Hydrophilic Interaction Anion Exchange HPLC

Hydrophilic interaction anion exchange (HIAX) HPLC was performed with an IonPac AS11 column (Dionex, Sunnyvale, USA; 4 × 250 mm, combined with a 4 × 50mm guard column) on a Shimadzu Nexera UPLC system as described previously 12 (link),13 (link). A two solvent gradient with buffer A (0.8 M ammonium acetate, pH 3) and buffer B (80% acetonitrile; LC-MS grade) 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. A pool of pyridylaminated (PA) oligomannosidic N-glycans from white beans (containing Man_3-9GlcNAc₂) was used to calibrate the column and test efficiency of separation. For RP-HPLC isomeric/isobaric comparison with wild-type glycans, an Ascentis Express RP-amide column (150 x 4.6 mm, 2.7 µm; Sigma Aldrich, calibrated in terms of glucose units with an oligoglucose standard) was employed with a gradient of 30% (v/v) methanol (buffer B) in 100 mM ammonium acetate, pH 4 (buffer A) being applied at a flow rate of 0.8 ml/min as follows: 0-4 min, 0% B; 4-14 min, 0-5% B; 14-24 min, 5-15% B; 24-34 min, 15-35% B; 34-35 min, return to starting conditions 20 (link). The fluorescence was measured at 320 nm (excitation) and 400 nm (emission) with a Shimadzu RF-20A XS detector.