (a), (b), (c), and (d) are continuously performed in the same reaction vessel.
- 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.
- 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—, —CONH—, R 3 , R 4 , R 5 Represents H, CH 3 , or a hydrocarbon chain having 2 to 5 carbon atoms, and m and n represent the number of monomer units.
- R 1 represents a hydrocarbon chain having 1 to 20 carbon atoms that may be interrupted by —O—, —S—, —NH—, —CO—, —CONH—, and examples thereof include the following. .
- a, b, and d represent an integer of 1 to 5
- c represents an integer of 1 to 10.
- R 2 represents a hydrocarbon chain having 1 to 20 carbon atoms that may be interrupted by —O—, —S—, —NH—, —CO—, —CONH—, and examples thereof include the following. .
- e and f represent an integer of 1 to 5
- g represents an integer of 1 to 10.
- R 3 , R 4 and R 5 may be the same or different and each represents H, CH 3 , or a hydrocarbon chain having 2 to 5 carbon atoms, and examples thereof include the following.
- h represents an integer of 1 to 4.
- 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), etc. Also good.
- a crosslinked polymer having a structure represented by the following (Formula 5) or (Formula 6) can be 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—, —CONH—, R 3 , R 4 , R 5 Represents H, CH 3 , or a hydrocarbon chain having 2 to 5 carbon atoms, and m and n represent the number of monomer units.
- Specific examples of R 1 to R 5 are the same as those described in the description of (Formula 1).
- polymer particles represented by (Formula 5) polymer particles having a structure represented by the following (Formula 6) can also be suitably used.
- substances containing hydrazide groups can be preferably used.
- the pH of the reaction system in which the sugar chain and the sugar chain-trapping substance are reacted is preferably acidic, preferably 2 to 9, more preferably 2 to 7, and further preferably 2 to 6.
- 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 2 hours.
- the reaction is preferably carried out in an open system to evaporate the solvent completely from the viewpoint of efficiently performing the sugar chain capture reaction.
- the sugar chain-trapping substance in which the sugar chain has been captured in the step (a) is washed to remove sugar chains not captured by the sugar chain-trapping substance and other biological samples.
- a protein denaturant solution such as an aqueous solution of guanidine or a surfactant, alcohols such as methanol and ethanol, water, an aqueous buffer, and the like are used.
- the pH of the aqueous solution is preferably near neutral, and the pH is 4 to 10, more preferably 6 to 8.
- the washing step (b) may not be performed depending on the state of the initial biological sample, for example, the degree of mixing of substances other than sugar chains.
- 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.
- a mixed solvent of an acid, water and an organic solvent the content of water is preferably 0.1% to 90%, more preferably 0.1% to 80%, and further 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 to 9, more preferably 2 to 7, and further preferably 2 to 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.
- sugar chain cleaving reaction can be carried out from weakly acidic to neutral, compared to the conventional cleaving reaction in the presence of a strong acid such as cleaving by 10% trifluoroacetic acid treatment, It is possible to suppress the occurrence of sugar chain hydrolysis such as elimination of acid residues.
- step (d) the free sugar chain obtained in step (c) is labeled.
- 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.
- 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 preferably 1 mM to 10M, and the concentration of the reducing agent is preferably 1 mM to 10M.
- the reaction time is 10 minutes to 24 hours, preferably 10 minutes to 8 hours, more preferably 10 minutes to 3 hours.
- the compound having an amino group preferably has ultraviolet-visible absorption characteristics or fluorescence characteristics, and is preferably at least one selected from the following group, for example.
- the pH is preferably 2 to 9, more preferably 2 to 8, and further preferably 2 to 7 under acidic to neutral conditions.
- the reaction temperature is 4 to 90 ° C, preferably 30 to 90 ° C, more preferably 40 to 80 ° C.
- the concentration of the amino compound is 1 mM to 10M, preferably 10 mM to 10M, 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, more preferably 1 hour to 3 hours.
- sodium cyanoborohydride 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.
- step (d) since the resulting solution contains a labeled sugar chain, 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 to prevent sialic acid from leaving is preferably neutral.
- the present invention provides, from another aspect, a sugar chain detection method including a step of detecting a sugar chain labeled by the sugar chain labeling method. Furthermore, from another aspect, there is provided a sugar chain sorting method including a step of sorting sugar chains labeled by the sugar chain labeling method.
- Example 1 Preparation of biological samples 1 mg of bovine serum-derived fetuin was dissolved in 50 ⁇ L of 100 mM ammonium bicarbonate, 5 ⁇ L of 120 mM DTT (dithiothreitol) was added, and the mixture was reacted at 60 ° C. for 30 minutes. After completion of the reaction, 10 ⁇ L of 123 mM IAA (iodoacetamide) was added and allowed to react at room temperature for 1 hour in the dark. Subsequently, protease treatment was performed with 400 U of trypsin, and the protein portion was fragmented into peptides. The reaction solution was treated at 90 ° C. for 5 minutes, and then treated with 5 U glycosidase F to release sugar chains from the peptide, thereby obtaining a pretreated biological sample.
- IAA iodoacetamide
- the beads were washed with a guanidine solution, water, methanol, and a triethylamine solution, 10% acetic anhydride / methanol was added, and the mixture was reacted at room temperature for 30 minutes to cap unreacted hydrazide groups. After capping, the beads were washed with methanol, aqueous hydrochloric acid and water.
- Example reagent removal step 50 ⁇ L of the reaction solution was recovered, diluted 10-fold with acetonitrile, and then added to a column packed with silica gel (IAtrobeads, 6RS-8060, manufactured by Mitsubishi Chemical Yatron) to adsorb the labeled sugar chain to the silica gel. After washing the column with acetonitrile / acetonitrile / water mixed solution (95: 5), the labeled sugar chain was recovered with 50 ⁇ L of ultrapure water.
- silica gel IAtrobeads, 6RS-8060, manufactured by Mitsubishi Chemical Yatron
- the obtained labeled sugar chain was measured by HPLC. 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. FIG. 1 shows the measurement results. A sugar chain labeled with 2AB was detected.
- 1 indicates a peak derived from a 1 sialic acid-containing sugar chain
- 2 indicates a peak derived from a 2 sialic acid-containing sugar chain
- 3 indicates a peak derived from a 3 sialic acid-containing sugar chain
- 4 indicates 4 sialic acid.
- a peak derived from a contained sugar chain is shown.
- Example 2 The same procedure as in Experimental Example 1 was performed except that bovine serum-derived IgG was used instead of bovine serum-derived fetuin.
- FIG. 2 shows the HPLC measurement results. A sugar chain labeled with 2AB was detected.
- 5 indicates a neutral sugar-derived peak
- 6 indicates an acidic sugar chain-derived peak
- Example 3 The experiment was performed in the same manner as in Experimental Example 1 except that Affigel Hz (BIO-RAD, 153-6047) was used as a bead having a hydrazide group as a carrier for capturing sugar chains. 50 ⁇ L of Affigel Hz was used.
- FIG. 3 shows the HPLC measurement results. In the figure, the solid line indicates the case where Affigel Hz is used, and the broken line indicates the case where BlotGlyco (R) of Experimental Example 1 is used. Even when Affigel Hz was used, the strength was weaker than when BlotGlyco (R) was used, but a sugar chain labeled with 2AB was detected.
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Abstract
Description
(1)生体試料中に含有する糖鎖を標識化する方法であって、
(a)生体試料から糖鎖を特異的に捕捉する物質である糖鎖捕捉物質に糖鎖を捕捉する工程、
(c)糖鎖捕捉物質から糖鎖を遊離させる工程、
(d)遊離された糖鎖を標識化する工程、
を含み、(a)、(c)、(d)の工程を同一の反応容器内で連続して行うことを特徴とする糖鎖標識方法。
(2)生体試料中に含有する糖鎖を標識化する方法であって、
(a)生体試料から糖鎖を特異的に捕捉する物質である糖鎖捕捉物質に糖鎖を捕捉する工程、
(b)糖鎖を捕捉した糖鎖捕捉物質を洗浄する工程
(c)糖鎖捕捉物質から糖鎖を遊離させる工程、
(d)遊離された糖鎖を標識化する工程、
を含み、(a)、(b)、(c)、(d)の工程を同一の反応容器内で連続して行うことを特徴とする糖鎖標識方法。
(3)(a)の工程において、糖鎖捕捉物質が糖鎖のアルデヒド基と特異的に反応する官能基を有する担体である(1)又は(2)記載の糖鎖標識方法。
(4)前記官能基が、ヒドラジド基又はアミノオキシ基である(3)記載の糖鎖標識方法。
(5)前記糖鎖捕捉物質が下記の(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)記載の糖鎖標識方法。
(6)前記糖鎖捕捉物質が下記の(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(5)記載の糖鎖標識方法。
(7)(c)の工程において、糖鎖捕捉物質に酸処理を行なう工程を有する(1)~(6)いずれか記載の糖鎖標識方法。
(8) (a)および(c)の工程において、反応溶媒を蒸発させる工程を有する(1)~(7)いずれか記載の糖鎖標識方法。
(9)(d)の工程において、糖鎖の標識化をアミノ基を有する化合物により行う(1)~(8)いずれか記載の糖鎖標識方法。
(10)前記アミノ基を有する化合物による糖鎖の標識化が還元的アミノ化反応によりなされるものである(9)記載の糖鎖標識方法。
(11)前記アミノ基を有する化合物が紫外可視吸収特性又は蛍光特性を有するものである(9)又は(10)記載の糖鎖標識方法。
(12)前記アミノ基を有する化合物が、
8-Aminopyrene-1,3,6-trisulfonate,8-Aminonaphthalene-1,3,6-trisulphonate,7-amino-1,3-naphtalenedisulfonic acid,2-Amino9(10H)-acridone,5-Aminofluorescein,Dansylethylenediamine,2-Aminopyridine,7-Amino-4-methylcoumarine,2-Aminobenzamide,2-Aminobenzoic acid,3-Aminobenzoic acid,7-Amino-1-naphthol,3-(Acetylamino)-6-aminoacridine,2-Amino-6-cyanoethylpyridine,Ethyl p-aminobenzoate,p-Aminobenzonitrile,及び7-aminonaphthalene-1,3-disulfonic acid、
から選ばれる少なくとも1つである(9)~(11)いずれか記載の糖鎖標識方法。
(13)(1)~(12)いずれか記載の糖鎖標識方法によって標識化した糖鎖を検出する工程を含む、糖鎖の検出方法。
(14)(1)~(12)いずれか記載の糖鎖標識方法によって標識化した糖鎖を分取する工程を含む、糖鎖の分取方法。
(a)生体試料から糖鎖を特異的に捕捉する物質である糖鎖捕捉物質に糖鎖を捕捉する工程、
(b)糖鎖を捕捉した糖鎖捕捉物質を洗浄する工程
(c)糖鎖捕捉物質から糖鎖を遊離させる工程、
(d)遊離された糖鎖を標識化する工程、
を含み、(a)、(b)、(c)、(d)の工程を同一の反応容器内で連続して行う糖鎖標識方法である。
本実施例では、モデルケースとして糖タンパク質であるウシ血清由来フェチュインおよびウシ血清由来IgGの糖鎖を分析試料として調製する方法およびその測定方法を示す。
(生体試料の予備処理)
ウシ血清由来フェチュイン1 mgを100mM重炭酸アンモニウム50μLに溶解させた後、120mM DTT(ジチオスレイトール)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)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を得た。
糖鎖捕捉用の担体であるヒドラジド基を有するビーズ5mg(BlotGlyco(R))、住友ベークライト株式会社製、BS-45601S、式2の構造を有し、モノマー仕込み比がm:n=20:1のポリマー)が入ったディスポカラムに予備処理済の生体試料の懸濁物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ビーズが乾固した状態であることを目視で確認した。続いて、グアニジン溶液、水、メタノール、トリエチルアミン溶液にてビーズを洗浄後、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、水にてビーズを洗浄した。
ビーズ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、60℃で2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ビーズが乾固した状態であることを目視で確認した。
ビーズの入ったディスポカラムに、2-aminobenzamide(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0.35M、1Mになるように30%酢酸/DMSO混合溶媒に溶解させて調整した溶液50μLを添加し、60℃で2時間反応させた。
上記、糖鎖捕捉工程(a)、洗浄工程(b)、糖鎖遊離工程(c)、及び標識化工程(d)は同一の容器であるディスポカラム内で行なった。
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、シリカゲル (Iatrobeads、6RS-8060、三菱化学ヤトロン製)を詰めたカラム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、アセトニトリル/水混合溶液(95:5)にてカラムを洗浄後、超純水50μLにて標識糖鎖を回収した。
得られた標識糖鎖をHPLC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。図1に測定結果を示す。2ABにより標識化された糖鎖が検出された。
ウシ血清由来フェチュインの代わりにウシ血清由来IgGを用いた以外は、実験例1と同様に行なった。図2にHPLCの測定結果を示す。2ABにより標識化された糖鎖が検出された。
糖鎖捕捉用の担体であるヒドラジド基を有するビーズとしてアフィゲルHz(BIO-RAD、153-6047)を用いた以外は、実験例1と同様に行った。アフィゲルHzは50μL使用した。図3にHPLCの測定結果を示す。図で実線は、アフィゲルHz使用の場合、破線は、実験例1のBlotGlyco(R)使用の場合を示す。アフィゲルHz使用の場合も、BlotGlyco(R)を用いた場合と比較すると強度が弱くなるが、2ABにより標識化された糖鎖が検出された。
Claims (14)
- 生体試料中に含有する糖鎖を標識化する方法であって、
(a)生体試料から糖鎖を特異的に捕捉する物質である糖鎖捕捉物質に糖鎖を捕捉する工程、
(c)糖鎖捕捉物質から糖鎖を遊離させる工程、
(d)遊離された糖鎖を標識化する工程、
を含み、(a)、(c)、(d)の工程を同一の反応容器内で連続して行うことを特徴とする糖鎖標識方法。 - 生体試料中に含有する糖鎖を標識化する方法であって、
(a)生体試料から糖鎖を特異的に捕捉する物質である糖鎖捕捉物質に糖鎖を捕捉する工程、
(b)糖鎖を捕捉した糖鎖捕捉物質を洗浄する工程
(c)糖鎖捕捉物質から糖鎖を遊離させる工程、
(d)遊離された糖鎖を標識化する工程、
を含み、(a)、(b)、(c)、(d)の工程を同一の反応容器内で連続して行うことを特徴とする糖鎖標識方法。 - (a)の工程において、糖鎖捕捉物質が糖鎖のアルデヒド基と特異的に反応する官能基を有する担体である請求項1又は2記載の糖鎖標識方法。
- 前記官能基が、ヒドラジド基又はアミノオキシ基である請求項3記載の糖鎖標識方法。
- (c)の工程において、糖鎖捕捉物質に酸処理を行なう工程を有する請求項1~6いずれか記載の糖鎖標識方法。
- (a)および(c)の工程において、反応溶媒を蒸発させる工程を有する請求項1~7いずれか記載の糖鎖標識方法。
- (d)の工程において、糖鎖の標識化をアミノ基を有する化合物により行う請求項1~8いずれか記載の糖鎖標識方法。
- 前記アミノ基を有する化合物による糖鎖の標識化が還元的アミノ化反応によりなされるものである請求項9記載の糖鎖標識方法。
- 前記アミノ基を有する化合物が紫外可視吸収特性又は蛍光特性を有するものである請求項9又は10記載の糖鎖標識方法。
- 前記アミノ基を有する化合物が、8-Aminopyrene-1,3,6-trisulfonate,8-Aminonaphthalene-1,3,6-trisulphonate,7-amino-1,3-naphtalenedisulfonic acid,2-Amino9(10H)-acridone,5-Aminofluorescein,Dansylethylenediamine,2-Aminopyridine,7-Amino-4-methylcoumarine,2-Aminobenzamide,2-Aminobenzoic acid,3-Aminobenzoic acid,7-Amino-1-naphthol,3-(Acetylamino)-6-aminoacridine,2-Amino-6-cyanoethylpyridine,Ethyl p-aminobenzoate,p-Aminobenzonitrile,及び7-aminonaphthalene-1,3-disulfonic acidから選ばれる少なくとも1つである請求項9~11いずれか記載の糖鎖標識方法。
- 請求項1~12いずれか記載の糖鎖標識方法によって標識化した糖鎖を検出する工程を含む、糖鎖の検出方法。
- 請求項1~12いずれか記載の糖鎖標識方法によって標識化した糖鎖を分取する工程を含む、糖鎖の分取方法。
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CN2009801154305A CN102144164A (zh) | 2008-04-30 | 2009-04-28 | 标记糖链的方法 |
US12/989,717 US8828732B2 (en) | 2008-04-30 | 2009-04-28 | Method of labeling sugar chain |
CA2721998A CA2721998A1 (en) | 2008-04-30 | 2009-04-28 | Method of labeling sugar chain |
AU2009241146A AU2009241146B2 (en) | 2008-04-30 | 2009-04-28 | Method of labelling sugar chain |
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JP2011195744A (ja) * | 2010-03-23 | 2011-10-06 | Sumitomo Bakelite Co Ltd | 酸性糖鎖捕捉用基材 |
JP2012158729A (ja) * | 2011-02-03 | 2012-08-23 | Sumitomo Bakelite Co Ltd | 酸性糖鎖試料調製方法 |
JP2012211817A (ja) * | 2011-03-31 | 2012-11-01 | Sumitomo Bakelite Co Ltd | 糖鎖測定方法および糖鎖固定化基材 |
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US20130310552A1 (en) * | 2011-03-11 | 2013-11-21 | Sumitomo Bakelite Co., Ltd. | Sugar chain fluorescent labeling method |
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US11293926B2 (en) | 2017-04-04 | 2022-04-05 | Sumitomo Bakelite Co., Ltd. | Method for preparing sugar chain |
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US20110046364A1 (en) | 2011-02-24 |
KR20110011633A (ko) | 2011-02-08 |
CA2721998A1 (en) | 2009-11-05 |
EP2282209A1 (en) | 2011-02-09 |
AU2009241146A1 (en) | 2009-11-05 |
CN102144164A (zh) | 2011-08-03 |
US8828732B2 (en) | 2014-09-09 |
AU2009241146B2 (en) | 2014-01-16 |
DK2282209T3 (da) | 2012-11-26 |
JP5500067B2 (ja) | 2014-05-21 |
EP2282209A4 (en) | 2011-06-15 |
EP2282209B1 (en) | 2012-10-03 |
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KR101578960B1 (ko) | 2015-12-18 |
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