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. UDP-glucose we received and Glucose 1 phosphate we ordered were permethyrated and profiled by NSI-MS as standards for the glycogen study. 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 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.
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