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: NMR Spectroscopy The sample was deuterium exchanged by dissolving in D2O and lyophilization. The sample was dissolved in 80 ?L D2O and placed in a 3 mm Shigemi NMR tube. 1-D Proton, TOCSY and NOESY NMR spectra, run with water presaturation, and gradient enhanced COSY and HSQC spectra were acquired on a Varian Inova-800 MHz spectrometer at 298 K (25 ?C). Chemical shifts were measured relative to internal acetone (?H=2.218 ppm, ?C=33.00 ppm). Permethylation For glycosyl linkage analysis, the sample was permethylated, depolymerized, reduced, and acetylated;and the resultant partially methylated alditol acetates (PMAAs) analyzed by gas chromatography-mass spectrometry (GC-MS) as described by York et al (1985) Methods Enzymol. 118:3-40. Initially, an aliquot of the sample was freeze-dried and suspended in about 200 ?l of dimethyl sulfoxide. The sample was then permethylated by the method of Ciukanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). The sample was subjected to the NaOH base for 10 minutes then methyl iodide was added and left for 40 minutes. The base was then added and left for 10 minutes and more methyl iodided was added and left for 40 minutes. The addition of more methyl iodide and NaOH base were to insure complete methylation of the polymer. Linkage analysis Following sample workup, the permethylated material was hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121?C), reduced with NaBD4, and acetylated using acetic anhydride/pyridine. The resulting PMAAs were analyzed on an Agilent 7890A GC interfaced to a 5975C MSD (mass selective detector, electron impact ionization mode);separation was performed on a 30 m EC-1 bonded phase fused silica capillary column. ESI Mass spectrometry The permethylated sample was analyzed by direct infusion in 50 % MeOH solution on a ThermoFisher LTQ. Experimental details are available upon request.
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