Dual Electron-Based Fragmentation with Ion Mobility to Advance Native Top-Down Proteomics

The identification and quantification of biological macromolecules remain challenging despite major advances in the speed, resolution and mass accuracy of modern mass spectrometers. A key weakness with current instrumentation lies in the methods used to induce fragmentation. The reliance in particular on collision-induced dissociation (CID) has limited such analyses to bottom-up workflows of trypsin-digested peptides of 10-30 residues. At e-MSion, we have developed an efficient electron-fragmentation technology called ExD now co-marketed with Agilent for their family of Q-TOFs and with Thermo for their QE Orbitraps. We succeeded with our phase I feasibility question to raise the fragmentation efficiency for doubly charged peptides from 1-3% to approaching 20%. This makes our ExD technology practical for peptide characterization and PTM localization in bottom up workflows -- the bread and butter for most proteomics laboratories. What has really captured the interest of the biopharma and the top-down communities in the past year is the exceptional sequence coverage of native proteins we obtain with the same ExD cell. The resulting spectra are less congested than those obtained with ETD/UVPD/CID fragmentation methodologies and it works for larger macromolecular protein complexes than has ever been possible before. Even with our simpler fragmentation patterns, the spectral congestion from proteoforms greater than ~30 kDa becomes too complex for many fragments to be distinguished even the highest resolution mass spectrometers. Our ExD technology is also faster than all other electron-based fragmentation methods. This speed allows entire proteins to be sequenced even after Ion Mobility Separations (IMS), which allows for spectra to be better resolved by adding a fourth dimension of resolution. Because of this unique capability, Waters recently purchased a prototype of our ExD cell adapted to fit at the exit of the IMS in their Synapt G2 mass spectrometer. Shortly after installation, we were able to sequence hemoglobin variants from native tetramers directly sprayed from human red blood cell lysates, FAB antibody subunits, and alcohol dehydrogenase (150 kDa). Some complexes such as GroEL and viral capsids still resist dissociation. We propose to overcome the challenges of both spectral congestion and dissociation of large native complexes by utilizing dual ExD cells with IMS. We will optimize the entrance-ExD cell to dissociate native protein complexes and use the exit- ExD cell to further fragment IMS-resolved subunits. We will develop the control electronics and software needed to coordinate the behavior of the two ExD cells with the IMS operation. Success will make possible characterization of larger proteoforms by top-down native proteomics than possible before. The adoption of our technology offers an extremely cost-effective solution that will accelerate the ability of many NIH investigators to probe disease mechanisms by characterizing complex macromolecules under native conditions with increased accuracy, speed, and fewer misidentifications.Project Narrative: Even with all of the scientific progress made to date, the complexity of disease- affected tissues still challenges our ability to probe what makes people sick. The goal of this Phase II SBIR project is to develop a powerful tool for characterize biological molecules into identifiable fragments that will improve the diagnosis and treatment of diseases ranging from arthritis, cancer, diabetes to heart disease and neurodegeneration.