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 Aliquots (to provide about 2.0 mg) of the sample (1317042) were placed in microcentrifuge tubes and lyophilized. The dried samples (1317042) were dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4) and denatured immediately by boiling at 100oC for 5min prior to trypsin digestion at 37oC for 20 hours. After tryptic digestion, the sample was heated at 100oC for 5 min to deactivate the enzyme, centrifuged for at 4oC for 15 min and washed with nanopure water and re-centrifuged. The sample was dried down in a speed vac. The sample then 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 to separate the N-linked glycans from the glycopeptides and peptides. The N-linked glycan fraction of the sample was eluted with 5% acetic acid and then lyophilized. A portion of the N-glycan fraction was incubated at 37oC with -glucosidase from Sacchromyces cerevisiae (Sigma-Aldrich) in 100 mM potassium phosphate buffer (pH 6.8) for 20 hours. Then the sample was dried down in a speed vac. Preparation of the per-O-methylated carbohydratesThe dried N-linked fractions (2.0mg of glycoproteins) 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 and then lyophilized in freeze-dryer. 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 the matrix. Full mass spectra of sample 1317042 were obtained initially using a MALDI TOF Mass Spectrometer (Applied Biosystems).
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