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 The dried cell pellets were dissolved with 200 L protease buffer (0.1 M Tris-HCl pH 8.2 containing 0.01 M CaCl2) and the tubes were placed in a heating block (1000C, 5 min) to denature the protein. After cooling to room temperature, 50 L trypsin (2 mg/mL) and 50 L chymotrypsin (2 mg/mL) were added to the tubes and incubated at 370C for about 21 h. Trypsin and chymotrypsin were deactivated by heating the tubes at 100oC for 5 min and digests were spun at 3000 rpm at room temperature for 15 min. Each of the supernatants was passed through a C18 sep pak cartridge. The pellets were added subsequently with 400 L of H2O, vortexed and spun as described previously and the supernatants were loaded into the respective C18 sep pak cartridges. The adsorbed peptides and glycopeptides were washed with 9 mL of 5% acetic acid and eluted in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid, and 100% isopropanol. The eluates were dried initially under N2 and the remaining solutions were lyophilized. The dried eluates were redissolved with 30 L of H2O and 20 L of 0.1 M sodium phosphate buffer (pH 7.5), treated with PNGase F, and incubated at 370C for 18 h to release the N-linked glycans. After the second enzymatic digestion (PNGase F), the digests were passed through a C18 sep pak cartridge to separate the carbohydrate fraction and the peptide-containing fraction. The N-linked glycans were eluted first with 5% acetic acid followed by the elution of O-glycopeptide and peptide fraction with 100% isopropanol. Both fractions were dried either by lyophilization (N-glycan) or under a stream of nitrogen gas (O-glycopeptide and peptide).Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridgeThe 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 further cleaned of contaminants. Briefly, the glycans were dissolved in methanol:water (1:1) 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.Oligosaccharide Profiling by Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF)Profiling of N-linked glycans was performed initially using MALDI/TOF-MS. The machine used was a 4700 Proteomics analyzer (Applied Biosystems), which was set in the reflector positive ion mode. Permethylated glycans were crystallized on a MALDI plate with -dihydroxybenzoic acid (DHBA, 20 mg/mL solution in 50% methanol:water) as a matrix.Oligosaccharide Profiling by Linear Ion Trap-Fourier transform Ion Cyclotron Resonance (FTICR) Mass Spectrometer (LTQ-FT, Thermo Scientific)The N-linked oligosaccharides detected by MALDI-TOF MS were confirmed by LTQ-FT MS. Mass spectrometric analysis was performed following the method developed by Atwood et al. (2007). Briefly, permethylated glycans were dissolved in 1 mM NaOH in 50% methanol and infused directly into the instrument. Total ion mapping, automated MS/MS analysis (at 29% collision energy), m/z range from 500 to 2000 was scanned in successive 2.8 mass unit window that overlapped the preceding window by 2 mass units.
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