The primary market focus for the high-end mass spectrometer industry is to fully characterize proteins and their post-translational modifications (PTMs) within the biopharmaceutical industry. These analyses remain challenging despite major advances in the speed, resolution and mass accuracy of modern mass spectrometers. A key weakness with current instrumentation for protein characterization 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. When subjected to CID, many fragile PTMs on these short peptides are lost in complex ways. An alternative fragmentation methodology called electron capture dissociation (ECD) is well known for producing exceptionally clean spectra of entire proteins while also preserving PTMs. However, this technology has been feasible only in expensive FTICR mass spectrometers. The difficulty arises from confining enough low-energy electrons to efficiently fragment peptide bonds, which has limited the application of ECD in other instruments. The e-MSion team has developed an efficient ECD technology to confine electrons with a carefully designed magnetic field that operates without affecting the ion flightpath in mass spectrometers. One major advantage of our technology over competing fragmentation techniques such as ETD is speed. We established Phase I feasibility by showing that our ECD technology is fast enough to be used in quadrupole-Time of Flight (Q- ToF) mass spectrometers at speeds compatible with UPLC and ion mobility-based separations of complex samples. Our technology also efficiently supports sequencing of proteins as large as 30 kDa in seconds while leaving even the most fragile PTMs intact. The proposed Phase II SBIR project will complete the optimization/integration of our ECD into Q-ToF's to make the operation seamless for two major manufacturers of Q-ToFs. The primary commercial goal is to become a value-added reseller for upgrading Q- Tofs in Phase III. To accomplish this, our first Aim is to refine the engineering, software integration and application to middle- and top-down protein characterization. Aim 2 is to work with early adopters in both Biopharma and in proteomics fields to demonstrate the capabilities of the technology. The third Aim is to further modify the design of the ECD cell to perform Electron-Induced Dissociation (EID) more efficiently for the characterization of singly charged peptides and glycoproteins. This entails subtle modifications to the current ECD cell that allows larger quantities of higher-energy electrons to flow through the system. Completion of Aim 3 will open the market for triple-quad mass spectrometers, which is five times larger than the more expensive Q-ToFs. The adoption of our technology will accelerate the ability of many NIH investigators as well as BioPharma to probe disease mechanisms by characterizing macromolecules in complex biological samples with increased accuracy and speed, while reducing false discoveries.