MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The sample was transferred from the original container to a screw cap tube, dried under N2,, redissolved with H2O and divided equally the solution into two aliquots, and lyophilized. One aliquot was designated for neutral and amino sugars analysis with the other aliquot for sialic acids analysis. The aliquot intended for neutral and amino sugars was hydrolyzed with 400 ¿L of 2.0 N trifluoroacetic acid (TFA) at 100¿C for 4 h, whereas the aliquot for sialic acids was hydrolyzed with400 ¿L of 2.0 M acetic acid at 80¿C for 3 h. The hydrolysates were lyophilized, resuspended in H2O, sonicated for 7 min in ice and transferred to injection vials. A mix of standards for neutral and amino sugars and for sialic acids with a known number of moles was hydrolyzed in the same manner and at the same time as the sample. Four concentration of standard mix (0.5, 1.0, 2.0, and 4.0 nmoles per injection) 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 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 individual neutral and amino sugars were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used eluents A - degassed nanopure water and B - 200 mM NaOH for neutral and amino sugars, and C - 100 mM NaOH and D - 1M sodium acetate in 100 mM NaOH for sialic acids. Injections were made every 40 minutes for neutral and amino sugars and every 35 min for sialic acids. 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).