Protocol for High-Throughput Analysis of the Plasma N -Glycome by UHPLC

1 Glycoprotein Denaturation

(a) Plasma (or serum) aliquots of 5 uL per sample are dispensed into a 96-well V-bottom PP plate (see Notes 2 and 3).

(b) Denaturation solution (55 uL per well, 100 mM ammonium bicarbonate, 12 mM DTT) is added and the plate is covered and placed on a robotic heater shaker and incubated at 65 °C with agitation at 700 rpm for 30 min.

(c) The plate is left to cool down for 10 min and then iodoacetamide solution (10 uL per well,120 mM) is added, the plate is covered with an anti-evaporation lid and incubated at room temperature with agitation at 700 rpm for 30 min.

(d) A trypsin solution (10 μL of a 40,000 U/mL solution per well) is added, the plate is covered with a foil seal and placed on a robotic heater shaker at 37 °C with agitation at 700 rpm for 120 min. Next, the temperature is increased to 105 °C and incubation is continued for 10 min.

(e) The plate is left to cool to room temperature, briefly spun down and the seal removed.

2 N-glycan Release

(a) To release glycans from glycoproteins prepare a PNGaseF solution (0.5 mU in 1.0 M ammonium bicarbonate).

(b) PNGaseF solution (10 μL) is added and the plate is sealed with an anti-evaporation lid and incubated at 37 °C with agitation at 700 rpm for 120 min.

3 Hydrazide- Mediated Glycan Cleanup

(a) A 96-well chemically inert filter plate is washed with 100 μL of MeOH.

(b) Ultralink hydrazide resin (40 μL of a suspension in water) is dispensed to each well.

(c) The resin is sequentially washed with MeOH (200 μL), H₂O (200 μL), and MeCN (200 μL).

(d) To seal the membranes plate is placed on a heater (80 °C, 10 min).

(e) MeCN–acetic acid (98:2) is added to the resin (180 μL), followed by 20 μL of the glycan solution (glycans released with PNGaseF, the remaining glycan sample is kept as a stock).

(f) The filter plate is incubated with shaking at 700 rpm at 80 °C for 60 min.

(g) MeCN–acetic acid (98:2) is added to the resin (50 μL) and shaking is continued at the same temperature (80 °C) for 10 min to disrupt resin aggregates.

(h) The resin is washed sequentially with MeOH (2 × 300 μL per well), 2.0 M guanidine (2 × 200 μL), H 2 O (2 × 200 μL), trimethylamine–MeOH (1:99, 2 × 200 μL), and MeOH (2 × 200 μL).

(i) Fresh MeOH (180 μL) and acetic anhydride (20 μL) are added and the plate is incubated for 30 min with agitation at 700 rpm at ambient temperature.

(j) Excess reagent is removed by fi ltration and the resin beads are washed sequentially with MeOH, H 2 O, and MeCN (400 μL).

(k) MeCN–acetic acid (98:2, 175 μL) and H₂O (25 μL) are sequentially added and the plate is incubated at 70 °C with agitation at 700 rpm for 90 min.

4 Fluorescent Labeling

(a) 2-AB fluorescent labeling mix (50 μL, 350 mM 2-aminobenzamide, 1.0 M sodium cyanoborohydride in acetic acid–dimethyl sulfoxide (30:70) is dispensed into each well.

(b) The plate is incubated at 65 °C with agitation at 800 rpm for 120 min.

(c) The labeling reaction is quenched by the addition of 400 μL MeCN–H 2 O (95:5).

5 Glycan Solid-Phase Extraction

(a) The suspension after 2-AB labeling is transferred to a 2 mL collection plate, the beads are left to settle and 200 μL of the supernatant is aspired and dispensed back into the filter plate.

(b) The suspension is mixed several times and transferred back into the 2 mL collection plate. This cycle is repeated once more to ensure a quantitative transfer of the resins.

(c) HyperSep Diol SPE cartridges are washed with 1 mL MeCN–H₂O (95:5), 1 mL H₂O, and 1 mL MeCN–H₂O (95:5).

(d) The supernatants of the quenched reaction mixtures without the beads are transferred onto the SPE cartridges (900 μL).

(e) Typically 5 min incubation leads to complete drainage of the solvent.

(f) The SPE cartridges are washed three times with 750 μL MeCN–H₂O (95:5) to remove excess of 2-AB.

(g) A collection plate is placed inside the robotic vacuum manifold and the SPE cartridges are washed under vacuum with 200 μL H₂O /MeCN (80:20), with an intermittent incubation period of 5 min to elute the retained glycans on the SPE cartridges.

(h) Following glycan elution, samples are concentrated to dryness in a vacuum evaporator.

6 HILIC-FLD-UHPLC

1. Prepare fresh solvents for UHPLC separation: solvent A (50 mM ammonium formate solution, pH 4.4) and solvent B (MeCN).

2. Set up the UHPLC system: prime the system, connect the column and turn on fluorescence detector.

3. Set up the instrument method in Empower software with the following parameters, flow: 0.56 mL/min, a linear gradient of 70–53 % MeCN in 30 min separation run: t = 0 min, 70 % solvent B; t = 24.81 min, 53 % solvent B; t = 25.5 min, 30 % solvent B and t = 26.55 min, 70 % solvent B.

4. Adjust the settings of the fluorescence detector: excitation wavelength 330 nm and emission wavelength 420 nm, data units: emission, date rate: 2 pts/s and PTM gain 20.0.

5. Run a blank sample of H₂O.

6. Run 2-AB labeled dextran ladder (standard) that is be used for calibration, dissolve sample in 30 μL MeCN–H₂O (70:30).

7. Run your samples of interest, bring the volume of your 2-AB labeled glycan samples to 30 μL with MeCN–H₂O (70:30).

You may be interested in:

N-Glycan Profiling Service

Reference

1. Lauc, G., & Wuhrer, M. (2017). High-throughput glycomics and glycoproteomics. Springer New York.