This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sample cleaning by washing with acetone:water The sample was cleaned with acetone:water (4:1) two times and 100% acetone once. The preceding cleaning step was performed by placing the tube of sample solution in ice for 15 min, centrifugation at 4oC for 15 min and removal of supernatant. The resulting pellet was quick-dried in a vacuum dessicator. Release of O-linked glycans by ?-elimination The sample was subjected directly to ?-elimination procedures to cleave the O-linked glycans from the glycoprotein. 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 were added to the sample, vortexed, and incubated overnight at 450C. The incubated sample then was neutralized with 10% acetic acid, desalted by passing through a packed column of Dowex resins, and lyophilized. After lyophilization, the dried sample was cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. Released O-linked oligosaccharides were recovered by passing the sample through a C18 sep pak cartridge. An aliquot of the released O-linked glycans fraction (to provide ~200 ?g of the original sample) was allocated for monosaccharide composition analysis. Preparation and purification of per-O-methylated carbohydrates The released O-linked glycans from the sample was dissolved in dimethyl sulfoxide 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. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and redissolved with methanol prior to analysis by MALDI-TOF. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) MALDI-MS was performed in the positive ion mode using ?-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) as a matrix. Full mass spectrum of the sample was obtained by using a 4700 Proteomics analyzer (Applied Biosystems). Monosaccharide composition analysis of O-Linked Oligosaccharides The O-linked glycan aliquot intended for monosaccharide composition analysis was hydrolyzed with 400 ?L of 2.5 N trifluoroacetic acid (TFA) at 100?C for 4 h. The hydrolysate was dried under a stream of nitrogen gas, resuspended in H2O, sonicated for 7 min in ice and transferred to an injection vial. A mix of standards for neutral and amino sugars with a known number of moles was hydrolyzed in the same manner and at the same time as the sample. Four concentration of standard mix (0.5, 1.0, 2.0, and 4.0 nmoles per injection) were prepared to establish a calibration equation. The number of moles of each residue in the sample was quantified by linear interpolation from the calibration equation. The monosaccharides were analyzed by HPAEC using a Dionex DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector, and a Thermo-Separation AS3500 autosampler containing a stainless steel needle. The sugars were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used eluents A - degassed nanopure water, B - 200 mM NaOH, and C - 100 mM NaOH. Injections were made every 40 minutes. All methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., """"""""High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates"""""""", 1994, Methods Enzymol. 230: 208-225). Instrument control and data acquisition were accomplished using Dionex PeakNet software, version 5.01.
Showing the most recent 10 out of 104 publications
