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 divided into two aliquots: one for neutral and amino sugars analysis and the other aliquot for mannose-6-phosphate analysis. The aliquot intended for neutral and amino sugars analysis was hydrolyzed with 2 N trifluoroacetic acid (TFA) at 100oC for 4 hours and the aliquot for mannose-6-phosphate analysis was hydrolyzed with 6.75 N TFA at 100oC for 1.5 hours. The hydrolysates were then dried under N2, redissolved in H2O, sonicated for 7 min in ice and transferred to an injection vial. A mix of standards for neutral and amino sugars, and for mannose-6-phosphate with a known number of moles was hydrolyzed in the same manner and at the same time as the sample. Four concentration of the neutral and amino sugar standard mix (0.5, 1.0, 2.0, and 4.0 nmoles per injection) and mannose-6-phosphate (640, 1280, 2560, 5120 picomoles per injection) were prepared to establish a calibration equation. The number of moles of each sugar in the sample was quantified by linear interpolation from the calibration equation. The neutral and amino sugars and mannose-6-phosphate 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, and mannose-6-phosphate 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, 1 M sodium acetate in 100 mM NaOH for mannose-6-phosphate. Injections were made every 40 minutes for neutral and amino sugar determination and every 35 minutes for mannose-6-phosphate determination. 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). Instrument control and data acquisition were accomplished using Dionex chromeleon software.
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