The capabilities of native mass spectrometry have improved dramatically in recent years due to advances in instrument speed, resolution, mass accuracy, and ion optics designed specifically to handle large protein complexes. Despite these advances, the depth of characterization achievable by native mass spectrometry is still limited due to inefficient dissociation and fragmentation of large protein complexes within the mass spectrometer. At e-MSion, Inc., we have developed an efficient electron-fragmentation technology called ExD now co-marketed with Agilent for their family of Q-TOFs, with Waters for their Q-IM-TOFs, and with Thermo for their Ultra High Mass Range (UHMR) Orbitraps. The ExD technology provides extensive fragmentation of denatured and native proteins enabling thorough sequencing and localization of posttranslational modifications. However, the large masses of many protein complexes now accessible by mass spectrometry make them particularly challenging to dissociate and fragment by electron-based ion activation methods alone. Surface induced dissociation (SID) is a complementary technique capable of dissociating large protein complexes to reveal higher order structure, such as subunit stoichiometry, topology, and interfaces, with minimal unfolding and fragmentation of the subunits. However, SID is incapable of separating intermolecular disulfide crosslinked proteins. We have shown our ExD technology is extremely effective at cutting multiple disulfide bonds in Cysteine Knot Proteins and monoclonal antibodies. Recent advances in the Wysocki lab at Ohio State University have resulted in a remarkable shortening of their SID design, which now makes it possible to combine the two complementary approaches of native protein dissociation and fragmentation. In this phase I proposal, we will evaluate the feasibility of developing a hybrid ExD-SID cell for the UHMR Orbitrap mass spectrometer to characterize disulfide-crosslinked native protein complexes. Ion optics and electronics required to perform SID will be integrated into the ExD cell and ExD controller for the UHMR Orbitrap platform. We will optimize the hybrid cell design to maximize ion transmission, ExD, SID, and ExD-SID experiments. The developed hybrid cell and methods will be applied the characterization of native antibodies as a model system. The hybrid ExD-SID cell will enable efficient fragmentation of disulfide bonds and dissociation of noncovalent interactions enabling separation of the intact heavy and light chains of the antibody. Success in addressing the feasibility question will yield a powerful tool for rapid characterization and discovery of monoclonal antibody therapeutics. More broadly, successful development of a hybrid ExD-SID cell will create a tool capable of bringing native mass spectrometry into the mainstream for structural biology approaches by greatly expanding the mass range of macromolecular complexes amenable to extensive characterization.

Even with all of the scientific progress made to date, the complexity of disease still challenges our ability to probe what makes people sick. This project will develop a hybrid technology to better characterizing antibodies as biotherapeutic molecules that will improve the treatment of diseases ranging from arthritis to neurodegeneration as well as respiratory viral infections and the body’s defensive reactions.