SIALIC ACIDS COMPOSITION ANALYSIS BY HPAEC

这个子项目是利用这些资源的众多研究子项目之一

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: An aliquot of about 150 ¿g each from NANA and saliva samples were prepared for sialic acids analysis. The aliquots were hydrolyzed with 500 ¿L of 2.0 M acetic acid at 80¿C for 3 h. All hydrolysates were dried under nitrogen gas, resuspended in H2O, sonicated for 7 min in ice and transferred to injection vials. A mix of standards for N-acetylneuraminic acid and N-glycolylneuraminic acid with a known number of moles was hydrolyzed in the same manner and at the same time as the samples. Four concentrations of standard mixture were prepared to establish a calibration equation. The number of moles of each residue in the sample was quantified by linear interpolation from the calibration equation. The sialic acids were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The sialic acids were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used the following mobile phase eluents: 100 mM NaOH, and 1M sodium acetate in 100 mM NaOH and injections were made every 40 min. 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).