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Keratan sulfate oligosaccharides
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Keratan sulfate oligosaccharides

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Introduction Protocol References Credit lines
Category
Glycosaminoglycans
Protocol Name

Keratan sulfate oligosaccharides

Authors
Toyoda, Hidenao
Laboratory of Bio-analytical Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University
KeyWords
Reagents

Keratanase II from Bacillus sp. (Seikagaku Corp., Tokyo, Japan)

Keratan sulfate from Bovine cornea (Seikagaku Corp.)

Instruments

Ultrafree MC (Biomax 30) (Merck Millipore, Billerica, MA)

Capcell Pak NH2 UG80 column (2.0 mm i.d. x 35 mm) (Shiseido Co., Ltd., Tokyo, Japan)

HPLC system (Alliance system: Waters Corp., Milford, MA)

API-365 mass spectrometer (Applied Biosystems/Life Technologies, Carlsbad, CA)

Methods
1.

LC/MS/MS analysis of saturated disaccharides from keratan sulfate

1) 

 Incubate KS (1-100 ng) with kratanase II (10 mIU) in a total volume of 40 μL of 5 mM sodium-acetate buffer (pH 6.0) at 37˚C for 24 h.

Comment 0
2) 

 Filter the mixture through an Ultrafree MC (Biomax 30).

Comment 0
3) 

 Inject a 10 μL portion of the filtrate into the LC/MS/MS. Typical chromatogram is shown in Fig. 2.

Comment 1
Figure & Legends

Figure & Legends

Fig. 1. Structure of disaccharide obtained by keratanase II digestion of KS

Reprinted from Anal Biochem., 290(1), Oguma T, Toyoda H. et al., Analytical method for keratan sulfates by high-performance liquid chromatography/turbo-ionspray tandem mass spectrometry, 68-73, 2001, with permission from Elsevier. doi:10.1006/abio.2000.4940.

Fig. 2. Typical MRM chromatogram of oligosaccharides from bovine nasal cartilage by keratanase II digestion

Peaks: 1, Galβ1-4GlcNAc(6S); 2, Gal(6S)β1-4GlcNAc(6S).

Reprinted from Anal Biochem., 290(1), Oguma T, Toyoda H. et al., Analytical method for keratan sulfates by high-performance liquid chromatography/turbo-ionspray tandem mass spectrometry, 68-73, 2001, with permission from Elsevier. doi:10.1006/abio.2000.4940.

 

Copyrights

Copyright 2001. Elsevier, for Fig. 1 & Fig. 2 in Figure & Legends

Copyright 2010. Ritsumeikan University, JCGGDB & AIST. for the rest of the contents

Date of registration:2014-03-31 11:45:13
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