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 Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. A 500 and 700ug aliquot was taken from the sample and added to separate tubes with 20ug of inositol as the internal standard. For chitin analysis, the tube with 700ug was hydrolyzed in 4M HCl for 4 hours at 100 degrees Celsius. The sample was then dried down and run in parallel with the 500ug sample. Methyl glycosides were then prepared from the two dry samples following the mild acid treatment by methanolysis in 1 M HCl in methanol at 80?C (18 hours), followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples were then per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80?C (0.5 hours). These procedures were carried out as previously described in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15;York, et al. (1985) Methods Enzymol. 118:3-40. GC/MS analysis of the TMS methyl glycosides was performed on an AT 6890N GC interfaced to a 5975B MSD, using a Supelco EC-1 fused silica capillary column (30m ? 0.25 mm ID).
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