The released N-glycans were labelled with 2-aminobenzamide (2-AB). The labelling mixture consisted of 2-AB (19.2 mg/ml; Sigma Aldrich, USA) and 2-picoline borane (44.8 mg/ml; Sigma Aldrich, USA) in dimethyl sulfoxide (Sigma Aldrich, USA) and glacial acetic acid (Merck, Germany) mixture (70:30 v/v). To each sample, 25 μl of labelling mixture was added, followed by 2 h incubation at 65 °C. Free label and reducing agent were removed from the samples using hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE). After incubation samples were brought to 96% of acetonitrile (ACN) by adding 700 μl of ACN (J.T. Baker, USA) and applied to each well of a 0.2 μm GHP filter plate (Pall Corporation, USA). Solvent was removed by application of vacuum using a vacuum manifold (Millipore Corporation, USA). All wells were prewashed with 70% ethanol (Sigma-Aldrich, St. Louis, MO, USA) and water, followed by equilibration with 96% ACN. Loaded samples were subsequently washed 5× with 96% ACN. N-glycans were eluted with water and stored at −20 °C until usage.

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