Permethylation and Mass Spectrometry Analysis of N-Glycans

Permethylation of glycans was carried out as described previously (45 (link)). Briefly, N-glycans were permethylated by treatment with 0.2 ml of methyl iodide (Merck) in NaOH-DMSO (3 g of NaOH in 2 ml of DMSO) slurry for 15 min at 37 °C with intermittent mixing. Permethylated N-glycans were extracted in chloroform, dried under nitrogen, and purified on a Sep-Pak® Classic C₁₈ column (Waters) using 3 ml each of 10, 50, and 75% acetonitrile in water. Eluted fractions were lyophilized and redissolved in 20 μl of methanol, mixed with equal volumes of Super-DHB (2,5-dihydroxybenzoic acid; 20 mg/ml in 70% methanol), and spotted on a MALDI plate. MS and MS/MS data were acquired in positive ion mode using an AB SCIEX TOF/TOF 5800 system. Calmix (Applied Biosystems) was used as an internal standard for calibration in both modes. MS/MS collision-induced dissociation was carried out with argon gas at a voltage of 1 kV. Data were acquired using a TOF/TOF Series Explorer (AB SCIEX). Data from 10,000 shots, collected from different areas of the spot (laser intensity, 4500 for MS and 5000–6000 for MS/MS), were summed up and analyzed using Data Explorer software (AB SCIEX). The observed peaks were annotated using GlycoWorkbench software.