JP2020173228A5 - - Google Patents
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- JP2020173228A5 JP2020173228A5 JP2019076685A JP2019076685A JP2020173228A5 JP 2020173228 A5 JP2020173228 A5 JP 2020173228A5 JP 2019076685 A JP2019076685 A JP 2019076685A JP 2019076685 A JP2019076685 A JP 2019076685A JP 2020173228 A5 JP2020173228 A5 JP 2020173228A5
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- liquid chromatography
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- 150000002016 disaccharides Chemical class 0.000 claims description 26
- 108090000790 Enzymes Proteins 0.000 claims description 20
- 102000004190 Enzymes Human genes 0.000 claims description 20
- 229920002683 Glycosaminoglycan Polymers 0.000 claims description 18
- 239000012472 biological sample Substances 0.000 claims description 15
- 238000004458 analytical method Methods 0.000 claims description 11
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 8
- 238000004811 liquid chromatography Methods 0.000 claims description 6
- 239000000969 carrier Substances 0.000 claims description 4
- 125000000524 functional group Chemical group 0.000 claims description 4
- 239000007790 solid phase Substances 0.000 claims description 4
- 125000005073 adamantyl group Chemical group C12(CC3CC(CC(C1)C3)C2)* 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims 2
- 125000003368 amide group Chemical group 0.000 claims 1
- 239000000047 product Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 210000004369 Blood Anatomy 0.000 description 5
- 210000002966 Serum Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 108010048429 chondroitinase B Proteins 0.000 description 4
- 108010015332 keratanase II Proteins 0.000 description 4
- 238000000034 method Methods 0.000 description 3
- WKPUACLQLIIVJJ-RHKLHVFKSA-M (2S,3R,4R,5S,6R)-4-hydroxy-3-methoxy-6-[(2S,3R,4S,5S,6R)-6-methoxy-4-oxido-5-(sulfooxyamino)-2-(sulfooxymethyl)oxan-3-yl]oxy-5-sulfooxyoxane-2-carboxylate Chemical compound [O-][C@H]1[C@H](NOS(O)(=O)=O)[C@H](OC)O[C@@H](COS(O)(=O)=O)[C@@H]1O[C@H]1[C@@H](OS(O)(=O)=O)[C@H](O)[C@@H](OC)[C@@H](C([O-])=O)O1 WKPUACLQLIIVJJ-RHKLHVFKSA-M 0.000 description 2
- 229920001287 Chondroitin sulfate Polymers 0.000 description 2
- KXKPYJOVDUMHGS-OSRGNVMNSA-N Chondroitin sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](OS(O)(=O)=O)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](C(O)=O)O1 KXKPYJOVDUMHGS-OSRGNVMNSA-N 0.000 description 2
- 229920000045 Dermatan sulfate Polymers 0.000 description 2
- AVJBPWGFOQAPRH-FWMKGIEWSA-L Dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 description 2
- 229920002971 Heparan sulfate Polymers 0.000 description 2
- KXCLCNHUUKTANI-RBIYJLQWSA-N Keratan Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@H](COS(O)(=O)=O)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H]([C@@H](COS(O)(=O)=O)O[C@@H](O)[C@@H]3O)O)[C@H](NC(C)=O)[C@H]2O)COS(O)(=O)=O)O[C@H](COS(O)(=O)=O)[C@@H]1O KXCLCNHUUKTANI-RBIYJLQWSA-N 0.000 description 2
- 229920000288 Keratan sulfate Polymers 0.000 description 2
- 206010028093 Mucopolysaccharidosis Diseases 0.000 description 2
- 208000002678 Mucopolysaccharidosis Diseases 0.000 description 2
- 229940059329 chondroitin sulfate Drugs 0.000 description 2
- 229940051593 dermatan sulfate Drugs 0.000 description 2
- -1 heparitinase Proteins 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 102100003684 HPSE Human genes 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 108010037536 heparanase Proteins 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
Description
表１中、「ＣＳ」はコンドロイチン硫酸、「ＤＳ」はデルマタン硫酸、「ＨＳ」はヘパラン硫酸、「ＫＳ」はケラタン硫酸の略号である。
また、ΔＤｉ−４Ｓ、Ｄｉ−０Ｓはそれぞれコンドロイチン硫酸、デルマタン硫酸がコンドロイチナーゼＢで分解（消化）されてできる二糖であり、ΔＤｉＨＳ−０Ｓ、ΔＤｉＨＳ−ＮＳ、ΔＤｉＨＳ−６Ｓはヘパラン硫酸がヘパリチナーゼにより分解されてできる二糖であり、Ｇａｌ−ＧｌｃＮａｃ（６Ｓ）、Ｇａｌ（６Ｓ）−ＧｌｃＮａｃ（６Ｓ）はケラタン硫酸がケラタナーゼＩＩで分解されてできる二糖である。
In Table 1, "CS" is an abbreviation for chondroitin sulfate, "DS" is an abbreviation for dermatan sulfate, "HS" is an abbreviation for heparan sulfate, and "KS" is an abbreviation for keratan sulfate.
In addition, ΔDi-4S and Di-0S are disaccharides formed by decomposing (digesting) chondroitin sulfate and dermatan sulfate with chondroitinase B, respectively, and ΔDiHS-0S, ΔDiHS-NS, and ΔDiHS-6S are heparan sulfate heparanase. Gal-GlcNac (6S) and Gal (6S) -GlcNac (6S) are disaccharides formed by decomposing keratan sulfate with keratanase II.
［実施例３］
生体試料として、ＧＡＧ特異的酵素であるコンドロイチナーゼＢ、ヘパリチナーゼ、ケラタナーゼＩＩの混合液を添加した血液（全血）をろ紙に含ませて乾燥させた乾燥ろ紙血（乾燥血液ろ紙（Dried Blood Spots（ＤＢＳ）））試料を用い、該ＤＢＳ試料から抽出した二糖を含む回収物をＬＣ／ＭＳ／ＭＳに導入してＧＡＧの定量分析を行った。ＬＣ分析条件、ＭＳ分析条件、ＭＲＭの測定条件は実施例２と同じ条件に設定した。この実施例では、１０個のＤＢＳ試料（ＤＢＳ１〜１０）を用意した。ＤＢＳ試料から回収物を得、ＬＣ／ＭＳ／ＭＳに導入するまでの手順は以下の通りである。
[Example 3]
As a biological sample, dried blood (Dried Blood Spots) obtained by impregnating filter paper with blood (whole blood) supplemented with a mixture of GAG-specific enzymes chondroitinase B, heparitinase, and keratanase II and drying it. (DBS)) ) Using the sample, the recovered product containing disaccharide extracted from the DBS sample was introduced into LC / MS / MS and quantitative analysis of GAG was performed. The LC analysis conditions, the MS analysis conditions, and the MRM measurement conditions were set to the same conditions as in Example 2. In this example, 10 DBS samples (DBS1-10) were prepared. The procedure for obtaining the recovered product from the DBS sample and introducing it into LC / MS / MS is as follows.
＜二糖の回収及びＬＣ／ＭＳ／ＭＳへの導入＞
１．各ＤＢＳ試料の血液が含浸している部分をＤＢＳパンチャー（PerkinElmer（登録商標）、株式会社パーキンエルマージャパン）で切り出してディスク（直径３．３ｍｍ）を得た。
２．ディスクを、100μLの0.1％ＢＳＡを含有する、９６ウェルフィルタープレート（Omega 10K、日本ポール株式会社）の各ウェルに入れた。
３．各ウェルにＧＡＧ特異的酵素（コンドロイチナーゼＢ、ヘパリチナーゼおよびケラタナーゼII）を添加し、３７℃で一晩インキュベートした。
４．得られたＧＡＧの消化物をフィルタープレートでろ過し、得られたろ過液を2500×gで１５分間、遠心分離した後、回収物をＬＣ ／ ＭＳ ／ ＭＳに導入した。
<Recovery of disaccharide and introduction to LC / MS / MS>
1. 1. The blood-impregnated portion of each DBS sample was cut out with a DBS puncher (PerkinElmer (registered trademark), PerkinElmer Japan Co., Ltd.) to obtain a disc (diameter 3.3 mm).
2. The disc was placed in each well of a 96-well filter plate (Omega 10K, Nippon Pole KK) containing 100 μL of 0.1% BSA.
3. 3. GAG-specific enzymes (chondroitinase B, heparitinase and keratanase II) were added to each well and incubated overnight at 37 ° C.
4. The obtained digested product of GAG was filtered through a filter plate, and the obtained filtrate was centrifuged at 2500 × g for 15 minutes, and then the recovered product was introduced into LC / MS / MS.
［実施例４］
生体試料として、ＧＡＧ特異的酵素であるコンドロイチナーゼＢ、ヘパリチナーゼ、ケラタナーゼＩＩの混合液を添加した血液（血清）試料を用い、各血清試料から抽出した二糖を含む回収物をＬＣ／ＭＳ／ＭＳに導入してＧＡＧの定量分析を行った。ＬＣ分析条件、ＭＳ分析条件、ＭＲＭの測定条件は実施例２と同じ条件に設定した。この実施例では、１０個の血清試料（Ｓｅｒｕｍ２、４、６、８、１０、１２、１４、１６、１８、２０）を用意した。血清試料から回収物を得、ＬＣ／ＭＳ／ＭＳに導入するまでの手順は、以下の通りである。
[Example 4]
As a biological sample, a blood (serum) sample to which a mixed solution of GAG-specific enzymes chondroitinase B, heparitinase, and keratanase II was added was used, and a recovered product containing disaccharide extracted from each serum sample was LC / MS /. It was introduced into MS and quantitative analysis of GAG was performed. The LC analysis conditions, the MS analysis conditions, and the MRM measurement conditions were set to the same conditions as in Example 2. In this example, 10 serum samples (Serum 2 , 4, 6, 8, 10, 12, 14, 16, 18, 20) were prepared. The procedure for obtaining the recovered product from the serum sample and introducing it into LC / MS / MS is as follows.
＜分析結果＞
ＬＣ／ＭＳ／ＭＳにより分析した結果、得られたＭＲＭクロマトグラムを図４に示す。図４には、それぞれ、ΔＤｉ−４Ｓ、ΔＤｉＨＳ−０Ｓ、ΔＤｉＨｓ−ＮＳ、Ｇａｌ−ＧｌｃＮＡｃ（６Ｓ）、Ｇａｌ（６Ｓ）−ＧｌｃＮＡｃ（６Ｓ）、コンドロシンのそれぞれのプリカーサーイオンのm/zとプロダクトイオンのm/zの組合せをＭＲＭトランジションとして設定し、ＬＣ／ＭＳ／ＭＳ分析した結果、得られたＭＲＭクロマトグラムが示されている。
<Analysis result>
The MRM chromatogram obtained as a result of analysis by LC / MS / MS is shown in FIG. In FIG. 4 , the m / z and product ions of the precursor ions of ΔDi-4S, ΔDiHS-0S, ΔDiHs-NS, Gal-GlcNAc (6S), Gal (6S) -GlcNAc (6S), and chondrosin, respectively, are shown. As a result of setting the combination of m / z as the MRM transition and performing LC / MS / MS analysis, the obtained MRM chromatogram is shown.
また、本発明の第２態様は、
生体試料に複数種類のグリコサミノグリカン特異的酵素を添加して、該生体試料に含まれるグリコサミノグリカン由来の複数種類の二糖を生成する第１工程と、
液体クロマトグラフィ質量分析法により、前記複数種類の二糖を分離して分析する第２工程と
を備え、
前記液体クロマトグラフィ質量分析法における液体クロマトグラフィで用いられるカラムが、官能基としてのアダマンチル基が結合された固相担体が充填されたカラムである、分析方法である。
The second aspect of the present invention is
The first step of adding a plurality of types of glycosaminoglycan-specific enzymes to a biological sample to produce a plurality of types of disaccharides derived from glycosaminoglycan contained in the biological sample.
A second step of separating and analyzing the plurality of types of disaccharides by a liquid chromatography mass analysis method is provided.
The column used in liquid chromatography in the liquid chromatography mass spectrometry is an analysis method in which a column is packed with a solid phase carrier to which an adamantyl group as a functional group is bonded.
本発明の第４態様は、第１態様のグリコサミノグリカンの分析方法において、前記第１酵素によって生成される二糖にΔＤｉ−４Ｓが含まれており、前記第２酵素によって生成される二糖にΔＤｉＨＳ−６Ｓが含まれているか、前記第１酵素によって生成される二糖にΔＤｉ−０Ｓが含まれており、前記第２酵素によって生成される二糖にΔＤｉＨＳ−０Ｓが含まれているものである。
In the fourth aspect of the present invention, in the method for analyzing glycosaminoglycan of the first aspect, ΔDi-4S is contained in the disaccharide produced by the first enzyme, and the disaccharide produced by the second enzyme is two. The sugar contains ΔDiHS-6S, or the disaccharide produced by the first enzyme contains ΔDi-0S, and the disaccharide produced by the second enzyme contains ΔDiHS-0S. It is a thing.
また、本発明の第２態様の別の側面は、
被検者から生体試料を取得する工程と、
前記生体試料に複数種類のグリコサミノグリカン特異的酵素を添加して、該生体試料に含まれるグリコサミノグリカン由来の複数種類の二糖を生成する工程と、
液体クロマトグラフィ質量分析法により、前記複数種類の二糖を分離して分析する工程と、
前記分析結果に基づき前記被検者におけるムコ多糖症の有無を検査する工程とを備え、
前記液体クロマトグラフィ質量分析法における液体クロマトグラフィで用いられるカラムが、官能基としてのアダマンチル基が結合された固相担体が充填されたカラムである、ムコ多糖症の検査方法である。
In addition, another aspect of the second aspect of the present invention is
The process of obtaining a biological sample from a subject and
A step of adding a plurality of types of glycosaminoglycan-specific enzymes to the biological sample to produce a plurality of types of disaccharides derived from glycosaminoglycan contained in the biological sample.
A step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry, and
A step of inspecting the subject for the presence or absence of mucopolysaccharidosis based on the analysis result is provided.
The column used in the liquid chromatography in the liquid chromatography mass spectrometry is a column packed with a solid phase carrier to which an adamantyl group as a functional group is bound, which is a method for testing mucopolysaccharidosis.
Claims (5)
液体クロマトグラフィ質量分析法により、前記複数種類の二糖を分離して分析する第２工程と
を備え、
前記液体クロマトグラフィ質量分析法における液体クロマトグラフィで用いられるカラムが、官能基としてのアミド基が結合された固相担体が充填されたカラムである、グリコサミノグリカンの分析方法。 The first step of adding a plurality of types of glycosaminoglycan-specific enzymes to a biological sample to produce a plurality of types of disaccharides derived from glycosaminoglycan contained in the biological sample.
A second step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry is provided.
A method for analyzing glycosaminoglycan, wherein the column used in liquid chromatography in the liquid chromatography mass spectrometry is a column packed with a solid phase carrier to which an amide group as a functional group is bonded.
前記第２工程において、前記第１酵素が添加された生体試料と、前記第２酵素が添加された生体試料を、それぞれ液体クロマトグラフィ質量分析する、請求項１に記載のグリコサミノグリカンの分析方法。 In the first step, among the plurality of types of glycosaminoglycan-specific enzymes, at least one of the plurality of types of glycosaminoglycan-derived disaccharides contained in the biological sample is produced. The step of adding an enzyme to the biological sample and one or more kinds of glycosaminoglycan-derived disaccharides other than the disaccharide produced by the first enzyme among the plurality of kinds of glycosaminoglycan-specific enzymes. It is equipped with a step of adding a second enzyme that produces
The method for analyzing glycosaminoglycan according to claim 1, wherein in the second step, the biological sample to which the first enzyme is added and the biological sample to which the second enzyme is added are each subjected to liquid chromatography mass analysis. ..
液体クロマトグラフィ質量分析法により、前記複数種類の二糖を分離して分析する第２工程と
を備え、
前記液体クロマトグラフィ質量分析法における液体クロマトグラフィで用いられるカラムが、官能基としてのアダマンチル基が結合された固相担体が充填されたカラムである、グリコサミノグリカンの分析方法。 The first step of adding a plurality of types of glycosaminoglycan-specific enzymes to a biological sample to produce a plurality of types of disaccharides derived from glycosaminoglycan contained in the biological sample.
A second step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry is provided.
A method for analyzing glycosaminoglycan, wherein the column used in liquid chromatography in the liquid chromatography mass spectrometry is a column packed with a solid phase carrier to which an adamantyl group as a functional group is bonded.
In the second step, when the obtained MRM chromatogram contains three or more peaks as a result of liquid chromatography mass spectrometry with one MRM transition, the peak having the shortest elution time or the peak having the longest elution time. The method for analyzing glycosaminoglycan according to claim 4, wherein the disaccharide is quantified using the above.
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| EP1746108A1 (en) | 2004-04-27 | 2007-01-24 | Maruha Corporation | Fish-origin chondroitin sulfate/dermatan sulfate hybrid chain |
| US20070161074A1 (en) | 2005-12-27 | 2007-07-12 | Daiichi Pharmaceutical Co., Ltd. | Diagnostic method of mucopolysaccharidoses |
| JP5927760B2 (en) | 2011-02-03 | 2016-06-01 | 住友ベークライト株式会社 | Acid glycan sample preparation method |
| US10317380B2 (en) | 2014-07-03 | 2019-06-11 | Shimadzu Corporation | Chromatograph mass spectrometer and program |
| JP6298206B1 (en) | 2017-08-08 | 2018-03-20 | 田中 正彦 | Medicine containing pyrazolone derivative |
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