O-LINKED OLIGOSACCHARIDE PROFILING AND LINKAGE ANALYSIS OF ONE SAMPLE

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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. ¿- elimination, Desalting, Borate removal, Cleaning by C18 sep-pak cartridge O-linked carbohydrate fractions were cleaved from the glycopeptides by ¿-elimination procedures. Briefly, 1M sodium borohydride (NaBH4) in 50 mM Sodiumhydroxide (NaOH) was added to the sample and incubated 17 h at 45oC. The incubated sample then was neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8  100, Sigma Aldrich) and then was lyophilized. Dried sample was cleaned of borate with methanol:acetic acid (9:1, v/v) under a stream of nitrogen gas. Then the sample was passed through a C18 reversed phase cartridge to purify the O-glycans. The carbohydrate fractions (O-linked glycans) were first eluted with 5% acetic acid and then the peptides 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 carbohydrate fraction was dried by lyophilization, whereas the peptide fractions were dried in the speed vacuum concentrator and then combined in one tube. Preparation of the per-O-methylated carbohydrates, Cleaning up by C18 sep-pak cartridge 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. Glycosyl linkage analysis For determination of sugar linkages, partially methylated alditol acectates were prepared from permethylated O-glycans. Briefly, permethylated glycans were hydrolysed with 2M TFA at 100oC for 4 h, followed by reduction with 1% NaBH4 in 30mM NaOH and acetylation with acetic anhydride/pyridine (1:1, v/v) at 100 ¿C for 15 min. The partially methylated alditol acetates thus obtained were analyzed by GC-MS. 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). Gas Chromatograph-Mass Spectrometry (GC-MS) The partially methylated alditol acetates were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode). The separations were performed on two different columns with different temperature as follows;