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.Glycosyl sialic acid 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 samples by acidic methanolysis.The dry sample provided by the client was pre-hydrolyzed by 0.1N trifluoroacetic acid at 80 C (3 hours). After drying to remove the TFA, methyl glycosides were then prepared by methanolysis in 0.05 M HCl in methanol at 80 C for 1 hour. The sample was 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 Sumi et al. (2001) Prep. Biochem. & Biotechnol. 31(2):135-146. GC/MS analysis of the TMS methyl glycosides was performed on an HP 5890 GC interfaced to a 5970 MSD, using a Supelco DB-1 fused silica capillary column (30m x 0.25 mm ID).Sialic Acid Determination by HPAEC The sample was analyzed for sialic acid composition by release of sialics by hydrolysis, and analysis by Dionex high pH-anion-exchange chromatography (HPAEC).Sialic acids were released from a portion of the sample by mild acid hydrolysis using 2M acetic acid at 80 C for 3 h. After hydrolysis the sample was dried using a centrifugal vacuum evaporator, and then resuspended in 200 l water and sonicated on ice. For analysis, 10 l was injected. Blanks (water) were injected and run between the samples (data not shown) to prevent carry-over. The calibration standards were hydrolyzed in parallel with your samples, and then injected at three levels (0.25, 0.75 and 1.5 nmol). The quantitation was based on a linear interpolation from these standards. Sialic acids were analyzed using a Dionex DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector and a Thermo-Separations AS3500 autosampler containing a stainless steel needle. Analyses were performed using a Dionex CarboPac PA10 (4 x 250 mm) analytical column with an Amino Trap and Borate trap column. The gradient program used C, 100 mM NaOH and D, 1 M sodium acetate in 100 mM NaOH for the sialic acids. Injections were made every 60 minutes for the sialic acid determinations. The methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates , 1994, Methods Enzymol. 230: 208-225).
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