Glycosaminoglycan Analysis in Carcinoma

Carcinoma tissues were lyophilized. Glycosaminoglycan preparation, lyase treatment, fluorescence disaccharide labeling and separation were performed according to [31 (link)]. Briefly, tumors were protease- and DNAase digested, and GAGs were purified on anion-exchange chromatography. Then, 500ng GAGs, as estimated by the carbazol method, were subjected to degradation by chondroitinase ABC (Sigma) or by a mixture of heparinases (in-house preparation, purified from E.coli transformed with the pET-15b expression vector carrying heparinase I, or pET-19b expression vector carrying heparinase II or III; provided by prof. Jian Liu, University of North Carolina). Fluorophore-labeling of the resulting disaccharides was performed by 2-aminoacridone (AMAC, Sigma). Pre-column AMAC-labeled disaccharides were analyzed by HPLC as described previously [31 (link)]. Quantification was done by comparison to known weight of mock-treated standard disaccharides (Iduron, UK).