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 Half of each sample (250 g) was transferred to a microcentrifuge tube and dried in a speed vac. The dried samples were dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4), and denatured immediately by boiling at 100 oC for 5min prior to trypsin digestion at 37 oC for 20 hours. After trypsin digestion, the samples were heated at 100 oC for 5 min to deactivate the enzyme. The samples were passed through a C18 reversed phase cartridge. And then samples were treated with a second enzyme, peptide N-glycosidase F (New England BioLabs) and incubated at 37 oC for 20 hours to release the N-linked glycans. After enzymatic digestion, the samples were passed through a C18 reversed phase cartridge to separate the N-linked glycans from the peptide. The N-linked glycan fractions of the samples were eluted with 5% acetic acid and then lyophilized. Preparation of the per-O-methylated carbohydratesThe lyophilized carbohydrate fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Analytical Biochemistry 203, 101-108 (1992)). The reaction was quenched by addition of water, and O- per-methylated carbohydrates were extracted with dichloromethane. The organic phase was concentrated to dryness and then the glycans were passed through a C18 Sep-Pak, eluted with 85 % acetonitrile, dried under a stream of nitrogen, and dissolved in methanol prior to analysis by mass spectrometry.Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) Profiling of N-linked glycans was performed initially using MALDI/TOF-MS on a 4700 Proteomics analyzer (Applied Biosystems). Permethylated glycans (~1 L) were crystallized on a MALDI plate with 1 L of 2, 3-dihydroxybenzoic acid (DHBA, 20 mg/mL solution in 50 % methanol: water) as matrix. All spectra were acquired in the reflector positive ion mode and averaged spectra of 50 laser shots.
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