This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Release of N-linked glycans The samples were dissolved with 100 L ammonium bicarbonate buffer (50 mM, pH 8.4) and followed immediately by reduction with 25 mM dithiothreitol (45 min at 50oC) and carboxyamidomethylation with 90 mM iodoacetamide (45 min at room temperature in the dark) prior to trypsin digestion (37oC, overnight). A second enzyme, peptide N-glycosidase F (New England BioLabs) was added to each of the tryptic digests and incubated at 37oC for 18 hours to release the N-linked glycans. After enzymatic digestions, the samples were passed through a C18 reversed phase cartridge to separate the N-linked, and O-glycopeptide and peptide fractions. The N-linked glycans from each sample was eluted first with 5% acetic acid and then lyophilized, whereas the O-glycopeptide and peptide fraction was eluted with isopropanol and dried under a stream of nitrogen.Release of O-linked glycans by -elimination The O-linked glycans were cleaved from the glycopeptide by -elimination procedures. Briefly, 250 L of 50 mM NaOH were added to each of the samples and then checked for pH. Upon determination that the pH was basic, another 250 L of 50 mM NaOH containing 19 mg of sodium borohydride were added to the samples, vortexed, and incubated overnight at 450C. The incubated samples then were neutralized with 10% acetic acid and desalted by passing through a packed column of Dowex resins and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. The samples were passed through C18 reversed phase cartridge to collect the cleaved O-glycans (eluted with 5% acetic acid). The O-glycan fraction was dried under a stream of nitrogen and dissolved in H2O and split into two aliquots for neutral and amino sugars, and sialic acids analyses.Composition analysis by HPAEC The samples were hydrolyzed to release the monosaccharides and were analyzed by High pH-Anion-Exchange Chromatography (HPAEC). The dried samples allocated for neutral and amino sugar analysis were hydrolyzed with 400 L of 2 N trifluoroacetic acid (TFA) at 100 C for 4 h. After hydrolysis, the samples were dried under a stream of N2 and resuspended in nanopure water, sonicated in cold water for 7 min and transferred to an injection vial. A mix of sugar standards with a known number of moles was hydrolyzed at the same time as the sample. Four concentrations of standards (0.5, 1.0, 2.0, and 4.0 nmoles per injection) were prepared and injected to establish a calibration equation. The number of moles of each sugar was quantified by linear interpolation from the calibration equation. The samples allocated for sialic acid analysis were hydrolyzed with 2 M acetic acid at 80 C for 3 h. After hydrolysis, the samples were lyophilized. The dried samples were then resuspended in water and sonicated in cold water for 7 min and transferred to an injection vial. A mix of sialic acid standards with a known number of moles was hydrolyzed at the same time as the samples. Four concentrations of standards (0.5, 1.0, 2.0, and 4.0 nmoles per injection) were prepared and injected to establish a calibration equation. The number of moles of each sialic acid was quantified by linear interpolation from the calibration equation The neutral and amino sugars and sialic acids were analyzed using a Dionex DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector, and a Thermo-Separations 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 the following eluents: A, 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 sialic acid analysis. Autoinjections were made every 40 min for the analysis of neutral and amino sugars and every 35 min for sialic acid analysis. The autosampler was set to deliver 10 L of sample or standard solution per injection. Blanks (water) were injected between standards and the samples to prevent carry-over contamination. 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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