This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Dialysis The sample solution (~6 mL) was transferred into 3 Tube-O-Dialyzers (4.0 kDa cut-off membrane;G BioSciences). Dialysis was performed against 4 L of nanopure water at 4oC for about 24 hours to remove salts and other contaminants. Nanopure water was replaced four times during the entire dialysis period. After dialysis, the sample was lyophilized and eventually combined in one microcentrifuge tube for enzyme digestion. Release of N-linked glycans The dried sample was dissolved with 50 mM ammonium bicarbonate and the tube was placed in a 100-degree Celsius heating block for 5 min to denature the protein. After cooling to room temperature, the sample was treated with 6 ?g of trypsin and 6 ?g of chymotrypsin and incubated at 37oC for 18 hr. Trypsin and chymotrypsin were deactivated thereafter by placing the tube in a 100-degree Celsius heating block for 5 min. The tryptic/chymotryptic digest was cleaned of any contaminants by passing it through a C18 sep pak cartridge. Once loaded in the cartridge, adsorbed sample was cleaned with 5% acetic acid and the glycopeptides/peptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid, and 100% iso-propanol. The eluate was dried initially under a stream of nitrogen to evaporate the iso-propanol and lyophilized eventually. The dried enzyme digest was dissolved with 50 mM NaPO4 buffer (pH~7.5), treated with PNGase F and incubated at 37oC for 18 hr to release the N-linked glycans. At the end of the N-glycanase digestion, the sample was passed through a C18 sep pak cartridge and N-linked glycans fraction was eluted with 5% acetic acid and lyophilized. Per-O-methylation of N-linked Glycans The PNGase-F released N-linked glycans from the sample was permethylated following the methods of Anumula and Taylor (1992). Briefly, the dried eluate was dissolved with dimethylsulfoxide and then methylated with NaOH and methyl iodide. The reaction was quenched by addition of water and per-O-methylated glycans were extracted with methylene chloride. Completion of permethylation is critical for linkage analysis, hence, a small aliquot of the permethylated glycans was analyzed by MALDI-TOF-TOF MS (Applied Biosystems 4700) to evaluate the profile. Preparation of Partially Methylated Alditol Acetates (PMAAs) For the determination of glycosyl linkages, PMAAs were prepared from the released permethylated N-linked glycans. Briefly, permethylated glycans were hydrolyzed with HCl:Water:Acetic acid (0.5:1.5:8, by vol.) at 80oC for 18h, followed by reduction with NaBD4. After hydrolysis and reduction steps, the free hydroxyls of the partially methylated alditols were acetylated with acetic anhydride:pyridine (1:1, v/v) in boiling water for 15 min to produce PMAAs. Gas Chromatograph-Mass Spectrometry (GC-MS) The PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode). The separation was performed on a 30 m EC 1 bonded phase fused silica capillary column (Altech). Electron impact mass spectra were obtained under the following conditions: oven temperature, 140?C (2.0?C/min) ? 220?C (20?C/min) ? 300?C (20?C/min);detector temperature, 280 ?C;inlet temperature, 250 ?C.
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