| Mice euthanized
||Blood samples collected
|Syringes and needles, EDTA-anticoagulant tubes, centrifuge
||IgG isolated from plasma
||Centrifuge, protein G monolithic plates
||Glycans released and labeled
||Vacuum concentrator and incubator
||Hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction (SPE) and HILIC ultra-performance liquid chromatography (UPLC)
||HILIC-SPE and HILIC-UPLC instruments
||NanoACQUITY UPLC system coupled to a Compact mass spectrometer
||Relative abundance of IgG glycans (26 chromatographic glycan peaks)
- CO2 chamber
- Syringes and needles
- EDTA-anticoagulant tubes
- 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)
- 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
- 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
- Mice are euthanized in a CO2 chamber.
- Blood is collected via cardiac puncture and transferred to EDTA-anticoagulant tubes.
- 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).
- Plasma samples are kept at -80°C and shipped on dry ice for further processing.
- After thawing, plasma samples are vortexed and centrifuged at 12,100 g for 3 min or 5000 g for 10 min.
- Prior to starting the IgG isolation, samples are randomized.
- 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.
- IgG concentrations are measured at 280 nm using a NanoDrop spectrophotometer.
Procedure: IgG glycan release and labeling
- 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.
- 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)
- After incubation at 65°C in the previous procedure, samples are left to cool to room temperature for 30 min.
- Samples (in a volume of 75 µL) are mixed with 700 µL of cold 100% acetonitrile.
- Free label and reducing agent are removed from the samples using HILIC-SPE on a 0.2 µm GHP filter plate.
- Solvent is removed by application of a vacuum.
- 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.
- Samples are loaded onto the GHP filter plate and incubated for 2 min before the vacuum application.
- Wells are subsequently washed five times using 200 µL of cold 96% acetonitrile.
- The last washing step is followed by centrifugation of 164 g for 5 min.
- 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.
- Combined eluates are stored at -20°C until use in the next procedure.
Procedure: Hydrophilic interaction liquid chromatography ultra-performance liquid chromatography (HILIC-UPLC)
- 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).
- 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.
- The separation method uses a linear gradient of 75-62% acetonitrile (v/v) at flow rate of 0.4 mL/min over 27 min.
- The gradient is maintained at 62% acetonitrile for an additional 5 min.
- The column is then washed for 2 min with 100% of solvent A.
- Initial conditions are restored in 1 min and held for an additional 5 min to ensure column re-equilibration.
- Samples are maintained at 10°C before injection, and the separation temperature is 60°C.
- 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.
- 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.
- 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.
- Dried HILIC-UPLC fractions are reconstituted in 5-20 µL (depending on the intensity of fluorescence) of ultrapure water.
- 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)).
- 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.
- Samples are injected on the trap column and washed for 1 min with solvent A at a flow rate of 40 µL/min.
- 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.
- 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.
- The NanoACQUITY UPLC system and the Bruker micrOTOF-Q are operated under HyStar software.
- Mass spectra are recorded from m/z 200 to 2,200 with two averages at a frequency of 0.5 Hz.
- 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.
- Glycan structures are assigned using software tools Glycomode and GlycoWorkbench according to obtained MS and MS/MS spectra.
- 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).