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.In-gel digestion, glycopeptide extraction from the gel The gels first were cut into smaller pieces and then subjected to repeated washing steps by soaking in 40mM ammonium bicarbonate (NH4HCO3) for 10 min followed by dehydration with 100% acetonitrile for 10 min until the color turned clear. After destaining, the gel pieces were subjected to a reduction reaction using 10 mM dithiothreitol in 40mM NH4HCO3 at 55 C for 1 hr followed by carboxyamidomethylation using 55 mM iodoacetamide (IAM) in the dark at room temperature for 45 min. After reduction-carboxyamidomethylation, the mixture was washed as described previously (40 mM NH4HCO3-100%acetonitrile) twice. The gel pieces then were re-swollen on ice with trypsin solution (10 L of trypsin in 1ml of 40mM Ambic) for 45 min and incubated overnight at 37 C. The supernatant were transferred to a new tube and then the peptides and the glycopeptides were extracted from the gel pieces with 20% acetonitrile in 5% formic acid, 50% acetonitrile in 5% formic acid and then 80% acetonitrile in 5% formic acid. The extracts were dried and then reconstituted with nanopure water and composited into one tube eventually.Cleaning up by C18, de-activation of trypsin, PNGase F treatment, and fractionation by C18 The glycopeptide/peptide extract 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 of any possible contaminant (salts, SDS, etc.) with 5% acetic acid. Glycopeptides and peptides then were eluted in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid and 100% isopropanol each into a microcentrifuge tube. The propanol fractions were dried, dissolved in H2O and composited in one tube, and dried on a speed vac. The dried sample was reconstituted with 50mM sodium phosphate buffer (pH 7.5) and heated at 100 C for 5 min to de-activate the trypsin. After cooling to room temperature, peptide N-glycosidase F (New England BioLabs) was added and incubated at 37oC overnight. Peptide N-glycosidase F (PNGase F) was deactivated at 100 C for 5 min and then dried on a speed vac. The dried tryptic-PNGase F digest was dissolved in 50 L citrate-phosphate buffer (pH ~5.5), treated with PNGase A, and incubated at 37oC overnight. After enzymatic digestions, the sample was passed through a C18 sep-pak cartridge to separate the N-linked glycans and O-linked glycopeptide and peptide fractions. The carbohydrate fraction (N-linked glycans) was eluted first with 5% acetic acid and then the O-linked glycopeptides and peptides were eluted with 100% 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 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 permethylated 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)Profiling of N-linked glycans was performed initially using MALDI/TOF-MS. 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.ElectroSpray Ionization Linear Ion Trap mass Spectrometry (ESI-LTQ/MSn)Mass analysis by LTQ-MS (Thermo Finnigan) was performed by direct infusion of permethylated glycans dissolved in 1 mM NaOH in 50% methanol (5 pmol/ L) into the LTQ instrument at a constant flow rate of 0.4 L/min. The capillary temperature was set to 210oC and MS analysis was performed in the positive ion mode. The collision energy was set at 28% to obtain fragmentation ions.
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