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 dried cell pellets and lysates 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, 25 L (trypsin 2 mg/mL) and 25 L chymotrypsin (2 mg/mL) were added to each tube and incubated at 370C for 18 h. The tryptic and chymotryptic digests were spun at 3000 rpm, 40C for 15 min and the supernatants were transferred into another tube. The pellets were added subsequently with 200 L of H2O, vortexed and spun as described previously and the supernatants were collected into their respective sample tubes. Both tryptic and chymotryptic digests, supernatant (from cells) and lysates were lyophilized. The dried digests were dissolved with 200 L of 5% acetic acid and passed through C18 sep pak cartridge to remove contaminants. The glycopeptides were eluted in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid, and 100% isopropanol. The eluates were dried under a stream of N2, redissolved with 25 L of H2O and 20 L of 0.1 M sodium phosphate buffer (pH 7.5), treated with 5 L of PNGase F, and incubated at 370C for 18 h. After the second enzymatic digestion (PNGase F), the mixture was 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 then lyophilized to dryness.Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridgeThe N-and O-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 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.Oligosaccharide Profiling by ElectroSpray Ionization-Linear Ion Trap-Fourier Transform Mass Spectrometry (ESI-LTQ-FT MS)The released N-linked oligosaccharides from the cells and lysates were profiled by ESI-LTQ-FT MS. 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 28 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.
Showing the most recent 10 out of 104 publications
