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 glycansOne milliliter of the sample, 6-051 was pipetted into a screw-cap tube and lyophilized. The dried sample then was dissolved in 125 L of 50 mM ammonium bicarbonate (pH 8.4), denatured at 100oC for 5 min prior to trypsin digestion (37oC, overnight). A second enzyme, peptide N-glycosidase F (New England BioLabs) was added to the tryptic digest and incubated at 37oC for 18 hours to release the N-linked glycans. After enzymatic digestions, the samples were passed through a C18 reversed phase cartridge. The N-linked glycan fraction was eluted first with 5% acetic acid and then lyophilized. The O-gycopeptide and peptide fraction was eluted in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid, and 100% isopropanol into a screw-cap tube.Release of O-linked glycans by -eliminationThe isopropanol in the O-glycopeptide and peptide eluate was evaporated under a stream of nitrogen gas and lyophilized. O-linked glycans were cleaved from the glycoprotein by -elimination procedures. Briefly, 250 L of 50 mM NaOH were added to the sample and then checked for pH. Upon determination that the pH was basic, another 250 L of 50 mM NaOH containing 19 mg of sodium borohydride was added to the sample, vortexed, and incubated overnight at 450C. The incubated sample then was neutralized with 10% acetic acid and desalted by passing through a packed column of Dowex resins and lyophilized. The dried sample was cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas before permethylation.Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridgeThe lyophilized N-linked and O-linked glycans were permethylated following the procedures of Ciucanu and Kerek (1984). Briefly, the samples were dissolved in dimethylsulfoxide and then methylated with NaOH and iodomethane (Ciucanu and Kerek, 1984). The reaction was quenched by the addition of water and per-O-methylated carbohydrates were extracted with dichloromethane.The per- O-methylated glycans were cleaned further 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 then 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 both N-linked and O-linked glycans was performed initially 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.Direct infusion of oligosaccharidesThe profile of glycan structures from both samples were confirmed by electrospray ionization mass spectrometry (ESI-MS) using an LCQ-MS (Thermo Finnigan) quadropole ion trap. Each sample (~5 pmol/ L) was infused directly into the instrument at a constant flow rate of 1 L/min via a syringe pump (Harvard Apparatus) and sprayed at 3.5 kV. A normalized collision energy of 35 and an isolation mass window of 2.2 Da was applied to obtain MSn.
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