This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Acetone precipitation Cold acetone was added to the sample, which was then centrifuged at 4 oC for 15 min and supernatant was removed. Cold acetone was added to the sample again and re-centrifuged. The sample was dried down in the speed vacuum. Release of N-linked glycans from glycopeptide The dried sample was dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4), and denatured immediately by heating at 100 ?C for 5min prior to trypsin digestion at 37 ?C for overnight. After trypsin digestion, the sample was heated at 100 ?C for 5 min to deactivate the enzyme. The sample was applied to a C18 sep-pak cartridge. Before elution of glycopeptides and peptides, the sample adsorbed in the C18 sep-pak cartridge was cleaned with 5% acetic acid. Peptides and glycopeptides then were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol. The propanol fraction was dried, and then sample was treated with a second and third enzyme, peptide N-glycosidase F (New England BioLabs) and peptide N-glycosidase A (Calbiochem) incubated at 37 ?C 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 peptides. The glycan fraction of the sample was eluted with 5% acetic acid and then lyophilized. Preparation of the per-O-methylated carbohydrates The 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 per-O-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) as matrix. All spectra were acquired in the reflector positive ion mode and averaged the spectra of 50 laser shots.
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