Next-generation Mass Spectrometry Technologies for Integrated Structural Proteomics- Renewal

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Project Summary Within each organism proteins are at work carrying out activities which impact every aspect of cellular function. A key factor in achieving such a wide range of protein functions involves the post translational modifications and sequence modifications that act to produce a vast array of functional protein states form a single gene product. Such proteoforms are further coupled directly to the 3D structures of the biomolecules created, which are further recruited into a wide array of dynamic multi-protein machines. Directly assessing the structures of these assembly states, along with the proteoforms that they contain is crucial for understanding human disease. Despite this, most structures remain unknown and are refractory to current technologies, and their proteoform compliment remains opaque. Standard structural biology approaches (X-ray, NMR, and Cryo-EM), while highly successful, require pure samples in large quantities, painstakingly optimized to produce monodisperse protein populations in every respect, and to remove spectral background. Furthermore, transient and polydisperse assemblies that exist within complex mixtures cannot be analyzed. Mass spectrometry (MS) approaches developed to attack this challenging problem can overcome many of these obstacles. While these tools are undergoing a rapid development phase, they currently lack the ability to discreetly assess the influence of proteoforms on multiprotein organization. Consequently there is a need to develop improved MS approaches capable of simultaneously assessing the structural proteome, enabling links between proteoform composition and 3D structure for the host of dynamic, heterogeneous macromolecular complexes of clear biomedical importance. This renewal application seeks to construct new, innovative MS techniques that 1) utilize new classes of chemical tagging reagents and mixed tagging methodologies to promote comprehensive sequencing of intact multi-protein complexes, 2) leverage next-generation cyclic ion mobility-mass spectrometry (IM-MS) technology to produce high-definition collision induced unfolding (CIU) and native top-down sequencing methods that enable improved identification of proteoforms within assemblies, 3) produce new techniques for the direct sequencing of membrane protein complexes, including laser-based activation of detergent clusters for improved data quality, 4) combine electron capture dissociation (ECD) with CIU for on-the-fly annotation of fingerprint data, and 5) automated methods for protein derivatization and clean-up compatible with native proteomics. This technology will be brought to bear to discover the structures of a series of selected proteoforms and complexes, each linked to human disease.