用于气相色谱/质谱分析的含半乳糖CS四糖衍生的部分甲基化糖醇乙酸酯的制备

Kyohei Higashi; Toshihiko Toida

doi:10.21769/BioProtoc.2600

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Preparation of the Partially Methylated Alditol Acetates Derived from CS Tetrasaccharides Containing Galactose for the Gas Chromatography/Mass Spectrometry Analysis

用于气相色谱/质谱分析的含半乳糖CS四糖衍生的部分甲基化糖醇乙酸酯的制备

KH Kyohei Higashi email

TT Toshihiko Toida

发布: 2017年11月05日第7卷第21期 DOI: 10.21769/BioProtoc.2600 浏览次数: 9466

评审: Anonymous reviewer(s)

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Abstract

Chondroitin sulfate (CS), a member of the glycosaminoglycan (GAG) family of carbohydrates, is composed of linear, sulfated repeating disaccharide sequences of N-acetyl-_D-galactosamine (GalNAc) and glucuronic acid (GlcA). Recently, a keratan sulfate (KS) disaccharide [GlcNAc6S(β1-3)Galactose(β1-]-branched CS-E was identified from the clam species M. chinensis. This protocol details a methodology to analyze the glycosidic linkages of galactose in KS disaccharide-branched CS by GC-MS analysis. A complementary method for the identification and characterization of KS-branched CS in M. chinensis can be found in Higashi et al. (2016).

Keywords: Mactra chinensis (中国蛤蜊)

Chondroitin sulfate (硫酸软骨素)

Keratan sulfate (硫酸角质素)

Partially methylated alditol acetates (部分甲基化糖醇乙酸酯)

GC-MS analysis (GC-MS分析)

Background

Gas chromatography/mass spectrometry (GC-MS) analysis of the reaction products of partially methylated alditol acetates (PMAA) derived from the polysaccharides has been shown to represent a powerful tool to investigate the glycosidic linkages. The PMAAs preparation from M. chinensis in this protocol was performed according to the method of Anumula and Tayler (1992) with minor modifications.

Materials and Reagents

Screw-cap tube (AGC techno glass, borosilicate Pyrex glass, 13 mm i.d. x 120 mm)
Glass measuring pipette (0.1, 0.5, 1 and 2 ml)
Pasteur pipette IK-PAS-5P (IWAKI, catalog number: 73-0001 )
Keratan sulfate from Bovine Cornea (SEIKAGAKU, catalog number: 400760 )
Dry tetrasaccharide (100 μg) is composed of ∆UA, N-acetyl-_D-galactosamine (4S, 6S), galactose, N-acetyl-_D-glucosamine (6S). Briefly, KS branched CS from M. chinensis was treated with chondroitinase ACII, and resulting tetrasaccharide was collected through the fractionation using HPLC with Docosil column. Please see details in Higashi et al. (2016)
Dimethyl sulfoxide, dehydrated (dry DMSO) (Wako Pure Chemical Industries, catalog number: 040-18032 )
Iodomethane (CH₃I) (Wako Pure Chemical Industries, catalog number: 139-02662 )
Chloroform
Nitrogen gas (> 99.995%) (Nippon Megacare)
Trifluoroacetic acid (TFA) (Wako Pure Chemical Industries, catalog number: 204-02743 )
Acetic acid (NACALAI TESQUE, catalog number: 00212-43 )
4-N,N-dimethylaminopyridine (Wako Pure Chemical Industries, catalog number: 042-19212 )
Pyridine (NACALAI TESQUE, catalog number: 29509-25 )
Acetic anhydride (Wako Pure Chemical Industries, catalog number: 011-00276 )
Hexane (NACALAI TESQUE, catalog number: 17935-05 )
Sodium hydroxide (NaOH) (NACALAI TESQUE, catalog number: 31511-05 )
Methanol (NACALAI TESQUE, catalog number: 21915-93 )
Dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, catalog number: 043-07216 )
0.5 mol/L hydrochloric acid methanolic solution (Wako Pure Chemical Industries, catalog number: 080-07725 )
Sodium tetrahydroborate (NaBH₄) (Wako Pure Chemical Industries, catalog number: 192-01472 )
NaOH-DMSO suspension (see Recipes)
5% (v/v) pyridine in 50% acetonitrile/water (see Recipes)
NaBH₄ (5 mg/ml) solution (see Recipes)

Equipment

Test tube mixer (SEIKAGAKU, model: TM-251 )
Centrifuge (KUBOTA, model: Model 5922 )
Sample concentrator (Hangzhou Allsheng Instruments, model: MD200-2 )
Time-of-flight mass spectrometer JMS-T100GCV (JEOL, model: JMS-T100GCV )
ZB-5ms column (0.25 µm film thickness, 0.25 µm i.d. x 30 m) (Phenomenex)
Agilent Technologies 7890A GC system (Agilent Technologies, model: Agilent 7890A GC )

Procedure

English

中文翻译

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如何引用

Higashi, K. and Toida, T. (2017). Preparation of the Partially Methylated Alditol Acetates Derived from CS Tetrasaccharides Containing Galactose for the Gas Chromatography/Mass Spectrometry Analysis. Bio-protocol 7(21): e2600. DOI: 10.21769/BioProtoc.2600.