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. The gel pieces were first washed with 40mM ammonium bicarbonate buffer. After dehydration with 100% acetonitrile, the gels were reduced and carboxymethylated with 10mM Dithiothreitol and 55 mM lodoacetamide. The gels were then washed with buffer and dehydrated with 100% acetonitrile. A solution containing 400ng of trypsin was added to the gels which were rehydrated in an ice bath for 45 minutes prior to incubation overnight at 37 C. The solution was removed and the gels were extracted three times with a mixture of formic acid and acetonitrile. All solutions for each sample were combined and dried on a speed-vac. After deactivation of the trypsin enzyme, N-linked oligosaccharides were released from the trysinized peptides 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 buffer. 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 carbohydrate 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 with methylene chloride and washed three times in waster in order to remove any impurities The resulting permethylated samples were resuspended in methanol/water and analyzed by MALDI-TOF mass spectrometry.
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