Glycan release and labeling of Korčula and Vis IgG samples was performed essentially as described by Royle and coworkers (46 (link)). Briefly, IgG was immobilized in a block of sodium dodecyl sulfate–polyacrylamide gel and N-glycans were released by digestion with PNGase F (ProZyme, Hayward, CA). Each step was done in a 96-well microtiter plate to achieve the best throughput of sample preparation. After deglycosylation, N-glycans were labeled with 2-AB fluorescent dye. Excess of label was removed by solid-phase extraction using Whatman 3MM chromatography paper. Finally, glycans were eluted with water and stored at −20°C until usage.
Orkney and TwinsUK IgG samples were first denatured with addition of 30 μL 1.33% sodium dodecyl sulfate (w/v) (Invitrogen, Carlsbad, CA) and by incubation at 65°C for 10min. Subsequently, 10 μL of 4% Igepal-CA630 (Sigma–Aldrich, St. Louis, MO) and 1.25 mU of PNGase F (ProZyme) in 10 μL 5× phosphate-buffered saline were added to the samples. The samples were incubated overnight at 37°C for N-glycan release. The released N-glycans were labeled with 2-AB. The labeling mixture was freshly prepared by dissolving 2-AB (Sigma–Aldrich) in dimethyl sulfoxide (Sigma–Aldrich) and glacial acetic acid (Merck) mixture (85:15, v/v) to a final concentration of 48mg/mL. A volume of 25 μL of labeling mixture was added to each N-glycan sample in the 96-well plate. Also, 25 μL of freshly prepared reducing agent solution (106.96mg/mL 2-picoline borane [Sigma–Aldrich] in dimethyl sulfoxide) was added and the plate was sealed using adhesive tape. Mixing was achieved by shaking for 10min, followed by 2-hour incubation at 65°C. Samples (in a volume of 100 μL) were brought to 80% acetonitrile (ACN) (v/v) by adding 400 μL of ACN (J.T. Baker, Phillipsburg, NJ). Free label and reducing agent were removed from the samples using hydrophilic interaction chromatography–solid-phase extraction. An amount of 200 μL of 0.1g/mL suspension of microcrystalline cellulose (Merck) in water was applied to each well of a 0.45 μm GHP filter plate (Pall Corporation, Ann Arbor, MI). Solvent was removed by application of vacuum using a vacuum manifold (Millipore Corporation, Billerica, MA). All wells were prewashed using 5×200 μL water, followed by equilibration using 3×200 μL acetonitrile/water (80:20, v/v). The samples were loaded to the wells. The wells were subsequently washed seven times using 200 μL acetonitrile/water (80:20, v/v). Glycans were eluted two times with 100 μL of water and combined eluates were stored at −20°C until usage.
Orkney and TwinsUK IgG samples were first denatured with addition of 30 μL 1.33% sodium dodecyl sulfate (w/v) (Invitrogen, Carlsbad, CA) and by incubation at 65°C for 10min. Subsequently, 10 μL of 4% Igepal-CA630 (Sigma–Aldrich, St. Louis, MO) and 1.25 mU of PNGase F (ProZyme) in 10 μL 5× phosphate-buffered saline were added to the samples. The samples were incubated overnight at 37°C for N-glycan release. The released N-glycans were labeled with 2-AB. The labeling mixture was freshly prepared by dissolving 2-AB (Sigma–Aldrich) in dimethyl sulfoxide (Sigma–Aldrich) and glacial acetic acid (Merck) mixture (85:15, v/v) to a final concentration of 48mg/mL. A volume of 25 μL of labeling mixture was added to each N-glycan sample in the 96-well plate. Also, 25 μL of freshly prepared reducing agent solution (106.96mg/mL 2-picoline borane [Sigma–Aldrich] in dimethyl sulfoxide) was added and the plate was sealed using adhesive tape. Mixing was achieved by shaking for 10min, followed by 2-hour incubation at 65°C. Samples (in a volume of 100 μL) were brought to 80% acetonitrile (ACN) (v/v) by adding 400 μL of ACN (J.T. Baker, Phillipsburg, NJ). Free label and reducing agent were removed from the samples using hydrophilic interaction chromatography–solid-phase extraction. An amount of 200 μL of 0.1g/mL suspension of microcrystalline cellulose (Merck) in water was applied to each well of a 0.45 μm GHP filter plate (Pall Corporation, Ann Arbor, MI). Solvent was removed by application of vacuum using a vacuum manifold (Millipore Corporation, Billerica, MA). All wells were prewashed using 5×200 μL water, followed by equilibration using 3×200 μL acetonitrile/water (80:20, v/v). The samples were loaded to the wells. The wells were subsequently washed seven times using 200 μL acetonitrile/water (80:20, v/v). Glycans were eluted two times with 100 μL of water and combined eluates were stored at −20°C until usage.
