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. Release of N-linked glycans An aliquot (70 ?L) of the sample was lyophilized and then the dried sample was dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4) and followed immediately by reduction with 25 mM dithiothreitol (45 min at 50oC) and carboxyamidomethylation with 90 mM iodoacetamide (45 min at room temperature in the dark) prior to trypsin digestion (37oC, overnight). A second enzyme, peptide N-glycosidase F (New England BioLabs) was added to the tryptic digest and incubated at 37oC for 18 hours to release the N-linked glycans. After enzymatic digestions, the sample was passed through a C18 reversed phase cartridge. The N-linked glycan fraction was eluted with 5% acetic acid and then lyophilized. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The N- linked glycans were permethylated for oligosaccharide profiling (Ciucanu and Kerek, 1984). The dried eluates were dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide. The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were cleaned of contaminants. Briefly, the glycans were dissolved in 1:1 methanol:water and loaded into a C18 sep pak cartridge and then washed with nanopure water. Per-O-methyl carbohydrates were eluted with 15% acetonitrile into a screw-cap tube, and with 85% acetonitrile into another screw-cap tube. The glycans eluted with 85% acetonitrile were dried under a stream of nitrogen gas and were dissolved with methanol for analysis by mass spectrometry. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) MALDI-MS was performed in the positive ion mode using ?-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) as a matrix. Full mass spectrum of the sample was obtained by using a 4700 Proteomics analyzer (Applied Biosystems). Profiling by ElectroSpray Ionization ?Linear Ion Trap mass Spectrometry (ESI-LCQ/MSn) The oligosaccharides detected by MALDI-TOF MS was confirmed by ESI-LCQ mass spectrometry. The remainder of the per-O-methylated glycans profiled for N-linked through MALDI-TOF machine were diluted in 1 mM NaOH in 50% methanol (~5 pmol/?L) and infused directly into the LCQ-Advantage (Thermo Finnigan) instrument at a constant flow rate of 1.0 ?L/min via a syringe pump (Harvard Apparatus) and sprayed at 3.5 kV. A normalized collision energy of 35 and an isolation mass window of 2 Da was applied to obtain MSn.
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