Middle‑Down MS for Disulfide Mapping in Multispecific Proteins

May 20, 2026

Disulfide bonds are critical for the folding, stability, and function of biotherapeutic proteins, yet conventional disulfide mapping workflows based on non‑reduced digestion can introduce scrambling artifacts and incomplete digestion. These effects can lead to false positive or false negative identifications, complicating accurate characterization of disulfide connectivity in complex protein constructs such as multispecific antibodies.  

This poster presents a middle‑down mass spectrometry workflow for disulfide mapping in multispecific protein subunits, combining limited enzymatic digestion with complementary HCD and ETD fragmentation to localize disulfide bonds without relying on non‑reduced protease digestion. In this work carried out by Amgen, you will learn how middle‑down MS/MS improves confidence in disulfide bond assignment, reduces the risk of method‑induced scrambling, and enables more reliable identification of distinct disulfide bond forms. It also demonstrates how integrating data across multiple fragmentation modes in Genedata Expressionist^® supports high‑confidence identification, improved sequence coverage, and enhanced data visualization. Designed for scientists working in peptide mapping and structural characterization of complex biotherapeutics.