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: For MALDI analysis, an aliquot (~2ul) of each sample was analyzed following the same method used for previous samples (PR010510Z-A). For glycosyl linkage analysis, 100 ul of sample B, those have been treated with and without laforin, were permethylated. An aliquot of the permethylated sample (~5%) was analyzed by MALDI and ESI. The rest of the permethylated materials were hydrolyzed, reduced and acetylated. The partially methylated alditol acetates thus obtained were profiled by GC-MS. Detailed procedures used for your sample analysis are described below. Glycosyl linkage analysis 1) Preparation of the per-O-methylated carbohydrates The sample was permethylated for the glycosyl linkage analysis. Briefly, the sample was dissolved in dimethylsulfoxide and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and the reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. An aliquot of permethylated glycans were examined by MALDI and ESI for confirming complete permethylation. 2) Preparation of partially methylated alditol acectates For determination of sugar linkages, partially methylated alditol acectates were prepared from fully permethylated glycans. Briefly, permethylated glycans were hydrolysed with HCl/water/acetic acid (0.5:1.5:8, by vol.) at 80oC for 18 h, followed by reduction with 1% NaBD4 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. MALDI/TOF-MS analysis For the analyses in negative ion mode, 22,42,62-Trihydroxyacetophenone monohydrate (THAP, 0.5M 2,4,6 THAP in ethanol : 0.1M diammonium hydrogen citrate in water = 2:1 (v/v)) were used as a matrix, whereas, ?-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol: water) were used for the analyses in positive ion mode. For the analysis of intact material, the matrix solution (1ul) was deposited first on the target, then an equal volume of the sample deposited. For the analysis of permethylated materials, the sample solution and an equal amount of matrix were mixed together then 1 uL of the mixture was deposited. All spectra was obtained by using a Microflex LRF (Bruker). MALDI/TOF-MS analysis was performed in the reflector positive or negative ion mode. NSI-MSn analysis Mass analysis was determined by using a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source (NSI-source). Permethylated glycans were dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument at a constant flow rate of 0.5 ?L/ min. A full FTMS spectrum was collected at 30 000 resolution. The capillary temperature was set at 210oC and MS analysis was performed in the positive ion mode. MSn analysis was performed with ITMS mode with 2.2 isolation width and the collision energy was set at 40. For total ion mapping (automated MS/MS analysis), m/z range, 300 to 2000 was scanned with ITMS mode in successive 2.8 mass unit windows that overlapped the preceding window by 2 mass units and the collision energy was set at 40. Gas Chromatograph-Mass Spectrometry (GC-MS) The glycosyl linkage analysis was performed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode). The separation of the partially methylated alditol acetates (glycosyl linkage analysis) was performed on a 30m EC1 bonded phase fused silica capillary column (Alletech, Deerfield, IL) using a temperature program of 80 oC (2 min)?180 oC (20 oC/min)?240 oC (4 oC/min). The detector temperature and the inlet temperature were set at 280 oC and 250 oC, respectively.
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