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 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 protease digest was passed through a C18 sep pak cartridge, cleaned with 5% acetic acid, and glycopeptides/peptides were eluted subsequently in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid and 100% isopropanol. The glycopeptide eluate was dried initially under a stream of nitrogen gas to evaporate the isopropanol and eventually lyophilized. The dried glycopeptides eluate was dissolved with sodium phosphate buffer, treated with PNGase F and incubated at 37oC overnight. After incubation, the enzyme (PNGase F) digest was passed through a C18 sep pak cartridge and the N-linked glycans fraction was eluted into a tube with 5% acetic acid, 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 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. Oligosaccharide Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF 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-TOF-MS using AB SCIEX TOF/TOF 5800 (Applied Biosystem MDS Analytical Technologies)
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