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.The samples were analyzed for monosaccharide composition by release of monosaccharides through hydrolysis and analysis using Dionex high pH-anion-exchange chromatography (HPAEC).N-linked oligosaccharide profiling by MALDI- and ESI-MSRelease of N-linked glycans An aliquot (2.0 mg) of the sample was lyophilized and then the dried sample was dissolved in 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 at 37oC for 20 hours. After tryptic digestion, the sample was heated for 5min at 100 oC, spun down for 15min at 4 oC, collected sample and dried down it in a speed vac. The sample was passed through a C18 reversed phase cartridge. A second enzyme, peptide N-glycosidase F (New England BioLabs) was added to the tryptic digest and incubated at 37oC for 20 hours to release the N-linked glycans. After enzymatic digestions, the sample was passed through a C18 reversed phase cartridge. The N-linked glycan was eluted with 5% acetic acid and then lyophilized.Preparation of the per-O-methylated carbohydrates The lyophilized N-linked fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Ciucanu and Kerek, 1984). The reaction was quenched by addition of water, and per-O-methylated carbohydrates were extracted with dichloromethane. The organic phase was concentrated to dryness and then dissolved with methanol. The glycans was passed through a C18 and dried down it in the lyophilizer. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI) MALDI-MS was performed in the positive ion mode using -dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol: water) as a matrix. Full mass spectra of sample (1317046) were obtained initially using a TOF-TOF Mass Spectrometer (Applied Biosystems). Direct infusion of oligosaccharides The profile of glycan structures from sample was confirmed by electrospray ionization mass spectrometry (ESI-MS) using an LCQ-MS (Thermo Finnigan). The permethylated glycans dissolved in 40 L of 1mM NaOH in 50% methanol (~5pmol/ L) was infused directly into the instrument at a constant flow rate of 1 L/min via a syringe pump (Harvard Apparatus) and sprayed at 3.5 kV. Mass spectra were obtained in the positive ion mode. The LCQ instrument was operated at this condition.Sheath Gas Flow Rate (arb) 20AUX / Sweep Gas Flow Rate (arb) 10Ion Spray Voltage (kV) 3.5Capillary Temperature (oC) 200Capillary Voltage (V) Automatically tuned using permethylated glycan (Fetuin)Tube lens Offset (V) Automatically tuned using permethylated glycan (Fetuin)
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