Zaytseva1 project protocol

IgG glycome: Glycan levels in 111 Collaborative Cross strains of mice   (2018)

Zaytseva OO, Krištić J, Morahan G, Lauc G
With: Ram R, Nguyen Q, Novokmet M, Vučković F, Vilaj M, Trbojević-Akmačić I, Pezer M, Davern KM

Project protocol - Contents

Workflow and sampling

Data collected
Mice euthanized CO2 chamber
2 Blood samples collected
Syringes and needles, EDTA-anticoagulant tubes, centrifuge -
3 IgG isolated from plasma Centrifuge, protein G monolithic plates -
4 Glycans released and labeled Vacuum concentrator and incubator -
5 Hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction (SPE) and HILIC ultra-performance liquid chromatography (UPLC) HILIC-SPE and HILIC-UPLC instruments -
6 Nano-LC-ESI-MS/MS NanoACQUITY UPLC system coupled to a Compact mass spectrometer Relative abundance of IgG glycans (26 chromatographic glycan peaks)

Equipment, software and supplies

  • CO2 chamber
  • Syringes and needles
  • EDTA-anticoagulant tubes
  • Centrifuge
  • Freezer (-80°C)
  • Freezer (-20°C)
  • Dry ice
  • Protein G Monolithic Plates (BIA Separations, Ajdovscina, Slovenia)
  • GHP Filter Plate (Pall Corporation, Ann Arbor MI)
  • NanoDrop Spectrophotometer (NanoDrop 8000, Thermo Scientific, USA)
  • Vacuum concentrator (Millipore Corporation, Billerica MA)
  • Incubator
  • Shaker
  • 96-well plates
  • Hydrophilic interaction chromatography ultra-performance liquid chromatography (HILIC-UPLC): Waters Acquity UPLC instrument (Milford MA)
  • Waters Empower 3 software, build 3471 (Milford MA)
  • Waters BEH Glycan chromatography column (Milford MA)
  • Waters ACQUITY UPLC system (Waters MA)
  • Compact Mass Spectrometer (Bruker Daltonics, Bremen, Germany)
  • Reverse-Phase Trap Column (Acclaim PepMap100 C8)
  • Analytical Column (Halo C18 nano-LC column) (Advanced Materials Technology, USA)
  • CE ESI-MS sprayer source (Agilent CA)
  • Bruker micrOTOF-Q
  • HyStar software, v 3.2
  • Glycomode software
  • GlycoWorkbench software

Reagents and solutions

  • Phosphate buffered saline (PBS)
  • Formic acid (Merck, Darmstadt Germany)
  • Ammonium bicarbonate (Merck, Darmstadt Germany)
  • SDS (Invitrogen, Carlsbad CA)
  • Igepal-CA630 (Sigma-Aldrich, St. Louis MO)
  • PNGase F (Promega, Madison WI)
  • 2-aminobenzamide (Sigma-Aldrich, St Louis MO)
  • Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis MO)
  • Glacial acetic acid (Merck, Darmstadt Germany)
  • 2-picoline borane (Sigma-Aldrich, St. Louis MO)
  • Acetonitrile (Sigma-Aldrich, St. Louis MO)
  • Ethanol (Carlo Erba Reagents, Val de Reuil, France)
  • Ultrapure water (MQ, Merck Millipore, Billerica MA)
  • Ammonium formate
  • Hydrolyzed and 2-aminobenzamide-labeled glucose oligomers

Procedure: Plasma preparation and isolation of IgG

  1. Mice are euthanized in a CO2 chamber.
  2. Blood is collected via cardiac puncture and transferred to EDTA-anticoagulant tubes.
  3. Blood samples are centrifuged at 3300 g for 6 min to obtain the plasma (to ensure a blinded study, the plasma samples are coded by number; a minimum of two plasma samples per strain are analyzed for glycans).
  4. Plasma samples are kept at -80°C and shipped on dry ice for further processing.
  5. After thawing, plasma samples are vortexed and centrifuged at 12,100 g for 3 min or 5000 g for 10 min.
  6. Prior to starting the IgG isolation, samples are randomized.
  7. IgG is isolated using protein G monolithic plates, as described previously (Pucic, M et al., 2011):
    • 100-500 µL of plasma is diluted with 700 µL of PBS, pH 7.4, and filtered through 0.45 µm GHP filter plate.
    • After filtration, samples are applied to the protein G plate and immediately washed with PBS, pH 7.4 to remove unbound proteins.
    • IgGs are eluted with 1 mL of 1M ammonium bicarbonate.
  8. IgG concentrations are measured at 280 nm using a NanoDrop spectrophotometer.

Procedure: IgG glycan release and labeling

  1. Release:
    • IgG samples are dried in a vacuum concentrator, dissolved in 30 µL of 1.33% SDS (w/v), and denatured by incubation at 65°C for 10 min.
    • After incubation, samples are left to cool to room temperature for 30 min.
    • Subsequently, 10 µL of 4% Igepal-CA630 is added and the samples are incubated on a shaker for 15 min.
    • After shaking, 1.2 U of PNGase F in 10 µL 5X PBS are added and incubated overnight at 37°C for N-glycan release.
  2. Labeling:
    • Released glycans are labeled with 2-aminobenzamide (labeling mixture is prepared fresh by dissolving 2-aminobenzamide in dimethyl sulfoxide and glacial acetic acid mixture (70:30, v/v) and adding 2-picoline borane to a final concentration of 19.2 mg/mL for 2-aminobenzamide and 44.8 mg/mL for 2-picoline borane).
    • A volume of 25 µL of labeling mixture is added to each N-glycan sample in a 96-well plate (sealed with adhesive tape).
    • Samples are mixed by shaking for 10 min, followed by 2h incubation at 65°C.

Procedure: Hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE)

  1. After incubation at 65°C in the previous procedure, samples are left to cool to room temperature for 30 min.
  2. Samples (in a volume of 75 µL) are mixed with 700 µL of cold 100% acetonitrile.
  3. Free label and reducing agent are removed from the samples using HILIC-SPE on a 0.2 µm GHP filter plate.
  4. Solvent is removed by application of a vacuum.
  5. All wells are prewashed using 200 µL of 70% ethanol, followed by 200 µL of ultrapure water and equilibrated with 200 µL of cold acetonitrile.
  6. Samples are loaded onto the GHP filter plate and incubated for 2 min before the vacuum application.
  7. Wells are subsequently washed five times using 200 µL of cold 96% acetonitrile.
  8. The last washing step is followed by centrifugation of 164 g for 5 min.
  9. Glycans are eluted twice with 90 µL if ultrapure water after 15 min of shaking at room temperature followed by centrifugation at 164 g for 5 min.
  10. Combined eluates are stored at -20°C until use in the next procedure.

Procedure: Hydrophilic interaction liquid chromatography ultra-performance liquid chromatography (HILIC-UPLC)

  1. Fluorescently labeled N-glycans are separated by HILIC on a UPLC instrument consisting of a quaternary solvent manager, sample manager, and a FLR fluorescence detector set with excitation and emission wavelengths of 250 and 428, respectively (the instrument is under the control of Empower 3 software).
  2. Labeled N-glycans are separated on a Waters BEH Glycan chromatography column (100 X 2.1mm i.d., 17 µm BEH particles, with 100 mM ammonium formate, pH 4.4 as solvent A and acetonitrile as solvent B.
  3. The separation method uses a linear gradient of 75-62% acetonitrile (v/v) at flow rate of 0.4 mL/min over 27 min.
  4. The gradient is maintained at 62% acetonitrile for an additional 5 min.
  5. The column is then washed for 2 min with 100% of solvent A.
  6. Initial conditions are restored in 1 min and held for an additional 5 min to ensure column re-equilibration.
  7. Samples are maintained at 10°C before injection, and the separation temperature is 60°C.
  8. The system is calibrated using an external standard of hydrolyzed and 2-aminobenzamide-labeled glucose oligomers from which the retention times for the individual glycans are converted to glucose units.
  9. Data processing is performed using an automatic processing method with a traditional integration algorithm, after which each chromatogram is manually corrected to maintain the same intervals of integration for all the samples.
  10. The chromatograms are all separated in the same manner into 26 peaks and the amount of glycans in each peak is expressed as a percentage of total integrated area.

Procedure: Nano-LC-ESI-MS/MS

  1. Dried HILIC-UPLC fractions are reconstituted in 5-20 µL (depending on the intensity of fluorescence) of ultrapure water.
  2. LC-MS analysis of the collected glycan fractions is performed using a nanoACQUITY UPLC system coupled to Compact mass spectrometer (the LC system consists of a reverse-phase trap column (5 mm X 300 µm i.d.) and an analytical column (150 mm X 75 µm i.d., 2.7 µm HALO fused core particles)).
  3. The method is based on one described previously by Pucic et al (2011) with the following modifications. The column is equilibrated at 30°C with solvent A (0.1% formic acid in water) at a flow rate of 1 µL/min.
  4. Samples are injected on the trap column and washed for 1 min with solvent A at a flow rate of 40 µL/min.
  5. After injection of the samples, a 5-min gradient at the 1 µL/min flow rate is applied to 40% eluent B (95% acetonitrile) followed by an isocratic elution with 40% eluent B for 5 min.
  6. The LC system is coupled via an online CE ESI-MS sprayer source to a Compact mass spectrometer operated in positive and negative ion mode.
  7. The NanoACQUITY UPLC system and the Bruker micrOTOF-Q are operated under HyStar software.
  8. Mass spectra are recorded from m/z 200 to 2,200 with two averages at a frequency of 0.5 Hz.
  9. Quadrupole ion energy and collision energy of the MS are set at 4 eV for MS analysis and at 14 to 23 eV for the MS/MS analysis.
  10. Glycan structures are assigned using software tools Glycomode and GlycoWorkbench according to obtained MS and MS/MS spectra.

Data collected by investigator

  • Relative abundance of each of 26 glycan peaks


Pucic, M. et al. High throughput isolation and glycosylation analysis of IgG-variabillity and heritability of the IgG glycome in three isolated human populations. Mol. Cell. Proteomics 10: M111.010090 (2011).