| Mice euthanized
||Blood samples collected
|Syringes and needles, EDTA-anticoagulant tubes, centrifuge
||IgG isolated from plasma
||Centrifuge, protein G monolithic plates
||Tryptic digests performed and Fc glycopeptides isolated and analyzed
||Reverse-phase solid phase extraction system, ultra-performance liquid chromatography system coupled to a Compact mass spectrometer
||IgG subclass abundance of glycopeptides
- CO2 chamber
- Syringes and needles
- EDTA-anticoagulant tubes
- Freezer (-80°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)
- Vacuum centrifuge
- 96-well plates (Fischer Scientific USA)
- Reverse-phase solid phase extraction system (Chromabond C18ec beads, Marcherey-Nagel, Germany)
- nanoACQUITY UPLC system (Waters, USA)
- Trap column (Acclaim PepMap 100 C8 (5mm x 300 µL i.d.)
- Separation column (HALO C18 nano-LC column (150 mm x 75 µL i.d., 2.7 µm HALO fused core particles), Advanced Materials Technology, USA)
- Compact Mass Spectrometer (Bruker Daltonics, Bremen, Germany)
- LaCyTools software, v. 1.0.1 b.7
- Phosphate buffered saline (PBS)
- Formic acid (Merck, Darmstadt Germany)
- Ammonium bicarbonate (Merck, Darmstadt Germany)
- Ultrapure water (MQ, Merck Millipore, Billerica MA)
- Trypsin (Worthington USA)
- Trifluoroacetic acid
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: Nano-LC-ESI-MS/MS of tryptic glycopeptides
- IgG samples are dried in a vacuum concentrator and dissolved in ultrapure water.
- A volume corresponding to ~10-15 µg of IgG is transferred to a 200 µL 96-well plate and cleaved with 200 ng trypsin.
- Resulting glycopeptides are purified by reverse-phase solid phase extraction using Chromabond C18ec beads.
- Purified glycopeptides are dried by vacuum centrifugation and then dissolved in 20 µL ultrapure water.
- Trypic digests are analyzed on a nanoACQUITY UPLC system coupled to a Compact mass spectrometer:
- 9 µL eluates containing IgG tryptic glycopeptides are loaded onto an Acclaim PepMap100 C8 trap column and washed 1 min with 0.1% trifluoroacetic acid (solvent A) at a flow rate of 40 µL/min.
- Separation is achieved on a HALO C18 nano-LC column using a 3.5 min gradient at a flow rate of 1 µL/min from 18.5% to 26% acetonitrile (80%) (solvent B).
- Glycopeptide compositions are assigned on the basis of m/z value and isotopic distribution pattern.
- Peak areas are calculated by summing areas for doubly and triply charged ions determined with LaCyTools and normalized to the total integrated area per IgG subclass.
- IgG subclass relative abundance (normalized to the total integrated area of each IgG subclass) of specific types of N-glycan structures
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).