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. Removal of contaminants by Acetone precipitation Acetone:water (4:1) was added to the samples (Als3 QCS #19186 and Als3 Pre-Q Absorber). The samples were placed on ice for 15 minutes and then centrifuged at 4 ?C for 15 minutes to pellet the protein. The supernatant was removed. Cold acetone:water (4:1) was added to the samples again and re-centrifuged. Furthermore, the pellets were washed with 100% Acetone. Finally, the pellets were dried down under a nitrogen stream. ?-Elimination, Desalting, Borate removal O-linked carbohydrates were cleaved from the glycopeptides by ?-elimination procedures. Briefly, 250 ?L of 50 mM NaOH were added to the samples (~100 ?g) 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 and voltexed and incubated overnight at 45 ?C. The incubated samples then were neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8 ?100, Sigma Aldrich) and then were lyophilized. Dried sample was cleaned of borate with methanol: acetic acid (9:1) solution under a stream of nitrogen gas. The samples were then passed through a C18 reversed phase cartridge to separate the O-linked glycans from the peptides. The O-linked glycan fraction of the samples were eluted with 5% acetic acid and then lyophilized. Monosaccharide composition of O-linked glycans We prepared two sets of each samples to check evidence of beta-glucans in the samples. A set is O-linked carbohydrates were cleaved from the glycopeptides by ?-elimination procedures without acetone precipitation and another set is with acetone precipitation. The monosaccharide composition of O-linked glycans of Als3 QCS #19186 and Als3 Pre-Q Absorber were analyzed by GC-MS. Methyl glycosides were prepared from a dried sample by methanolysis with 1 M HCl in methanol at 80?C (18 h), followed by re-N-acetylation with pyridine and acetic anhydride in methanol for detection of amino sugars. The samples then were O-per-trimethylsilylated (TMS) with Tri-Sil (Pierce) at 80?C. These procedures were carried out as described previously in Merkle and Poppe (1994);York, et al. (1985). GC/MS analysis of the TMS methyl glycosides was performed on a Hewlett Packard 5890 series ??GC interfaced to a 5970 mass selective detector (MSD), electron impact ionization mode at 70 eV, and ions were scanned from 50 to 550 m/z, using a 0.25 mm ? 30 m fused silica capillary column (EC1, Alltech Associates, Deerfield, IL). The carrier gas was Helium. GC was started at 80?C and held for 2 min and then increased temperature to 160?C at a rate of 20?C/min and held for 2 min;to 200?C at a rate of 2?C/min;to 250?C at a rate of 5?C/min. GC was held at the final temperature for 11 min. Preparation of the per-O-methylated carbohydrates The lyophilized carbohydrate fractions were dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Analytical Biochemistry 203, 101-108 (1992). The reaction was quenched by addition of water, and O- per-methylated carbohydrates were extracted with dichloromethane. The organic phase was concentrated to dryness and then the glycans were passed through a C18 Sep-Pak, eluted with 85 % acetonitrile, dried under a stream of nitrogen, and dissolved in methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) Profiling of N-linked and O-linked glycans was performed initially using MALDI/TOF-MS on a 4700 Proteomics analyzer (Applied Biosystems). Permethylated glycans (~1 ?L) were crystallized on a MALDI plate with 1 ?L of 2, 3-dihydroxybenzoic acid (DHBA, 20 mg/mL solution in 50 % methanol: water) as matrix. All spectra were acquired in the reflector positive ion mode and averaged spectra of 50 laser shots.
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