In contrast to other glycosaminoglycans, keratin sulfate (KS) does not contain uronic acids and its repeating disaccharide unit is composed of alternating D-galactose (Gal) and N-acetyl-D-glucosamine (GlcNAc). In most cases, the hydroxyl group at the C-6 position of GlcNAc residues and/or Gal residues are sulfated. Keratanase II is a kind of endo-β-N-acetylglucosaminidase and requires sulfation of the respective GlcNAc residues at the C-6 positions. The major hydrolysis products are shown in Fig. 1. The oligosaccharides can be determined by LS/MS/MS method. |
Category | Glycosaminoglycans |
Protocol Name | Keratan sulfate oligosaccharides |
Authors
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Toyoda, Hidenao
Laboratory of Bio-analytical Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University
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KeyWords |
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Reagents
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Keratanase II from Bacillus sp. (Seikagaku Corp., Tokyo, Japan) |
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Keratan sulfate from Bovine cornea (Seikagaku Corp.) |
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Instruments
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Ultrafree MC (Biomax 30) (Merck Millipore, Billerica, MA) |
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Capcell Pak NH2 UG80 column (2.0 mm i.d. x 35 mm) (Shiseido Co., Ltd., Tokyo, Japan) |
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HPLC system (Alliance system: Waters Corp., Milford, MA) |
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API-365 mass spectrometer (Applied Biosystems/Life Technologies, Carlsbad, CA) |
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Methods |
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LC/MS/MS analysis of saturated disaccharides from keratan sulfate
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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
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Filter the mixture through an Ultrafree MC (Biomax 30). |
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Inject a 10 μL portion of the filtrate into the LC/MS/MS. Typical chromatogram is shown in Fig. 2. |
Comment 1
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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.
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Copyrights |
Copyright 2001. Elsevier, for Fig. 1 & Fig. 2 in Figure & Legends
Copyright 2010. Ritsumeikan University, JCGGDB & AIST. for the rest of the contents
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Date of registration:2014-03-31 11:45:13 |
- Oguma, T., Toyoda, H., Toida, T., and Imanari, T. (2001) Analytical method for keratin sulfates by high-performance liquid chromatography/turbo-ionspray tandem mass spectrometry. Anal. Biochem. 290, 68-73 [PMID : 11180938] doi:10.1006/abio.2000.4940
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For those who wish to reuse the work, please contact JCGGDB management office (jcggdb-ml@aist.go.jp).
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