Protocol for High-Throughput Analysis of IgG Fc Glycopeptides by LC-MS

1 Affinity Purification

(a) Spin down 2 mL of ProtG beads in a 15 mL tube (3000 × g, 10 s).

(b) Carefully pipette the supernatant from the beads.

(c) Add 2 mL of PBS to the beads.

(d) Vortex thoroughly and repeat spinning and removal of supernatant.

(e) Resuspcnd the beads in 4.67 mL PBS.

(f) Using a 200 uL. multichannel pipette, apply 50 uL of slurry to each well of a 96-well filter plate.

(g) Wash the beads three times with 200 uL PBS using the vacuum manifold.

(h) Add 100 uL PBS and leave it on the beads.

(i) Add 2 uL of serum or plasma.

(j) Place the filter plate on a polypropylene 96-well V-bottom plate and seal wells with tape.

(k) Incubate the filter plate on a plate shaker for 1 h at 1000 rpm.

(l) Wash the samples three times with 200 uL of PBS using the vacuum manifold.

(m) Wash the samples three times with 200 uL of MQ using the vacuum manifold.

(n) Remove the filter plate from the vacuum manifold, tap the bottom carefully on lint-free paper to remove an excess of liquid, and place the filter plate on a new polypropylene 96-wellv-bottom plate.

(o) Add 100 uL 100 mM formic acid to the beads in each well and seal the wells with tape.

(p) Incubate on the plate shaker for 5 min at 1000 rpm.

(q) Remove the tape and elute the IgGs from the filter plate by centrifugation for 1 min at 100 × g.

(r) Dry the samples using a vacuum concentrator at 60 °C to complete dryness, which will take approximately 2-3 h.

(s) Samples can be digested directly or sealed with adhesive tape and stored at −20 °C.

2 Denaturation of Pure Antibody Samples

(a) Add 5 μg of antibody (sample) in an arbitrary volume to a 100 mM FA solution, but maintain a minimum final concentration of 75 mM FA. Vortex.

(b) Incubate for 15 min at room temperature (RT).

(c) Dry in a centrifugal vacuum concentrator set to 60 °C until complete dryness.

3 Protease Treatment

(a) Add 40 μL (1 μg) of the TPCK treated trypsin solution. Incubate for 5 min on a shaker at RT and low rpm (e.g., 400 rpm).

(b) Incubate at 37 °C for 18 h.

(c) Samples can be stored short term in a cooled (4 °C) autosampler or long term at −20 °C

4 LC-MS

(a) Inject 250 nL of sample into a flow of 25 μL/min of solvent A. The glycopeptides are trapped on the trap column.

(b) Switching to the gradient pump flow of 900 nL/min, the glycopeptides are eluted onto the analytical column where they are separated. Both trap and analytical column are kept at 30 °C. Changes in solvent B concentrations occur as linear gradients.

(c) Nebulization and ionization is achieved in the CaptiveSpray™ ESI source assisted by ACN-doped nebulizing gas from the nanoBooster™.

(d) Using highly optimized ion transfer settings, the glycopeptides are declustered and desolvated at high efficiency and with minimal fragmentation (between 0.1 % and 1 % compared to the expected parent peak).

(e) During the 5-minute analysis period, MS spectra are acquired every second in full spectrum analysis mode, with a mass range of m/z 550 to m/z 1800. It is crucial to save the MS data as profile spectra to ensure accurate mass determination and MS peak area integration for quantification. As different glycoforms of the same IgG isoform often co-elute, the separation will display three distinct "clusters" of glycopeptides.

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Glycopeptides Analysis

Reference

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