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. An aliquot of released N-linked glycans was hydrolyzed with 400 ?L of 2.0 N trifluoroacetic acid (TFA) at 100?C for 4 h. After hydrolysis, the digest was dried under a stream of nitrogen gas, resuspended in H2O, sonicated for 5 min in ice and transferred to an injection vial. A mix of standards for neutral and amino sugars 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 monosaccharides 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 program used the following mobile phase eluents: degassed nanopure water and 200 mM NaOH. Injection was made every 45 minutes. 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).
Showing the most recent 10 out of 104 publications
