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. The samples were dialyzed using a Tube-O-Dialyzer (4.0 kDa cut-off membrane;G BioSciences) against nanopure water at 4oC for about 18 hours to remove salts and other contaminants. Nanopure water was replaced three times during the entire dialysis period. Release of N-linked glycans After dialysis, an aliquot of each sample (to provide ~125 mg based on original sample information) was taken for monosaccharide composition analysis. The remainder was used for N-linked and O-linked glycans analysis by mass spectrometry. Briefly, the samples were 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 each sample and incubated at 37oC overnight. At the end of enzyme digestion, the tubes were heated at 100oC for 5 min to inactivate the trypsin. The tryptic digests were further cleaned of contaminants by passing through a C18 sep pak cartridge. Once loaded in the cartridge, the samples were cleaned with 5% acetic acid, and the glycopeptides/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 sodium phosphate, 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 fractions were eluted with 5% acetic acid. The O-linked glycopeptides/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 carbohydrate (N-linked glycans) fractions were dried by lyophilization, whereas the O-linked glycopeptides/peptides fractions were dried initially under a stream of nitrogen gas and eventually lyophilized. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The PNGase-F released N- linked glycans 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 with 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% 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. Analysis of glycans present in the samples was performed in the positive ion mode by MALDI-TOF-TOF-MS using 4700 Proteomics Analyzer (Applied Biosystems).
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