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: Glycosyl composition by GC-MS The samples (~20 ?g each) were placed in screw-cap tubes, 2 ?L 1 mg/mL inositol was added as internal standard, and the samples were lyophilized. Methyl glycosides then were prepared from the dried samples 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. 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. 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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