N hydroxysuccinimide activated sepharose

For N-glycan analysis, 1–2 μg of purified BmVAL-1 was reduced and denatured for 10 min at 95 °C in PBS containing 1.3% (w/v) SDS and 0.1% (v/v) β-mercaptoethanol. SDS was neutralized by adding 2% (v/v) NP-40 prior to overnight digestion at 37°C with trypsin (Sigma-Aldrich, USA), immobilized to N-hydroxysuccinimide-activated sepharose (GE Healthcare). trypsin beads were removed from the digestion mix by centrifugation and the pH of the mix was adjusted to 5 using 1 M sodium acetate. PNGase A (0.5 mU; Roche, Switzerland) was used to release N-glycans from BmVAL-1 while incubating overnight at 37°C. The incubation mixture was applied to C18 Bakerbond^™ SPE cartridges (JT Baker, USA) and the N-glycans were extracted from the flow-through on Extract Clean^™ Carbo SPE columns. Eluted N-glycans were labeled with anthranilic acid (Sigma-Aldrich) and desalted by hydrophilic interaction chromatography on Biogel P10 (BioRad, USA). Samples in 75% acetonitrile were mixed with 1 μl of matrix solution (20 mg/ml 2,5-dihydroxybenzoic acid in 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid) and were dried under a stream of warm air. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra (MS) were obtained using an Ultraflex II mass spectrometer (Bruker Daltonics, USA).

For N-glycan analysis, 1–2 μg of purified HpVAL-4 was reduced and denatured for 10 min at 95 °C in PBS containing 1.3% (w/v) SDS and 0.1% (v/v) β-mercaptoethanol. SDS was neutralized by adding 2% (v/v) NP-40 prior to overnight digestion at 37°C with trypsin (Sigma-Aldrich, USA) immobilized to N-hydroxysuccinimide-activated sepharose (GE Healthcare). trypsin beads were removed from the digestion mix by centrifugation and the pH of the mix was adjusted to 5 using 1 M sodium acetate. PNGase A (0.5 mU; Roche, Switzerland) was used to release N-glycans from HpVAL-4 while incubating overnight at 37°C. The incubation mixture was applied to C18 Bakerbond™ SPE cartridges (JT Baker, USA) and the N-glycans were extracted from the flow-through on Extract Clean™ Carbo SPE columns. Eluted N-glycans were labeled with anthranilic acid (Sigma-Aldrich) and desalted by hydrophilic interaction chromatography on Biogel P10 (BioRad). Samples in 75% acetonitrile were mixed with 1 μl of matrix solution (20 mg/ml of 2,5-dihydroxybenzoic acid in 50% acetonitrile, 0.1% (v/v) trifluoroacetic) and were dried under a stream of warm air. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra (MS) were obtained using an Ultraflex II mass spectrometer (Bruker Daltonics, USA).