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: The samples from 2 eppendorf tubes were combined into a screw-cap glass tube and dried under N2. The sample was redissolved thereafter with 300 ?L of nanopure water;150 ?L was pipetted into another screw-cap glass tube for sialic acids analysis and the remainder (150 ?L) was allocated for neutral and amino sugars analysis. Both aliquots were dried under a stream of nitrogen gas. The aliquot for neutral and amino sugars analysis was hydrolyzed with 2N trifluoroacetic acid at 100oC for 4 hr, whereas the aliquot for sialic acids analysis was hydrolyzed with 2M acetic acid at 80oC for 3 hr. All hydrolysates were lyophilized thereafter, resuspended in H2O, sonicated for 5 min in ice and transferred to injection vials. A mix of standards for neutral and amino sugars, and N-acetylneuraminic acid and N-glycolylneuraminic acid were hydrolyzed in the same manner and at the same time as the sample. Four concentrations of the standard mixtures were prepared to establish calibration equations. The number of moles of each analyte in the sample was quantified by linear interpolation from the calibration equation. The neutral and amino sugars, and sialic acids were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The monosaccharide residues were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The neutral and amino sugars, and sialic acids were analyzed in 2 different pairs of mobile phase eluents and gradient programs. The mobile phase eluents were degassed nanopure water and 200 mM NaOH for neutral and amino sugars, and 100 mM NaOH and 1M sodium acetate in 100 mM NaOH for sialic acids. Injections were made every 45 min for neutral and amino sugars, and every 40 min for sialic acids analysis. All 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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