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 glycansThe samples (5mg as glycol protein amount of each) were dried and resuspended in 65 l of nanopure H2O and then 20 ul of 100 mM Sodium phosphate buffer (pH 7.5) and 5ul of denaturing buffer (1% SDS and 1M -mercaptoethanol in Nanopure H2O) were added. The samples then were denatured by heating for 5 minutes at 100 C. After cooling, 10 ul 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 minutes and then were spun at maximum speed in a refrigerated microcentrifuge for 10 minutes to pellet the potassium salts of SDS. Ninety microliters of supernatant of each sample were transferred into fresh tube and then 5ul of PNGase F (New English BioLabs) were added to each sample solution, 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 was eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and then 100% iso-propanol. The carbohydrate fraction was dried by lyophilization, whereas the peptide fractions were dried in a Speed Vacuum Concentrator and then were combined into one tube. Preparation of the per-O-methylated carbohydrates, cleaning up by C18The lyophilized carbohydrate fraction 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 loaded into a C18 sep pak cartridge and then washed with nanopure water and 15% acetonitrile. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and were dissolved with methanol prior to analysis by mass spectrometry.Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF)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. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems).
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