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: Purification of samples The samples were transferred into screw-cap tubes and the proteins were precipitated with cold acetone:water to remove detergents, free sugars, and other contaminants. Purification of sample was accomplished by refrigerated centrifugation and removal of supernatant. The protein pellets were dried under N2 initially and subsequently dried under vacuum. Release of N-linked glycans The samples were dissolved with 0.1 M Tris-HCl buffer (pH ~8.0) and heated at 100?C for 5 min to denature the proteins. After cooling to room temperature, the samples were treated with trypsin and chymotypsin and incubated at 37oC overnight. The tryptic-chymotryptic digests were passed through C18 sep pak cartridges, cleaned with 5% acetic acid, and the glycopeptides/peptides were eluted subsequently in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid and 100% isopropanol. The eluates was dried initially under a stream of nitrogen gas and eventually lyophilized. The dried eluates were dissolved with sodium phosphate buffer, treated with PNGase F and incubated at 37oC for 18 hours to release N-linked glycans from the polypeptide chains. After incubation, each of the enzyme (PNGase F) digests was passed through a C18 sep pak cartridge and the N-linked glycans were eluted with 5% acetic acid into screw-cap tubes. The eluates were frozen in dry ice and lyophilized. Per-O-methylation of carbohydrates The N-linked glycans were permethylated for structural characterization by mass spectrometry (Anumula and Taylor, 1992). The dried eluates were dissolved with dimethylsulfoxide and methylated with NaOH and methyl iodide. The reaction was quenched with water and per-O-methylated carbohydrates were extracted with methylene chloride and dried under N2. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) The permethylated 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-MS using Bruker microflex .
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