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. - elimination, Desalting, Borate removalO-linked carbohydrate fractions were cleaved from the glycoprotein by -elimination procedures. Briefly, 250 L of 50 mM NaOH were added to each of the samples 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 were added to the samples, vortexed, and incubated overnight at 450C. The incubated samples then were neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8 100, Sigma Aldrich) and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas before permethylation.Preparation of the per-O-methylated carbohydrates, cleaning up by C18The 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). ElectroSpray Ionization Linear Ion Trap mass Spectrometry (ESI-LCQ/MSn)Mass analysis by LCQ-MS (Thermo Finnigan) was performed by direct infusion of permethylated glycans dissolved in 1 mM NaOH in 50% methanol ( 5 pmol/ L) into the LCQ instrument at a constant flow rate of 1 L/min via a syringe pump (Harvard Apparatus). The capillary temperature was set to 200oC and MS analysis was performed in positive ion mode. The collision energy was set at 35% for fragmentation in MS/MS.
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