Separation and Identification of PA-Labeled Glycans

Separation of PA-labelled glycans was carried out on a Shimadzu HPLC system equipped with a fluorescence detector (RF 20 AXS; excitation at 320 nm and emission at 400 nm). For RP-HPLC, a Hypersil ODS column (Agilent; a C18 column of the dimensions 250 × 4.6 mm) was used with 100 mM ammonium acetate, pH 4.0 (buffer A) and 30% (v/v) methanol (buffer B); after 5 minutes at 0% buffer B, a gradient of increasing buffer B (0.5% per minute) was programmed for 30 minutes. A pyridylaminated partial dextran hydrolysate was used for calibration in terms of glucose units; also commercial Man_5,9GlcNAc₂ (Takara) and previously-defined Man₈GlcNAc₂A and B isomers were also chromatographed to verify elution times on the shallower gradient employed in this study. Selected RP-HPLC fractions were then subject to hydrophilic-interaction/anion-exchange (HIAX) using an IonPac AS11 column (Dionex) as previously described ^{(15 (link))}. Buffer A was 0.8 M ammonium acetate, pH 3 and buffer B 80 % acetonitrile. The following gradient was applied at a flow rate of 1 ml/min: 0-5 min, 99 % B; 5-50 min, 90 % B; 50-65 min, 80 % B; 65-85 min, 75 % B. The HIAX column was calibrated using a mixture of oligomannosidic glycans (Man_3,6,7,9GlcNAc₂) derived from white beans.