Graphitized Carbon LC-MS Characterization of the Chondroitin Sulfate Oligosaccharides of Aggrecan

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• 作者：Ruby P. Estrella ; John M. Whitelock ; Nicolle H. Packer ; Niclas G. Karlsson
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：May 15, 2007
• 年：2007
• 卷：79
• 期：10
• 页码：3597 - 3606
• 全文大小：386K
• 年卷期：v.79,no.10(May 15, 2007)
• ISSN：1520-6882

文摘

A novel in-gel endoglycosidase technique to study oligosaccharides with graphitized carbon LC-MS has revealed differences in the sulfation profile between thelinkage and repeat regions of chondroitin sulfate onaggrecan. Bovine articular cartilage aggrecan was isolatedin a composite agarose PAGE gel or diluted in ammoniumacetate buffer and was digested overnight with chondroitinase ABC. Including a chemical release/reduction protocol after digestion, we could separate and detect threedifferentially sulfated chondroitin sulfate disaccharides ofthe repeat region (ges/gifchars/Delta.gif" BORDER=0 >UA1-3GalNAc0/4/6S-ol) from thethree differentially sulfated linkage region hexasaccharides (ges/gifchars/Delta.gif" BORDER=0 >UA1-3GalNAc0/4/6Sges/gifchars/beta2.gif" BORDER=0 ALIGN="middle">1-4GlcAges/gifchars/beta2.gif" BORDER=0 ALIGN="middle">1-3Galges/gifchars/beta2.gif" BORDER=0 ALIGN="middle">1-3Galges/gifchars/beta2.gif" BORDER=0 ALIGN="middle">1-4Xylitol). Graphitized carbon LC-MS in the negative ionmode was able to resolve isomeric disaccharides andlinkage region hexasaccharides. Specific MS² and MS³enabled us to confirm the sulfate location on all oligosaccharides by comparing their fragmentation with sulfateddisaccharide standards. The presence of unsulfated,6-sulfated, and 4-sulfated linkage regions was correlatedwith positive Western blot staining with the respective CSlinkage region neoepitope antibodies (1B5, 3B3, 2B6)on digested aggrecan. Our strategy of examining linkageregion and repeat region profiles is applicable to screeningGAGs from various biological samples in order to detectdifferences between normal and disease states.