ASMS 2026: Structural Characterization of IgG2 Disulfide Isoforms by Native CEX-MS and Peptide Mapping
A Servier–Protein Metrics collaboration on native cation-exchange MS for IgG2 hinge-region disulfide isoforms
About this Poster
IgG2 antibodies carry four hinge-region cysteines (two of them consecutive) prone to disulfide rearrangement — yielding three isoforms (A, A/B, B) that conventional analytical methods often fail to cleanly separate. This Servier–Protein Metrics collaboration develops a native cation-exchange-MS (CEX-MS) method using volatile salts, combined with a middle-up F(ab')2 strategy that eliminates Fc heterogeneity. Isoform elution order was assigned via redox treatments and site-directed mutagenesis. An optimised non-reduced peptide mapping workflow — enhanced by a novel Isotope Envelope Confidence (IEC) score — confidently identifies the complex interchain hinge disulfide patterns that traditional MS/MS often struggles to resolve.
Key Learnings:
- See how native CEX-MS with volatile salts (ammonium acetate) achieves chromatographic resolution of IgG2 isoforms equal to or better than reversed-phase.
- Understand how middle-up F(ab')2 analysis eliminates Fc-related heterogeneity, producing high-quality MS spectra for clean isoform assignment.
- Learn how redox treatments and site-directed mutagenesis pinned down the elution order of the A, A/B, and B isoforms — and characterised an engineered pseudo-isoform-B mAb designed for agonistic activity.
- Discover how an Isotope Envelope Confidence (IEC) score in Byos non-reduced peptide mapping confidently identifies complex interchain disulfide patterns that conventional MS/MS interpretation misses.
Published in Anal. Chem. 2025, 97, 17, 9395–9404. A Servier–Protein Metrics collaboration.