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. Oligosaccharide composition analysis by HPAEC Each of the five samples was dissolved with 400 ?L of nanopure H2O initially and further diluted upon observation that the peaks of the initial sample solutions were large. A standard, 6 ?-Mannobiose (Sigma-Aldrich), was used and prepared at four concentrations (75, 150, 300, and 600 picomoles per 10 ?L) to establish a calibration equation. The number of moles of oligosaccharide peak in each of the samples was quantified by linear interpolation from the calibration equation. The oligosaccharides were analyzed by HPAEC using a Dionex DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector, and a Thermo-Separation AS3500 autosampler containing a stainless steel needle. A CarboPac PA200 (3 x 250 mm) analytical column with an appropriate guard column was installed to the machine to separate the oligosaccharide peaks. The eluents used in the gradient program were: A, degassed nanopure water and;B, 200 mM NaOH. Injection volume was set to 10 ?L and 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). Instrument control and data acquisition were accomplished using Dionex PeakNet software, version 5.01.
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