This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The soil nematode Caenorhabditis elegans has a short life-cycle and provides an important model for the study of glycan functions in a high throughput fashion. Glycosaminoglycans (GAGs) are essential for growth and development. For example, chondroitin sulfate (CS) is indispensable for cytokinesis in C. elegans and heparan sulfate (HS) is required for neuronal migration and axon outgrowth. Although many genetic and biochemical studies have demonstrated the functional roles of CS and HS in C. elegans, more detailed knowledge has been limited by lack of methods for determining GAG structure. To date, much of the structural information has been limited to disaccharide compositions inferred using chromatography with fluorometric detection. The work presented herein describes a glycan preparation and LC/MS platform for analyzing the structure of C. elegans GAGs. C. elegans N2-Bristol nematodes were grown in liquid culture, cleaned, and then disrupted by bead beating. Disrupted nematodes were lyophilized, delipidated and GAGs released with alkaline borohydride. The GAG-rich fraction was precipitated with ethanol and then purified by C-18 solid phase extraction. Samples were applied to a DEAE spin column, eluted, and exhaustively digested with chondroitinases. An aliquot was saved for CS analysis. The remaining sample was applied to a second DEAE spin column, eluted, and digested with heparin lyases. Products of both digests were reductively aminated with 2-anthranilic acid. An aliquot of each preparation was then injected onto a 250 m x 15 cm capillary column packed in-house with Amide-80 resin (Tosoh) connected online to an Applied Biosystems/MDS Sciex QSTAR mass spectrometer.
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