Glycan labeling with different labels

Eleven different fluorescent glycan labels were tested, all in pentaplicates: 2-aminobenzamide (2AB), 2-aminobenzoic acid (2AA), 3-aminobenzoic acid (3AA), 4-aminobenzoic acid (4AA), aniline (AN), 4-aminophenol (4AP), benzocaine (BC), 1-naphthylamine (1NA), 3-amino-2-naphthoic acid (32ANA), 6-amino-2-naphthoic acid (62ANA) and 8-amino-2-naphthalenesulfonic acid (82 ANSA) (Table 1). All labels were obtained from Sigma-Aldrich, St. Louis, MO, United States. LogP values were calculated using SwissADME online platform (http://www.swissadme.ch/) (Daina et al., 2017)—consensus logP calculated as an arithmetic mean of logP values generated using five different methods was used. pKa values were calculated using Chemicalize online platform (https://chemicalize.com/) (Chemaxon, Budapest, Hungary). Percentage of ionization was calculated based on the pKa values using the Henderson-Hasselbalch equation: pH=pKa+log⁡⁡[A−]log[HA] .

Glycan fluorescent labels used in the experiment and their chemo-physical properties. LogP values were calculated using SwissADME online platform, while the pKa values were calculated using Chemicalize online platform (Chemaxon).

For each sample, 50 μg of dried IgG sample was resuspended and denatured by incubation with 30 μl SDS (1.33% wt/vol; Invitrogen, Carlsbad, CA, United States) at 65°C for 10 min. Subsequently, 10 μl of 4% Igepal-CA630 (Sigma-Aldrich, St Louis, MO, United States) and 1.2 u PNGase F (Promega, Madison, WI, United States) in 10 μl 5× PBS were added. The samples were incubated overnight at 37°C to allow release of N-glycans. The released N-glycans were labeled with the corresponding label. The labeling mixture was freshly prepared by dissolving the label (0.14 M) and 2-picoline borane (0.42 M) in a mixture of DMSO (Sigma-Aldrich) and glacial acetic acid (Merck, Darmstadt, Germany) (70:30, vol/vol). Labeling mixture (25 μl) was added to each N-glycan sample in the 96-well plate, which was then sealed using adhesive seal. Mixing was achieved by shaking for 10 min, followed by incubation at 65°C for 2 h. To each sample (75 μl), 700 μl of acetonitrile (ACN) (J. T. Baker, Phillipsburg, NJ, United States) was added. Free label and reducing agent were removed from the samples using HILIC solid-phase extraction (SPE). A GHP filter plate, 0.2 μm, (Pall Corporation, Ann Arbor, MI, United States) was used as the stationary phase. All wells were prewashed using 1 μl × 200 μl of ethanol/water (70:30, vol/vol) and 1 μl × 200 μl water, followed by equilibration using 1 μl × 200 μl of ACN/water (96:4, vol/vol). Solvent was removed by the application of a vacuum using a vacuum manifold (Millipore Corporation, Billerica, MA, United States). The samples were loaded into the wells, which were subsequently washed five times using 200 μl of ACN/water (96:4, vol/vol). Glycans were eluted with 2 μl × 90 μl of water and combined eluates were stored at −20°C until usage.