N-LINKED OLIGOSACCHARIDE PROFILING OF TWO SAMPLES

University Of Georgia, Athens GA

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Abstract

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. Methods: First of all, each sample (original one and modified one) was cleaned up by Acetone precipitation. Then each sample was denatured and treated with Trypsin and PNGase A. The released N-glycans were permethylated and analyzed by MALDI/TOF-MS. The procedures are shown in detail below. Removal of contaminants by Acetone precipitation Acetone:Water (4:1) was added to the dried sample. The sample solution was placed on ice for 15 minutes and then spun at 3000 rpm in a refrigerated centrifuge for 15 minutes to pellet the protein. The supernatant was removed. The preceding washing steps were repeated twice. Release of N-linked glycans The dried sample was dissolved in 0.1 M Tris-HCl buffer, pH 8.2 containing 0.01 M CaCl2. The sample then was denatured by heating for 5 minutes at 100[unreadable]C. After cooling, the sample was digested with the trypsin (37oC, overnight). The sample was then heated at 100[unreadable] C for 5 min to inactivate trypsin and spun at 3000 rpm in a refrigerated centrifuge for 15 minutes. The supernatant was collected and dried. The sample was then passed through a C18 sep-pak cartridge and washed with 5% acetic acid to remove contaminants (salts, free sugar, etc.). Peptides and glycopeptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol and dried in a speed vacuum concentrator. The dried samples were combined and incubated with PNGase A at 37[unreadable] C overnight to release N-glycans. After digestion, the sample was passed through a C18 sep-pak cartridge and the carbohydrate fraction was eluted with 5% acetic acid and dried by lyophilization. Preparation of the per-O-methylated carbohydrates The carbohydrate fraction was dissolved in dimethylsulfoxide and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992). 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 and profiled 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 [unreadable]-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol:water) as a matrix. The spectrum was obtained by using a Microflex LRF (Bruker).