Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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
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: 10471

评审: Anonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Biochemical Journal, featuring study using the protocol.

Sep 2016

Advertisement

Special Issue on Protocols for Organoid Research
Technologies for RNA Detection
“科研可重复性与创新”系列网络研讨会
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

中国蛤蜊中硫酸角质素二糖-支链硫酸软骨素E的分离

中国蛤蜊中硫酸角质素二糖-支链硫酸软骨素E的分离

Kyohei Higashi and Toshihiko Toida

2017年08月05日 7567 阅读

几丁质酶解的色谱分析

几丁质酶解的色谱分析

Sophanit Mekasha [...] Vincent G. H. Eijsink

2021年05月05日 5684 阅读

拟南芥中鼠李半乳糖醛酸聚糖 I（RG-I）的纯化方法详述

拟南芥中鼠李半乳糖醛酸聚糖 I（RG-I）的纯化方法详述

Liang Zhang [...] Breeanna R. Urbanowicz

2026年02月05日 544 阅读

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) search Chondroitin sulfate (硫酸软骨素) search Keratan sulfate (硫酸角质素) search Partially methylated alditol acetates (部分甲基化糖醇乙酸酯) search GC-MS analysis (GC-MS分析) search

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

  1. Screw-cap tube (AGC techno glass, borosilicate Pyrex glass, 13 mm i.d. x 120 mm)
  2. Glass measuring pipette (0.1, 0.5, 1 and 2 ml)
  3. Pasteur pipette IK-PAS-5P (IWAKI, catalog number: 73-0001 )
  4. Keratan sulfate from Bovine Cornea (SEIKAGAKU, catalog number: 400760 )
  5. 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)
  6. Dimethyl sulfoxide, dehydrated (dry DMSO) (Wako Pure Chemical Industries, catalog number: 040-18032 )
  7. Iodomethane (CH3I) (Wako Pure Chemical Industries, catalog number: 139-02662 )
  8. Chloroform
  9. Nitrogen gas (> 99.995%) (Nippon Megacare)
  10. Trifluoroacetic acid (TFA) (Wako Pure Chemical Industries, catalog number: 204-02743 )
  11. Acetic acid (NACALAI TESQUE, catalog number: 00212-43 )
  12. 4-N,N-dimethylaminopyridine (Wako Pure Chemical Industries, catalog number: 042-19212 )
  13. Pyridine (NACALAI TESQUE, catalog number: 29509-25 )
  14. Acetic anhydride (Wako Pure Chemical Industries, catalog number: 011-00276 )
  15. Hexane (NACALAI TESQUE, catalog number: 17935-05 )
  16. Sodium hydroxide (NaOH) (NACALAI TESQUE, catalog number: 31511-05 )
  17. Methanol (NACALAI TESQUE, catalog number: 21915-93 )
  18. Dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, catalog number: 043-07216 )
  19. 0.5 mol/L hydrochloric acid methanolic solution (Wako Pure Chemical Industries, catalog number: 080-07725 )
  20. Sodium tetrahydroborate (NaBH4) (Wako Pure Chemical Industries, catalog number: 192-01472 )
  21. NaOH-DMSO suspension (see Recipes)
  22. 5% (v/v) pyridine in 50% acetonitrile/water (see Recipes)
  23. NaBH4 (5 mg/ml) solution (see Recipes)

Equipment

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

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

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.

ris ris Download Citation in RIS Format

分类

生物化学 > 糖类 > 多糖

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件