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. Glycosyl composition by GC-MS The sample was transferred to a pre-rinsed screw-cap tube, added with 3 ?g of inositol as an internal standard, and lyophilized. Methyl glycosides then were prepared from the dried sample by methanolysis with 1 M HCl in methanol at 80?C overnight followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The methyl glycosides of the sample 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. Since the absolute content of the analytes was expected to be low, the final derivatized sample was dissolved in a small volume of hexane and had to be injected manually.
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