The primary approach for glycomics research requires the isolation of glycans from cells/tissues for structural characterization and functional studies of the glycans.   A method for cell-based micro-scale isolation and quantification of high, moderate and non-sulfated glycosaminoglycans (GAGs) was developed using Chinese hamster ovary (CHO) cells. This micro-scale isolation relied on a strong anion-exchange spin column stepwise eluted with different concentrations of NaCl. Hyaluronic acid, chondroitin sulfate and heparin were used to optimize the isolation of the endogenous GAGs in CHO-S cells. The presence of added protein did not adversely impact the isolation of hyaluronic acid. The recovered GAGs were subjected to disaccharide composition by treating GAGs with polysaccharide lyases, fluorescently labeling the resulting disaccharides with 2-aminoacridone, and analysis by ultra-performance liquid chromatography-mass spectrometry. Disaccharide analysis of recovered GAGs from CHO-S cells showed the presence of four major disaccharide products, the 0SHS, NSHS, NS2SHS disaccharides from heparin sulfate and 4SCS disaccharide from chondroitin sulfate. Surprisingly, 28.8% of the CHO-S cell GAGs was non-sulfated heparosan. This method can also be used to determine the presence of other unsulfated GAGs including hyaluronic acid and unsulfated chondroitin.

Cell based microscale isolation of glycoaminoglycans for glycomics study

244th National Meeting (2012)

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