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.Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis.N-linked oligosaccharide profiling by MALDI- and ESI-MSRelease of N-linked glycansAn aliquot (to provide about 2.0 mg) of the samples (1317042 and PD06004) were placed in microcentrifuge tubes and lyophilized. Dried PD06004 sample was dialyzed first in nanopure water using a Chemincon tube-O-dialyzer overnight and then lyophilized. The dried samples (1317042 and PD06004) 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 trypsin digestion, the samples were 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 samples were dried down in a speed vac. The samples then were passed through a C18 reversed phase cartridge. A second enzyme, peptide N-glycosidase F (New England BioLabs) was added to the tryptic digests and incubated at 37oC for 20 hours to release the N-linked glycans. After enzymatic digestions, each of 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 each sample was eluted with 5% acetic acid and then lyophilized.Preparation of the per-O-methylated carbohydratesThe lyophilized N-linked fractions (2.0mg of each 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 permethylated 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 and PD06004) were obtained initially using a MALDI TOF Mass Spectrometer (Applied Biosystems).Direct infusion of oligosaccharidesThe profile of glycan structures detected by MALDI-TOF from the samples were confirmed by electrospray ionization mass spectrometry (ESI-MS) using an LCQ-MS (Thermo Finnigan). The permethylated glycans used for MALDI-TOF were dried and redissolved in 40 L of 1mM NaOH in 50% methanol (~5pmol/ L) and infused directly into the instrument at a constant flow rate of 1 L/min via a syringe pump (Harvard Apparatus) and sprayed at 3.5 kV. Mass spectra were obtained in the positive ion mode. The LCQ instrument was operated at this condition.
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