The capabilities of native mass spectrometry have improved dramatically in recent years due to advances ininstrument speed, resolution, mass accuracy, and ion optics designed specifically to handle large proteincomplexes. Despite these advances, the depth of characterization achievable by native mass spectrometry isstill limited due to inefficient dissociation and fragmentation of large protein complexes within the massspectrometer. At e-MSion, Inc., we have developed an efficient electron-fragmentation technology called ExDnow co-marketed with Agilent for their family of Q-TOFs, with Waters for their Q-IM-TOFs, and withThermo for their Ultra High Mass Range (UHMR) Orbitraps. The ExD technology provides extensivefragmentation of denatured and native proteins enabling thorough sequencing and localization ofposttranslational modifications. However, the large masses of many protein complexes now accessible bymass spectrometry make them particularly challenging to dissociate and fragment by electron-based ionactivation methods alone. Surface induced dissociation (SID) is a complementary technique capable ofdissociating 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 isincapable of separating intermolecular disulfide crosslinked proteins. We have shown our ExD technology isextremely 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 oftheir SID design, which now makes it possible to combine the two complementary approaches of nativeprotein dissociation and fragmentation. In this phase I proposal, we will evaluate the feasibility of developinga hybrid ExD-SID cell for the UHMR Orbitrap mass spectrometer to characterize disulfide-crosslinked nativeprotein complexes. Ion optics and electronics required to perform SID will be integrated into the ExD celland ExD controller for the UHMR Orbitrap platform. We will optimize the hybrid cell design to maximize iontransmission, ExD, SID, and ExD-SID experiments. The developed hybrid cell and methods will be appliedthe characterization of native antibodies as a model system. The hybrid ExD-SID cell will enable efficientfragmentation of disulfide bonds and dissociation of noncovalent interactions enabling separation of theintact heavy and light chains of the antibody. Success in addressing the feasibility question will yield apowerful 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 massspectrometry into the mainstream for structural biology approaches by greatly expanding the mass range ofmacromolecular complexes amenable to extensive characterization.
Public Health Relevance Statement: 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.
Project Terms: Antibodies ; Monoclonal Antibodies ; Clinical Treatment Moab ; mAbs ; Arthritis ; arthritic ; Biological Products ; Biologic Products ; Biological Agent ; biopharmaceutical ; biotherapeutic agent ; Biological Response Modifier Therapy ; Biologic Therapy ; Biological Therapy ; biological therapeutic ; biological treatment ; biotherapeutics ; biotherapy ; Capsid ; Cells ; Cell Body ; Cysteine ; Half-Cystine ; L-Cysteine ; Disease ; Disorder ; Disulfides ; Electronics ; electronic device ; Electrons ; Negative Beta Particle ; Negatrons ; Family ; Hybrid Cells ; Somatic Cell Hybrids ; Hybrids ; Ions ; Laboratories ; Light ; Photoradiation ; Mainstreaming ; Educational Mainstreaming ; achievement Mainstream Education ; Marriage ; Methods ; Biological Models ; Biologic Models ; Model System ; Nerve Degeneration ; Neuron Degeneration ; neural degeneration ; neurodegeneration ; neurodegenerative ; neurological degeneration ; neuronal degeneration ; Ohio ; Optics ; optical ; Periodicity ; Cyclicity ; Rhythmicity ; Post-Translational Protein Processing ; Post-Translational Modification Protein/Amino Acid Biochemistry ; Post-Translational Modifications ; Post-Translational Protein Modification ; Posttranslational Modifications ; Posttranslational Protein Processing ; Protein Modification ; Proteins ; Mass Spectrum Analysis ; Mass Photometry/Spectrum Analysis ; Mass Spectrometry ; Mass Spectroscopy ; Mass Spectrum ; Mass Spectrum Analyses ; Sulfhydryl Compounds ; Mercaptans ; Mercapto Compounds ; Thiols ; sulfhydryl group ; Technology ; Thinness ; Leanness ; Universities ; Water ; Hydrogen Oxide ; Work ; link protein ; cartilage link protein ; Businesses ; Dissociation ; base ; crosslink ; cross-link ; improved ; Left ; Surface ; Phase ; Individual ; tool ; instrument ; Nature ; Hour ; Complex ; Reaction ; Techniques ; Viral ; disulfide bond ; Performance ; stoichiometry ; structural biology ; success ; Speed ; novel ; Cell surface ; Devices ; Reporting ; Position ; Positioning Attribute ; Macromolecular Complexes ; design and construct ; design and construction ; Proteomics ; miniaturize ; viral respiratory infection ; Viral Respiratory Tract Infection ; protein complex ; Address ; Higher Order Chromatin Folding ; Higher Order Structure ; Higher Order Chromatin Structure ; Protein Analysis ; Resolution ; Small Business Innovation Research Grant ; SBIR ; Small Business Innovation Research ; transmission process ; Transmission ; Development ; developmental ; mass spectrometer ; design ; designing ; cost effective ; frontier ; Therapeutic antibodies ; Therapeutic Monoclonal Antibodies ; MAb Therapeutics ; monoclonal antibody drugs ; therapeutic mAbs ; protein crosslink ; lens ; lenses ; flexibility ; flexible ; experimental study ; experiment ; experimental research ; preservation ; detection sensitivity ;
