The 2DE gel spots were excised and destained using 40% methanol 7% acetic acid. The gel plugs were dehydrated with acetonitrile, the acetonitrile was removed and 25mM DTT was allowed to absorb into the plug. The plug was heated to 100°C for 5 min, allowed to cool, and alkylated in the dark for 30 min with 75mM iodoacetamide. The gel plugs were washed using two repeats of acetonitrile dehydration and 20 mM ammonium bicarbonate rehydration. After a final dehydration, the gel plugs were dried in a speed vac. Peptide∶N-glycosidase F (PNGase F) was diluted with 20 mM ammonium bicarbonate pH 7 and allowed to adsorb into the gel plug. The gel plug was then covered with the same solution and allowed to incubate overnight at 37°C. The glycans were eluted from the gel plug by sonication in Milli-Q water three times; the elutant was pooled, dried down, and labeled with a 2AB dye (Ludger, Oxford, UK) according to the manufacturer's instructions. The glycans were then cleaned up using paper chromatography and filtered using a 0.22-µm syringe filter. Fluorescently labeled glycans were subsequently analyzed using the Waters Alliance high-performance liquid chromatography system with a normal phase column (TSK amide 80 columns) complemented with a Waters fluorescence detector and quantified using the Millennium Chromatography Manager (Waters Corporation, Milford, MA). The mobile phase consisted of solvent A (50 mM ammonium formate, pH 4.4) and solvent B (acetonitrile). The gradient used was as follows: linear gradient from 20% to 58% solvent A at 0.4 mL/minute for 152 min followed by a linear gradient from 58% to 100% solvent A for the next 3 min. The flow rate was increased to 1.0 mL/minute; the column was washed in 100% solvent A for 5 min. Following the wash step, the column was equilibrated in 20% solvent A for 22 min in preparation for the next sample. Glycan structures were identified by calculating the glucose uptake value and exoglycosidase digestion, as described previously [21] (link).
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