From Congestion to Clarity: On the Complementarity of Resolving Power and Spectral Simplification for Intact Protein...

Top-down mass spectrometry (TDMS) is a powerful platform for the structural and functional analysis of intact proteins, enabling the detailed characterization of proteoforms and precise localization of post-translational modifications. The incorporation of alternative fragmentation techniques, such as electron transfer dissociation, electron transfer higher-energy collisional dissociation, and ultraviolet photodissociation, in instruments such as Tribrid Orbitrap mass spectrometers enhances sequence coverage and improves the confidence in PTM assignment. However, tandem mass spectrometry of intact proteins >30 kDa presents substantial challenges. The resulting spectra are often highly complex with overlapping product ion signals that complicate spectral interpretation. Although increasing the mass resolution can help resolve closely spaced product ions, it is often insufficient to fully alleviate spectral congestion for large proteins. In such cases, proton transfer charge reduction (PTCR) can simplify mass spectra by dispersing product ions across a wider mass-over-charge (m/z) range. In this study, we evaluated the impact on TDMS of increasing resolving power and PTCR-enabled spectral simplification using four intact proteins: enolase (46.6 kDa), carbonic anhydrase (29 kDa), myoglobin (16.9 kDa), and ubiquitin (8.6 kDa). For carbonic anhydrase, combined MS² fragmentation at low resolving power (60,000 at m/z 200) yielded 50.5% sequence coverage, which increased to 92.6% at high resolving power (480,000 at m/z 200) and further to 97.7% when PTCR was applied. This approach was applied to the characterization of biopharmaceuticals by analyzing the three digested and disulfide-reduced ∼25 kDa subunits of the NIST monoclonal antibody (mAb) on a liquid chromatography time scale.