A comprehensive solution for native top down mass spectrometry data analyses across structural biology and biopharma

PROJECT SUMMARY Native top down mass spectrometry is continuing to evolve into a powerhouse analytical technique that can generate transformative data on the native state of proteoforms and protein complexes. Technological advances in mass spectrometers have spurred the rapid rise of nTDMS over recent years, paving impressive avenues for structural biology research and protein therapeutics development. For instance, new insights into non-covalent ligand binding and localization can be revealed by nTDMS data that are complementary to traditional structural biology approaches such as electron microscopy and NMR. Drug developers can apply nTDMS to study therapeutic proteins in preclinical and clinical settings to learn more about modifications that are present and the complexes being formed. The number of novel protein and protein complex drug molecules is ever increasing, each with a host of difficult analytical and data analysis challenges. On the bioinformatics side of nTDMS, strides have been made in algorithmic development for mass determination of large molecules; however, no fully featured analysis platform for nTDMS currently exists that also integrates search. Here, Proteinaceous is developing and commercializing a new software named ProSight Native to fill this analysis void. In Phase I of the development, a first-of-its-kind platform was introduced for analyzing targeted protein complex data from the classic nTDMS complex-down experiment. The platform offers deconvolution for intact complexes and their dissociated subunits, as well as search for subunit identification and a stoichiometry calculation for complex composition determination. For the Phase II grant, a host of new research will be undertaken to enable a comprehensive platform to be commercialized that features an improved deconvolution algorithm for native proteoforms, the first-ever nTDMS-specific high-throughput search, and robust quantitation workflows for biopharma applications. Major development effort will be spent here on new techniques for nTDMS searches of both proteoforms and complexes, including a multi-pronged approach for scoring automated stoichiometry assignments. Additionally, structural biology elements will be integrated alongside proteoform fragmentation data to bring these important components together and facilitate connections between the two. Lastly, the results will be displayed in an intuitive, structural biology-centric viewer that will make analysis approachable for mass spectrometrists and non-mass spectrometrist alike. In total, ProSight Native will aim to significantly advance nTDMS with these novel features, while also increasing accessibility to the powerful data that can be generated using nTDMS techniques.