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: The 16 fractions we received (PR080409B) and the left-over sample, which we received and analyzed previously were combined based on HPLC chromatogram and it resulted in five fractions as follows; Fraction 1: A and B from the 1g glycogen and 600mg glycogen (PR090409) The first batch A and B (PR070709) Fraction 2: 1g glycogen C, 600mg glycogen C,D (PR080409) The first batch C and D (PR070709) Fraction 3: 1g glycogen D,E, 600mg glycogen E (PR080409) E from the first batch (PR070709) Fraction 4: F from the 1g glycogen and 600mg glycogen (PR080409) F from the first batch (PR070709) Fraction 5: G and H from the 1g glycogen and 600mg glycogen (PR080409) G, H from the first batch (PR070709) The five fractions were permethylated and profiled by mass spectrometry. The detailed procedures used for your sample analysis are described below. 1) Preparation of the per-O-methylated carbohydrates The sample was permethylated prior to mass spec 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. Permethylated sample was dried under a stream of nitrogen gas and dissolved in methanol for mass spec analysis. 2) Mass spectrometry MALDI/TOF-MS analysis 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). NSI-MSn analysis was performed by using on a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. Permethylated sample was dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument at a constant flow rate of 0.5 ?L/ min. The capillary temperature was set at 210oC and MS analysis was performed in the positive ion mode. For total ion mapping, automated MS/MS analysis, m/z range, 150 to 2000 was scanned with ITMS mode in successive 2.8 mass unit windows that overlapped the preceding window by 2 mass units.
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