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: Sample Preparation The sample was dissolved with nanopure water in its original dram vial to a concentration of 1 ?g/?L. From this stock solution, an aliquot was obtained for permethylation. Permethylation of the sugar was performed according to the method of Anumula and Taylor (1992). Briefly, the aliquot was dried under a stream of N2, redissolved with dimethylsulfoxide, and methylated with NaOH and methyl iodide. The reaction was quenched with water and per-O-methylated sugar was extracted with methylene chloride and dried under N2. Analysis by LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) The dried permethylated GlcNDAz was dissolved with methanol and an aliquot was diluted with 1 mM NaOH in 50% methanol. Also, from the original dram vial, an aliquot was diluted with 1 mM NaOH in 50% methanol. Each of the prepared solutions (native and permethylated) was infused directly into an LTQ Orbitrap Discovery mass spectrometer (Thermo Scientific) at a flow rate of 0.4 ?L/min to obtain a nanospray ionization (NSI). Full mass spectrum of each sample was obtained in the positive ion mode.
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