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. First of all, the sample was cleaned up by dialysis and acetone precipitation to remove sucrose and PS-80. Then the sample was split into six, three for neutral / amino sugar (triplicate), and the other three for Sialic acid (triplicate). Each sample were then hydrolyzed and injected to HPAEC for monosaccharide composition analysis. The detailed procedures are shown below. Removal of sucrose by Dialysis The sample was dialyzed against de-ionized water overnight to remove sucrose. Tube-O-DIALYZERTM (7.5 kDa cut-off, large tube, CHEMICON international) was used for dialysis. After dialysis, the sample solution was dried in the speed vacuum concentrator. Removal of detergents by Acetone precipitation Cold acetone was added to the dried sample. The sample solution was placed on ice for 15 minutes and then spun at maximum speed in a refrigerated microcentrifuge for 15 minutes to pellet the protein. The supernatant was removed. The preceding washing steps were repeated twice. Finally, the pellet was dried in the speed vacuum concentrator. Composition analysis by HPAEC Aliquots of the sample were prepared in triplicate (~100 ?g each for neutral and amino sugars, and sialic acid analyses) for monosaccharide composition analysis. The samples intended for neutral and amino sugars were hydrolyzed with 400 ?L of 2 N trifluoroacetic acid (TFA) at 100?C for 4 h, whereas aliquots for sialic acids were hydrolyzed with 2M acetic acid at 80?C for 3 h. The hydrolysates were lyophilized, resuspended 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 sialic acids with a known number of moles was hydrolyzed in the same manner and at the same time as the samples. Four concentration of standards (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 sugar 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 DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector, and a Thermo-Separation AS3500 autosampler containing a stainless steel needle. 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;B, 200 mM NaOH for the neutral and amino sugars;C, 100 mM NaOH;and D, 1 M sodium acetate in 100 mM NaOH for the sialic acids. Injections were made every 40 minutes for the neutral and amino sugar determinations and every 35 minutes for the sialic acid determinations. 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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