Isolated mucins were sent to the Complex Carbohydrate Research Center (Athens, GA) for analysis. Mucin samples were further dialyzed (3 kDa MWCO; G-Biosciences, 786611) and freeze-dried before treatment with 1.0 M sodium borohydride (NaBH4; ACROS,USA, 20005–1000) in a 50 mM NaOH solution (JT Baker, 3727–01). Samples were then heated to 45°C for 18 h to release O-glycans, followed by neutralization by 10% acetic acid (JT Baker, 9508–03), passed through a hydrogen foam resin column (AG 50w-x2 H+ resin, Bio-Rad, 142–1241) and lyophilized. Excess borate crystals were removed under a stream of nitrogen.
Dried glycans were derivatized (permethylated) for structural characterization by mass spectrometry as described previously 40 (link), 42 (link), 63 (link). Briefly, reduced glycans were dissolved in 0.2 mL dimethyl sulfoxide (DMSO; Thermo Fisher, D128–4) and methylated using 0.1 mL methyl iodide (Sigma- Aldrich, 289566) under alkaline conditions in DMSO/NaOH (50 % vol/vol). After vigorous mixing for 10 min, the reaction was quenched with water, extracted with 2 mL dichloromethane (DCM; Sigma-Aldrich, 650463) and excess methyl iodide was removed by sparging with nitrogen. Permethylated glycans were dissolved in 20 μL of methanol (Sigma-Aldrich, 900688) and stored at −20°C until further use.
Dried glycans were derivatized (permethylated) for structural characterization by mass spectrometry as described previously 40 (link), 42 (link), 63 (link). Briefly, reduced glycans were dissolved in 0.2 mL dimethyl sulfoxide (DMSO; Thermo Fisher, D128–4) and methylated using 0.1 mL methyl iodide (Sigma- Aldrich, 289566) under alkaline conditions in DMSO/NaOH (50 % vol/vol). After vigorous mixing for 10 min, the reaction was quenched with water, extracted with 2 mL dichloromethane (DCM; Sigma-Aldrich, 650463) and excess methyl iodide was removed by sparging with nitrogen. Permethylated glycans were dissolved in 20 μL of methanol (Sigma-Aldrich, 900688) and stored at −20°C until further use.