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: Dialysis The sample was first lyophilized but upon determination that the solution was not drying completely and upon being informed that the sample was stabilized in glycerol, dialysis was undertaken. The sample was transferred into a Tube-O-Dialyzer (4.0 kDa cut-off membrane;G BioSciences). Dialysis was performed against 4 L of nanopure water at 4oC for about 24 hours to remove glycerol and any other contaminants. Nanopure water was replaced four times during the entire dialysis period. After dialysis, the sample was lyophilized and transferred into a microcentrifuge tube for enzyme digestion. Release of N-linked glycans The sample was dissolved with 0.1 M Tris-HCl buffer (pH ~8.0) and heated at 100?C for 5 min to denature the protein. After cooling to room temperature, the sample was treated with trypsin and chymotypsin and incubated at 37oC overnight. The tryptic-chymotryptic digest was passed through a C18 sep pak cartridge, 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 eluate was dried initially under a stream of nitrogen gas to evaporate the isopropanol and lyophilized eventually. The dried eluate was 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, the enzyme (PNGase F) digest was passed through a C18 sep pak cartridge and the N-linked glycans fraction was eluted with 5% acetic acid and collected into a pre-rinsed (with methanol) screw-cap tube. The N-linked glycans eluate was 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). Briefly, the dried eluate was 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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