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 50% acetonitrile and transferred to a microcentrifuge tube and dried in a speed vac. The dried sample was dissolved in sodium phosphate buffer (100 mM, pH 7.5), treated with peptide N-glycosidase F (New England BioLabs) and incubated at 37oC for 20 hours to release the N-linked glycans. After enzymatic digestion, the sample was passed through a C18 reversed phase cartridge to separate the N-linked glycans from the peptide. 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 per-O-methylated carbohydratesThe dried N-linked fractions were 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. 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 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 peptides was performed by using the same LTQ-MS instrument as with the glycans. The peptides were dissolved in 25 L of 0.1% formic acid and 25 L of 0.1% formic acid/80% 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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