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. N-linked oligosaccharides were released from the samples by treatment with peptide N-glycosidase F (PNGase F, N-glycanase) using the enzyme digestion protocol supplier by the manufacturer (New England Biolabs, Beverly, MA). The samples were resuspended in 400 l d H20 and 40 l of 10X denaturing buffer (5% SDS and 10% beta-mercaptoethanol). The sample and standards were then denatured by heating for 5 minutes at 100 C. After cooling 40 l of 10X buffer and 40% NP-40 were added. The samples were mixed, and 4 l of enzyme (30U) were added. Samples were incubated overnight at 37 C. The digested samples were acidified and applied to a C18 classic SEP-PAK. This was eluted with 5 ml of 5% acetic acid to collect the N-linked sugars. The sample fractions were dried and permethylated by the methods of Ciukanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). After permethylation, the samples were extracted three times with methylene chloride/water in order to remove any impurities The resulting permethylated samples were resuspended in methanol and analyzed by MALDI-TOF. MALDI-TOF-MS was performed with a Voyager mass spectrometer operated in the positive ion mode. The mass spectrometer was calibrated with a mixture of maltooligosaccharides. Methanolic solutions of samples were diluted 1:1 with 2,5-dihydroxybenzoic acid (DHB) and a portion (1.0 l) was applied to the sample plate of the MS. Samples were desorbed from the sample plate with a nitrogen laser having a guide wire reading of 0.05% and a laser intensity of approximately 2100.
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