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 from glycopeptide The HPLC fraction of EPO Asn83 was dissolved in 100 L of 50% acetonitrile, transferred to a microcentrifuge tube, and dried in a speed vac. The dried sample was dissolved in 100 L sodium phosphate buffer (100 mM, pH 7.5), treated with 5 L (~38 units) peptide N-glycosidase F (New England BioLabs), and incubated at 37oC for 20 hours to release the N-linked glycans. After the enzymatic digestion, the sample was passed through a C18 reversed phase cartridge to separate the N-linked glycans from the peptide(s). The N-linked glycan fraction of the sample was eluted with 5% acetic acid and then lyophilized. The peptide fraction was eluted with isopropanol and dried under a stream of nitrogen. Preparation of the O-permethylated carbohydratesPermethylation was performed following the method developed at the Complex Carbohydrates Research Center (our previous report of 'Oligosaccharide structure analysis of EPO alfa drug substance batch 42p822').The dried N-linked fractions were dissolved in 200 L dimethylsulfoxide and then methylated with 300 L of the prepared base and 150 L of methyl iodide (99.5% ) (Analytical biochemistry 203, 101-108 (1992)). The reaction was quenched by addition of 2 mL water, and O-permethylated carbohydrates were extracted with 2 mL dichloromethane. The organic phase was concentrated to dryness. After permethylation, the glycans were passed through a C18, eluted with 85% acetonitrile and dried under a stream of nitrogen. NanoelectroSpray Ionization Linear Ion Trap mass Spectrometry (NSI-LTQ/MSn) Mass analysis of oligosaccharides was performed by direct infusion of the permethylated glycans dissolved in 1 mM NaOH in 50% methanol (5 pmol/ L) into an LTQ-MS (Thermo Finnigan) instrument using a nanoelectrospray source at syringe flow rate of 0.40 L/min. The capillary temperature was set to 210oC and MS analysis was performed in positive ion mode. The collision energy was set at 28% for fragmentation in MS/MS. The analysis of the peptides was performed by using the same LTQ-MS instrument as with the glycans. The peptides were dissolved in 50 L of 0.1% formic acid in 40% acetonitrile and injected into the LTQ instrument using same flow rate and capillary temperature as with the glycans. The collision energy for fragmentation in MS/MS was individually optimized for each MS signal.
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