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 About 2.5 to 2.8 mg of each glycoprotein was dissolved with protease buffer (0.1 M Tris-HCl, 0.01 M CaCl2, pH 8.2), and heated at 100oC for 5 min to denature the protein. After cooling to room temperature, trypsin was added to the sample and incubated at 37oC overnight. At the end of enzyme digestion, the tube was heated at 100oC for 5 min to inactivate the trypsin. The tryptic digests were cleaned of contaminants by passing through a C18 sep pak cartridge. Once loaded in the cartridge, each of the three samples was cleaned with 5% acetic acid and the glycopeptides and peptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol. The eluates were dried initially under a stream of nitrogen and then lyophilized. The dried tryptic digests were dissolved with 50 mM NaPO4 buffer (pH~7.5), treated with PNGase F and incubated at 37oC overnight to release the N-linked glycans. At the end of the second enzyme digestion, the samples were passed through a C18 sep pak cartridge and N-linked glycans fraction was eluted with 5% acetic acid and lyophilized. Two aliquots of the released N-linked glycans to represent about 140 ?g each of the original sample were partitioned for monosaccharide composition analysis. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The PNGase-F released N- linked glycans from the three samples were permethylated for structural characterization by mass spectrometry (Anumula and Taylor, 1992). The dried eluates were dissolved with 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 methylene chloride. Per- O-methylated glycans were further purified by passing through a C18 sep pak cartridge, washed with nanopure water and 15% acetonitrile. Finally, cleaned permethylated glycans were eluted with 85% acetonitrile and dried under a stream of nitrogen gas. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) The dried purified glycans were dissolved with methanol and crystallized with ?-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) matrix. Full mass spectrum of glycans present in each sample was performed in the positive ion mode by MALDI-TOF-TOF-MS using 4700 Proteomics Analyzer (Applied Biosystems).
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