This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Release of N-linked glycans The samples were transferred to microcentrifuge tubes and dried in a vacuum centrifuge. The dried samples were resuspended in 65 L of nanopure H2O and then 20 L of 100 mM sodium phosphate buffer (pH 7.5) and 5 L of denaturing buffer (1% SDS and 1 M -mercaptoethanol in nanopure H2O) were added. Denaturation of protein was accomplished by heating for 5 min at 100oC. After cooling, 10 L of 1M KCl in nanopure H2O was added to each sample solution and then was mixed by vortexing the tube. The sample tubes were placed on ice for 15 min and then were spun at maximum speed in a refrigerated microcentrifuge for 10 min to pellet the potassium salts of SDS. Ninety microliters of supernatant of each sample were transferred into another tube and then 5 L of PNGase F (New England BioLabs) were added to each sample solution (supernatant), mixed and then incubated at 37oC overnight. After enzymatic digestion, the sample was passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycan) was eluted first with 5% acetic acid, and then the O-linked glycopeptide and peptides fraction was eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and then 100% iso-propanol. Since both samples were less than 5 mg (4611-154A, 1.5 mg; 4611-154B, 3.1 mg), about 60% of the N-linked fraction was allocated for permethylation. The carbohydrate fractions were dried by lyophilization, whereas the peptide fractions were dried in a vacuum centrifuge and then were combined into one microcentrifuge tube.Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridgeThe lyophilized eluate intended for N-linked oligosaccharide profiling was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Ciucanu and Kerek, 1984). The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were dissolved in 1:1 methanol:water and loaded into a C18 sep pak cartridge and then washed with nanopure water. Per-O-methyl carbohydrates were eluted with 15% acetonitrile into a screw-cap tube, and with 85% acetonitrile into another screw-cap tube. The glycans eluted with 85% acetonitrile were dried under a stream of nitrogen gas and were dissolved with methanol for analysis by mass spectrometry.Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF)Profiling of N-linked glycans was performed by MALDI/TOF mass spectrometry. The machine used was a 4700 Proteomics analyzer (Applied Biosystems), which was set in the reflector positive ion mode. Permethylated glycans were crystallized on a MALDI plate with -dihydroxybenzoic acid (DHBA, 20 mg/mL solution in 50% methanol:water) as a matrix.
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