Deep Proteoform Sequencing with Top-Down Direct Mass Technology

Individual Ion Mass Spectrometry (I²MS) using Direct Mass Technology mode on an Orbitrap mass spectrometer (DMTm) increases sensitivity, resolution, and mass range for protein analysis. Here, we present an end-to-end workflow for deep proteoform sequencing using top-down mass spectrometry with DMTm. By assigning the charge of individual fragment ions and converting spectra from the m/z to the mass domain, DMTm resolves overlapping isotopic distributions that have limited conventional top-down mass spectrometry. Across different fragmentation modes on Orbitrap mass spectrometers, top-down DMTm significantly outperformed conventional top-down mass spectrometry methods. For a glycosylated 50.8 kDa antibody heavy chain, sequence coverage was greatly increased, from 27.5% to 83.3%, in 10 minutes of acquisition using a single fragmentation mode. Coverage of the middle 350 residues improved from 0% to >95%, demonstrating near-complete coverage of the difficult-to-characterize internal region of a large protein. The fragmentation patterns of DMTm were found to be complementary to conventional top-down, with higher internal coverage for DMTm and higher terminal coverage for conventional. Accordingly, aggregation of the data from the two modes further increased heavy chain sequence coverage to 90.2%. A new software platform, Proteoform Studio, provided optimized ion processing for improved sequence coverage and enabled real-time experimental monitoring as individual ions were accumulated. The platform automatically integrates conventional and DMTm data to provide the most comprehensive sequence coverage possible. Together, these advances enable substantially deeper proteoform sequencing and establish a straightforward, complete top-down DMTm workflow to confidently define proteoforms in biological systems and biotherapeutic development.