This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Release of N-linked glycans About 3.0 mg of each glycoprotein was dissolved with 0.1 M Tris-HCl and heated at 100oC for 5 min to denature the protein. After cooling to room temperature, trypsin and chymotrypsin were added to the samples and incubated at 37oC overnight. At the end of enzyme digestion, the samples were heated at 100oC for 5 min to deactivate the enzymes. The tryptic-chymotryptic digests were cleaned of contaminants by passing through a C18 sep pak cartridge. Once loaded in the cartridge, each of the two samples was 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 glycopeptides 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. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The PNGase-F released N- linked glycans from the glycoproteins 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. Oligosaccharide Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF TOF-MS) The permethylated N-linked 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 AB SCIEX TOF/TOF 5800 (Applied Biosystem MDS Analytical Technologies).
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