In order to fulfill their functions many protein complexes must first form dynamic complexes with multiple other proteins or binding partners. Characterization of the overall stoichiometry, topology, and inter- and intra-subunit contacts of protein and nucleoprotein complexes, and their assembly/disassembly, is critical because these complexes regulate key biological processes. Native mass spectrometry (nMS), particularly in combination with ion mobility (IM), and activation methods such as collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and surface induced dissociation (SID), is emerging as a powerful technique with which to study these complex systems and for guiding appropriate application of other structural biology tools. Despite the promise of nMS for structural biology, commercial instruments lack many of the tools necessary to fully characterize these complexes. SID, which is not yet commercialized, has proven to be an incredibly useful tool in the study of protein complexes, cleaving the weakest interfaces in the complex and producing sub- complexes that are reflective of the structure?s connectivity. While IM is commercially available on some platforms, the resolution is often insufficient for detailed structural studies. In TR&D1, we propose to enable higher energy SID to be performed, with more efficient fragment ion collection on multiple different instrument platforms. In TR&D2 we propose to develop high-resolution IM on a high-resolution Orbitrap instrument. In this TR&D we propose to couple the technologies developed in TR&Ds1 and 2, in addition to vendor prototype IM devices, in order to enable the full characterization of protein complexes using integrated, efficient workflows. Coupling of SID and IM is essential because when SID is placed before IM it allows conformational information to be obtained on the intact complex and the subcomplexes produced from SID even when the peaks overlap in m/z space, enabling structural models to be built. When SID is placed after IM, it allows different conformations of the intact complex (if present) to be individually mobility-selected for fragmentation. In addition to coupling SID and IM, we propose to combine these approaches with UVPD. This allows for the interrogation of complex assembly and subunit connectivity with SID and IM, with covalent fragmentation (sequencing of the peptide backbone) from UVPD. This approach will be beneficial in discerning ligand binding sites along with the sites of any post-translational modifications (PTMs). We propose to do this on multiple instrumental platforms, including the Waters Synapt G2(S), Thermo (Q) Exactive, and Bruker FTICR. The use of multiple platforms is necessary as each platform has different mass resolution, sensitivity, and speed, and certain platforms will be better suited to certain complexes. Hence incorporation with multiple platforms allows the experiments to be customized to the complex of interest. The use of multiple platforms is also essential as it allows better dissemination to the wider community, who may have access to only one of these platforms.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Biotechnology Resource Grants (P41)
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Special Emphasis Panel (ZRG1)
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Ohio State University
United States
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