Leveraging complementary ion activation methods with proton transfer charge reduction reactions for comprehensive characterization of monoclonal antibody heavy chain subunits

Characterization of large proteins by top-down mass spectrometry is challenged by low S/N of fragment ions and spectral congestion. Proton transfer charge reduction (PTCR) is one strategy that has shown great potential for addressing spectral congestion and enhancing sequence coverage of large proteins, but low S/N remains an obstacle, requiring extensive spectral averaging. Here we advance the characterization of large proteins, including an antibody (mAb) and an antibody drug conjugate (ADC), on a liquid chromatography timescale by implementing a hybrid strategy that combines ultraviolet photodissociation (UVPD), electron transfer higher collision energy dissociation (EThcD), PTCR, and gas-phase fractionation. By leveraging purposeful chromatographic peak broadening, fractionation + PTCR strategies, and the complementary nature of multiple activation methods, sequence coverages as a high as 83% and 79% were achieved for 50 kDa heavy chain (Hc) subunits of an mAb and ADC, respectively. Furthermore, unambiguous differentiation of two payload positional isomers of the ADC Hc was achieved.