Development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans.
An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method.
Matsuno
Yu-Ki
YK
Faculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka, Japan.
Yamada
Keita
K
Tanabe
Ayumi
A
Kinoshita
Mitsuhiro
M
Maruyama
Shu-zou
SZ
Osaka
Yu-suke
YS
Masuko
Takashi
T
Kakehi
Kazuaki
K
D002236:Carbohydrate Conformation; D011508:Chondroitin Sulfate Proteoglycans / Q000737:chemistry* / Q000378:metabolism; D019075:Electrophoresis, Capillary; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; D009844:Oligosaccharides