Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Plasma preparation and isolation of IgG
• Procedure: Nano-LC-ESI-MS/MS of tryptic glycopeptides
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	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	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

Equipment, software and supplies

• CO₂ chamber
• Syringes and needles
• EDTA-anticoagulant tubes
• Centrifuge
• 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)
• Incubator
• 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

Reagents and solutions

• 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
• Acetonitrile

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: Nano-LC-ESI-MS/MS of tryptic glycopeptides

1. IgG samples are dried in a vacuum concentrator and dissolved in ultrapure water.
2. A volume corresponding to ~10-15 µg of IgG is transferred to a 200 µL 96-well plate and cleaved with 200 ng trypsin.
3. Resulting glycopeptides are purified by reverse-phase solid phase extraction using Chromabond C18ec beads.
4. Purified glycopeptides are dried by vacuum centrifugation and then dissolved in 20 µL ultrapure water.
5. 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).
6. Glycopeptide compositions are assigned on the basis of m/z value and isotopic distribution pattern.
7. 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.

Data collected by investigator

• IgG subclass relative abundance (normalized to the total integrated area of each IgG subclass) of specific types of N-glycan structures

Reference

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).