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 dried sample was dissolved in Trypsin buffer (0.1M Tris-HCl, pH 8.2 containing 0.01M CaCl2). The sample then was denatured by heating for 5 minutes at 100 C. After cooling, the sample was digested with the trypsin (37oC, overnight). After tryptic digestion, the sample was heated at 100 C for 5 minutes to de-activate the trypsin. After cooling to room temperature, the sample was applied to a C18 sep-pak cartridge. Before elution of glycopeptides and peptides, the sample adsorbed in the C18 sep-pak cartridge was cleaned with 5% acetic acid to remove any possible contaminants (salts, etc.). Peptides and glycopeptides then were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol each into a microcentrifuge tube. The propanol fractions were dried and then combined in one tube, and then reconstituted with 50mM sodium phosphate buffer (pH 7.5), and then the N-glycans were released using PNGase F (New England BioLabs). After digestion, the sample was passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycan) was first eluted with 5% acetic acid and then the O-linked glycopeptides and peptides were eluted with 2-PrOH. The carbohydrate fraction was dried by lyophilization, whereas the other fraction was dried under a stream of air at low temperature.Preparation of the per-O-methylated carbohydratesThe 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. The spectrum was obtained by using a 4700 Proteomics analyzer (Applied Biosystems).
Showing the most recent 10 out of 104 publications
