High-performance liquid chromatography (HPLC) and MALDI-TOF MS analyses of 2-aminobenzamide (2-AB)-labeled N-glycans released from RNase B with or without EfMan-I treatment

2-AB solution (A) was prepared by adding 88 mg of 2-AB to 1 ml of DMSO/AcOH (7:3 by volume). The solution was stored at −20°C. Sodium cyanoborohydride solution (B) was freshly prepared by adding 64 mg of NaCNBH₃ to 1 ml of a solution mixture of DMSO/AcOH (7:3 by volume). To a dried free reducing glycan sample (0.01 to 1 mg), solution A (5 to 50 μl) and an equal volume of solution B were added. The mixture was heated at 65°C for 2 h. A 10-fold volume of acetonitrile was then added to precipitate the glycan-2AB conjugates. The mixture was cooled down at −20°C for 1 h and centrifuged at 13000 rpm for 2 min. The precipitate was collected and dissolved in H₂O (100–1000 μl). The obtained mixture was centrifuged at 13000 rpm for 2 min. The supernatant was collected, diluted, and the glycan-2AB conjugates were analyzed using high performance liquid chromatography (HPLC) with an AdvanceBio Glycan Map column (Agilent) using a slow gradient of Solvent A (ammonium formate, 100 mM, pH 4.5) and Solvent B (acetonitrile) and monitoring at A_{254 nm}.