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. First of all, the sample (1.2 mg as a glycoprotein amount) was cleaned up by dialysis and acetone precipitation to remove sucrose and PS-80. Then the sample was denatured and then treated with Trypsin and PNGase F to release N- glycans from a glycoprotein. Released N-glycans were separated from residual peptides/O-glycopeptides by C18 sep-pak column. An aliquot of the released N-glycans thus obtained (300 ug as a glycoprotein amount) were permethylated and analyzed by MALDI/TOF-MS. The reminder was used for HPAEC profiling. The 150 ug (as a glycoprotein amount) was injected and profiled per an injection and the analysis was repeated three times. The procedures are shown in detail below. Removal of sucrose by Dialysis The sample was dialyzed against de-ionized water overnight to remove sucrose. Tube-O-DIALYZERTM (7.5 kDa cut-off, large tube, CHEMICON international) was used for dialysis. After dialysis, the sample solution was dried in the speed vacuum concentrator. Removal of detergents by Acetone precipitation Cold acetone was added to the dried sample. The sample solution was placed on ice for 15 minutes and then spun at maximum speed in a refrigerated microcentrifuge for 15 minutes to pellet the protein. The supernatant was removed. The preceding washing steps were repeated twice. Finally, the pellet was dried in the speed vacuum concentrator. Release of N-linked glycans The 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 carbohydrates The lyophilized carbohydrate fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide. 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). N-linked oligosaccharide profiling by HPAEC The released N-glycans were resuspended in nanopure water and transferred to an injection vial and 40 ul of the aliquot was injected per a run. For the reference of the retention time of each N-glycan component, the N-glycans of fetuin and a glycoprotein, which we already know the N-glycan components of each peak in the HPAEC chromatogram, were released in the same manner and at the same time. The standards thus obtained were profiled just before sample analysis in the same condition. The profiling was performed by 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 separation was performed by a Dionex CarboPac PA100 analytical column. The gradient programs used eluents A, degassed nanopure water;B, 1M sodium hydroxide and C, 1M sodium acetate. Instrument control was accomplished using Dionex PeakNet software, version 5.01.
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