Graphitized carbon LC-MS characterization of the chondroitin sulfate oligosaccharides of aggrecan.
A novel in-gel endoglycosidase technique to study oligosaccharides with graphitized carbon LC-MS has revealed differences in the sulfation profile between the linkage and repeat regions of chondroitin sulfate on aggrecan. Bovine articular cartilage aggrecan was isolated in a composite agarose PAGE gel or diluted in ammonium acetate buffer and was digested overnight with chondroitinase ABC. Including a chemical release/reduction protocol after digestion, we could separate and detect three differentially sulfated chondroitin sulfate disaccharides of the repeat region (DeltaUA1-3GalNAc0/4/6S-ol) from the three differentially sulfated linkage region hexasaccharides (DeltaUA1-3GalNAc0/4/6Sbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylitol). Graphitized carbon LC-MS in the negative ion mode was able to resolve isomeric disaccharides and linkage region hexasaccharides. Specific MS2 and MS3 enabled us to confirm the sulfate location on all oligosaccharides by comparing their fragmentation with sulfated disaccharide standards. The presence of unsulfated, 6-sulfated, and 4-sulfated linkage regions was correlated with positive Western blot staining with the respective CS linkage region neoepitope antibodies (1B5, 3B3, 2B6) on digested aggrecan. Our strategy of examining linkage region and repeat region profiles is applicable to screening GAGs from various biological samples in order to detect differences between normal and disease states.
Estrella
Ruby P
RP
Proteome Systems Ltd., Locked Bag 2073, North Ryde Sydney, NSW 1670 Australia.
Whitelock
John M
JM
Packer
Nicolle H
NH
Karlsson
Niclas G
NG
D053674:Aggrecans / Q000737:chemistry*; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002358:Cartilage, Articular / Q000737:chemistry; D002417:Cattle; D002809:Chondroitin Sulfates / Q000737:chemistry*; D002853:Chromatography, Liquid; D006108:Graphite; D013058:Mass Spectrometry / Q000379:methods*; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry*
D053674:Aggrecans; D009844:Oligosaccharides; D006108:Graphite; D002809:Chondroitin Sulfates