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 glycansThe protein-rich powder was dissolved with 200 L protease buffer (0.1 M Tris-HCl pH 8.2 containing 0.01 M CaCl2) and the tube was 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 tube and incubated at 370C for about 21 h. Trypsin and chymotrypsin were deactivated by heating the tube at 100oC for 5 min and digest was spun at 3000 rpm under room temperature for 15 min. The supernatant was passed through a C18 sep pak cartridge. The pellet was added subsequently with 400 L of H2O, vortexed and spun as described previously and the supernatant was loaded into the same C18 sep pak cartridge. The adsorbed peptides and glycopeptides were washed with 8 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 eluate was dried initially under N2 and the remaining solution was lyophilized. The dried eluate was 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 digest was passed through a C18 sep pak cartridge to separate the carbohydrate fraction and the peptide-containing fraction. The N-linked glycans fraction was 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).Glucosidase digestion of released N-glycansThe lyophilized released N-glycan was dissolved with 200 L of NaPO4 buffer (pH~6.8) and was treated with 50 L of alpha-glucosidase (B. stearothermophilus; 300 units/mL in 10 mM NaPO4 buffer). After 48 h of digestion at 37oC, the mixture was added again with 50 L of alpha-glucosidase (same as above) and incubated under the same condition for another 48 h. Enzymatic digestion was terminated subsequently and the mixture was lyophilized.Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridgeThe enzymatic digest was permethylated for N-linked glycan profiling (Ciucanu and Kerek, 1984). The dried digest was 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 ElectroSpray Ionization-Mass Spectrometry (ESI-MS)The objective of the current experiment was to determine the fate of the glycan, 6 hexoses and 2 HexNAcs proposed as GlcNAc2Man6 (m/z 1784) in the previous report after glucosidase digestion. The dried per-O-methylated glycans were dissolved in 20 L methanol and mixed with 30 L of 1 mM NaOH in 50% methanol. The mixture was infused directly by a pump (Harvard Apparatus 22) into an LCQ Advantage (Thermo-Finnigan) instrument. Full mass spectrum of glycans, m/z 200 to 2000 was obtained by scanning for 2 min and fragmentation (MS/MS) analysis of the selected ion was undertaken to confirm the presence of an identified glycan. For MS/MS analysis, the selected ion was scanned with an isolation width of 2.2 and at 35% collision energy.
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