This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Dialysis The sample was dialyzed using a Tube-O-Dialyzer (4.0 kDa cut-off membrane;G BioSciences) against nanopure water at 4oC for about 24 hours to remove salts and other contaminants. Nanopure water was replaced four times during the entire dialysis period. After dialysis, the sample was transferred into a screw-cap tube and lyophilized. Glycosyl composition by GC-MS The sample was added with 5 ?g inositol as internal standard. The dried sample, monosaccharides standard mixture, and iduronic acid standard were placed under vacuum to attain complete dryness before methanolysis. Methyl glycosides then were prepared from the dried sample by methanolysis with 3 M HCl in methanol at 100?C for 2 h followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The preceding methanolysis and re-N-acetylation steps were repeated two times. The samples then were per-O-trimethylsilylated (TMS) with a Tri-Sil reagent (Thermo Scientific) at 80?C for 0.5 h. These procedures were carried out as described previously in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15;York, et al. (1985) Methods Enzymol. 118:3-40. Analysis of the TMS methyl glycosides was performed on a Hewlett Packard Series II 5890 gas chromatograph equipped with a Supelco EC-1 fused silica capillary column (30m ? 0.25 mm ID) and interfaced to a Hewlett Packard 5970 MSD.
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