Middle-Down Mass Spectrometry Characterization of Monoclonal Antibody Enabled by Electron-Activated Dissociation on a Time-of-Flight Platform: A Case Study of Methionine Oxidation PTM Identification and Quantification.

Characterizing post-translational modifications (PTMs) in monoclonal antibody products is critical for ensuring product quality attributes understanding and monitoring. While the bottom-up approach is widely adopted, it suffers from the loss of molecular connectivity and is susceptible to artifact generation. Conversely, top-down approaches face limitations due to the high complexity and poor gas-phase fragmentation of large intact molecules to get site-specific post-translational information profiling. Middle-down MS methods offer a complementary middle-ground approach to both bottom-up and top-down, with broader sequence coverage, and facilitate the simultaneous identification of multiple attributes and proteoforms. In this study, we optimized the electron-activated dissociation (EAD) approach for middle-down sequencing of NISTmAb subunits and present a potential MS and MS/MS strategy to rapidly characterize PTMs on antibodies, using methionine oxidation as an example. Peroxide-stressed and control mAb samples were digested and reduced into subunits using the IdeS enzyme and analyzed via LC-MS, with the optimized MRMHR EAD method. The resulting data enabled high-resolution subunit mass analysis, sequence confirmation, and potential for site-specific oxidation localization. Methionine oxidation localization in NISTmAb was achieved in light chain (M4), Fd' (M34, M101), and Fc/2 (M16, M122, M192), with these locations confirmed with bottom-up peptide mapping data. Quantification using the Fc/2 subunit enabled detection of oxidation levels up to 1% relative, with strong correlation (R2 > 0.99) between expected and observed values using MS1 data. This middle-down electron-activated dissociation (EAD) approach provides a potential method for site-specific characterization of PTMs in antibody products, offering valuable complementary mapping to support formulation development and production.