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. Preparation of Samples of oligosaccharide profiling, glycosyl linkage analysis, and monosaccharide composition analysis Release of N-linked glycans from the glycoproteins About 2.2 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 five 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 first with 5% acetic acid followed by the elution of O-glycopeptides/peptides fraction with 100% isopropanol. The carbohydrate (N-linked glycans) fraction was dried by lyophilization, whereas the isopropanol fraction was dried under a stream of nitrogen gas and stored. The dried N-linked glycans were redissolved with nanopure H2O and aliquot (representing ~150 ?g of the original material) was allocated for monosaccharide composition analysis and the remainder was utilized for oligosaccharide profiling and glycosyl linkage analysis. Experiment 1. N-linked Oligosaccharide Profiling by MALDI-TOF MS Methods: Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The released N-linked glycans intented for oligosaccharide profiling were permethylated according to the procedures of Ciucanu and Kerek (1984). 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 dichloromethane. Per- O-methylated glycans were 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. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) MALDI-TOF-MS was performed in the reflector positive ion mode using ?-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) as a matrix. Full mass spectrum of the sample was obtained by using a 4700 Proteomics analyzer (Applied Biosystems).
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