N-LINKED OLIGOSACCHARIDE PROFILING 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. 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. Methods: Release of N-linked glycans The lyophilized samples (Samples 1 thru 7, ~1 mg; Sample A ~0.4 mg; and Sample B ~0.7 mg) were dissolved with nanopure water, added with 100 mM sodium phosphate buffer (pH 7.5) and 1% SDS w/v/1M ¿-mercaptoethanol and denatured at 100oC for 5 min. After cooling to room temperature, the mixture was added with ice-cold 1 M potassium chloride and placed on ice for 1.0 hr. Subsequently, the tubes were spun at 4oC, maximum speed for 10 min to pellet the potassium salts of SDS. The supernatant of each sample was transferred into another clean microcentrifuge tube, treated with PNGase F and incubated at 37oC overnight. After enzymatic digestion, each of the digests was passed through a C18 sep pak cartridge and the carbohydrate fraction (containing N-linked glycans) was eluted with 5% acetic acid and lyophilized. Oligosaccharide Profiling by HPAEC-PAD The dried N-linked oligosaccharides of each sample were dissolved with nanopure water [20 ¿g/¿L] and transferred into vials for injection. The oligosaccharides were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The oligosaccharides were separated by a Dionex CarboPac PA100 (4 x 250 mm) analytical column with a guard column. The mobile phase eluents used were 160 mM NaOH (A) and 1 M sodium acetate in 160 mM NaOH (B) at a flow rate of 1 ml/min. Separation of oligosaccharides was accomplished with the following gradient program: 0 min, A=100% & B=0; 90 min, A=80% & B=20%; 91 min, A=50% & B=50%; 100 min, A=50% & B=50%; 101 min, A=100% & B=0; and 110 min, A=100% & B=0. Injection volume of the autosampler was set at 15 ¿L. Instrument control and data acquisition were accomplished using Dionex chromeleon software.