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. First of all, the sample (~3.5mg) was dialyzed with deionized water at 4 oC to exchange buffer. The sample were then dried and dissolved in 50mM Sodium Citrate buffer (pH 6.0) and Neuroraminidase digestion was performed. After Neuraminidase digestion, N-linked profiling was performed using a little aliquot (~225ug) to confirm complete exoglycosidase digestion. The remainder was dried and stored at 4 oC and then sent back to your lab. The procedures are shown in detail below. Neuroraminidase digestion The sample (~3.5mg) was dialyzed with a 7.5kDa cut-off membrane (Milipore) using nanopure water at 4 oC overnight to remove salts and then dried down in a Speed Vac. The sample was reconstituted with 50mM Sodium Citrate buffer (pH 6.0) and Neuroraminidase (New England Biolabs) is added to the sample and incubated at 37 ?C overnight. N-linked Glycan analysis Acetone:Water (4:1) was added to the dried sample. The sample solution was placed on ice for 15 minutes and then spun at maximum speed in a refrigerated microcentrifuge for 15 minutes to pellet the protein. The supernatant was removed. The sample was washed twice. The sample was resuspended in 20mM Sodium phosphate buffer (pH 7.5) / 0.05%SDS, 50mM ?-mercaptoethanol. The sample was then denatured by heating for 5 minutes at 100o C. After cooling, 10% (v/v) of 1M KCl was added to the solution and the sample tube was placed on ice for 15 minutes followed by centrifugation at maximum speed in a refrigerated microcentrifuge for 10 minutes to pellet the potassium salts of SDS. Supernatant was transferred into a fresh tube and then PNGase F (New England BioLabs) was added to the sample and then incubated at 37 oC overnight. After enzymatic digestion, the sample was passed through a C18 reversed phase cartridge and the carbohydrate fraction (N-linked glycan) was eluted with 5% acetic acid and dried by lyophilization. The N-linked glycans thus obtained were permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and profiled by MALDI-TOF/MS. MALDI/TOF-MS was performed in the reflector positive ion mode using ?-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol:water) as a matrix. The spectrum was obtained by using a 4700 Proteomics analyzer (Applied Biosystems).
