Acquity h class uplc instrument

The enzymatic activity of wild type EnvSia156 and H134A mutant were tested by incubating 0.05, 0.15, 0.3, and 0.6 µg of enzyme with procainamide labeled 3’sialyllactose substrate in 50 mM sodium acetate pH 5.5, for 1 h at 37 °C. After incubation, 2.4 μL of sample were mixed with 17.6 µL acetonitrile for a 12:88 ratio. Sixteen microliters were injected into a Waters Acquity BEH glycan amide column (2.1 × 150 mm, 1.7 μm) on a Waters ACQUITY UPLC H-Class instrument (Waters Corporation, Milford, MA) equipped with a quaternary solvent manager and a fluorescence detector. Solvents used were A: 50 mM ammonium formate buffer pH 4.4 and B: 100% acetonitrile. The gradient used was 0–1.50 min, 12% solvent A; 1.5–35 min, 47% solvent A; 35–36.5 min, 70% solvent A; 36.5–42 min, 12% solvent A with a flow rate of 0.561 mL/min. Samples were kept at 5 °C prior to injection and separation was performed at 30 °C. The fluorescence detection wavelengths were λ_ex = 308 nm and λ_em = 359 nm with a data collection rate of 20 Hz. Data were analyzed with Empower 3 chromatography workstation software (Waters Corporation).

The fluorescently labeled N-glycans were separated with hydrophilic interaction chromatography (HILIC) on an Acquity UPLC H-class instrument (Waters, Milford, MA, USA). The excitation and emission wavelengths were set to 250 nm and 428 nm, respectively. The plasma N-glycans and IgG N-glycans were separated on a 150 mm and 100 mm Glycan BEH Amide column (Waters, USA), respectively. 100 mmol/L solution of ammonium formate in water, with a pH of 4.4, was used as solvent A, and ACN was used as solvent B. For the plasma analysis, a linear elution gradient of 30–47% of solvent A, at a 0.56 mL/min flow rate in a 25 min analytical run, was used. The IgG was analyzed using a linear gradient of 25–38% of solvent A, at 0.4 mL/min flow rate in a 29 min analytical run. The data were processed using an automatic processing method, after which, each chromatogram was manually corrected to maintain consistent intervals of integration between the samples. The chromatograms were separated into 24 peaks (IGP1-IGP24) for the IgG N-glycans and 39 peaks (GP1-GP39) for the plasma N-glycans. The glycan composition of each peak was confirmed in previous studies using LC-MS [32 (link),34 (link)]. The sample chromatograms, with major glycan structures in each peak for the total plasma and IgG N-glycans, are given in Supplementary Figures S1 and S2.

Fluorescently labelled N-glycans were separated by hydrophilic interaction liquid chromatography (HILIC) on a Waters Acquity UPLC H-class instrument (Waters, Milford, MA) equipped with FLR fluorescence detector set to 330 nm for excitation and 420 nm for emission wavelength. Separation was achieved on a Waters bridged ethylene hybrid (BEH) Glycan chromatography column, 100 × 2.1 mm i.d., 1.7 μm BEH particles with 100 mM ammonium formate (pH 4.4) as a solvent A and ACN as a solvent B. Separation method used linear gradient from 75 % to 62 % solvent B (v/v) at a flow rate of 0.4 mL/min in a 25-minute analytical run. Column temperature was maintained at 60 °C. Obtained chromatograms were manually separated into 24 peaks using Empower 3 software, from which, using the total area normalization, relative abundances of 24 directly measured glycan traits were obtained (Supplementary Table 2). In-depth characterization of each of 24 chromatographic peaks was performed as previously described [23 (link)]. The most abundant glycan structure in each peak was chosen to represent that glycan peak. An example of chromatogram integration with the most abundant glycan structures in each peak of IgG glycome is shown in Supplementary Figure 1.