2 aminobenzamide

Glycans were released from total plasma proteins and labeled as previously described (65 (link)). In brief, 10 µl of plasma was denatured by adding 20 µl 2% (w/v) SDS (Invitrogen, USA) and the N-glycans were released by adding 1.2 U of PNGase F (Promega, USA). The released N-glycans were labeled with 2-aminobenzamide (Sigma-Aldrich, USA). Hydrophilic interaction liquid chromatography solid-phase extraction was used to remove free labels and the reducing agent from the samples. In the stationary phase, 0.2 µm 96-well GHP filter-plates (Pall Corporation, USA) were used. After a short incubation and washing five times with cold 90% ACN, the samples were loaded into the wells. After 15 min of shaking at room temperature, glycans were eluted with 2 × 90 µl of ultrapure water and then the combined eluates were stored at −20°C until usage.

Xylohexaose (Megazyme
Ireland) was labeled with the fluorescent dye 2-aminobenzamide (2AB,
Sigma-Aldrich), as previously described.^{11 (link)} Xylohexaose-2AB (0.5 mM) was then incubated with KfXYS1 and 30 mM UDP-Xyl in HEPES sodium salt pH 7.3 for 12 h, before
pelleting and washing as described previously. Alternatively, xylan
microparticles generated via a large-scale synthesis were labeled
with 2AB after formation as follows: 0.2 mg of XX-type microparticles
was suspended in 100 μL of 0.5 M sodium cyanoborohydride (NaCNBH₄, Sigma-Aldrich) and 0.25 M 2AB (Sigma-Aldrich). Reaction
pH was adjusted to between 5 and 5.5 with 1 M acetic acid. The mixture
was then incubated at 45 °C for 12 h. Following incubation, the
reaction was pelleted by centrifugation at 5,000g for 5 min, the supernatant was removed, and the microparticles were
washed 10× with 1 mL of dH₂O. Control samples were
also prepared following the same procedure but lacking NaCNBH₄ in the reaction mixture.
Xylan microparticles (∼20
μg) were labeled with propidium iodide by incubation for 30
min in 100 μg/mL aq. propidium iodide (Sigma-Aldrich). Following
incubation, the microparticles were pelleted and washed with 50 μL
of water. The microparticles were then pelleted again, and the supernatant
was removed, before resuspension in 5 μL of 1% agarose solution
(60 °C), and mounting on a glass microscopic slide.

In order to verify the results of the enzymatic desialylation of the native protein and pH-gradient separation of different sialoforms, the complete N-glycan profiling of Tf+S and Tf-S was performed. Briefly, the protein N-glycans were released with the addition of 1.2 U of PNGase F (Promega, USA) and overnight incubation at 37° C. The released N-glycans were labeled with 2-aminobenzamide (Sigma Aldrich, USA) and purified using hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE). Fluorescently labeled N-glycans were separated by Acquity UPLC H-Class instrument (Waters, USA) using BEH Glycan chromatography column (Waters, USA). All glycan structures were annotated with MS/MS analysis using Synapt G2-Si ESI-QTOF-MS system (Waters, USA). Glycan compositions and structural features were assigned using software tools GlycoWorkbench and Glycomode, according to obtained MS and MS/MS spectra [22 (link), 23 (link)]. Full details of the protein characterization by UPLC-MS have been described elsewhere [24 ].