Over the entire project period, work focussed mainly on objectives two and four. Due to the COVID-19 pandemic, the focus shifted further to investigating especially coronaviral protein complexes using mainly the basic, unmodified mass spectrometric setup.
Different components to modify an orbitrap UHMR have been obtained and tested. Multi-stage fragmentation and MS^N is now enabled by an omnitrap interfaced with the commercial system. This allows for slow CID, ECD and UVPD fragmentation. The setup will be further explored in follow-up projects and represents a unique resource for native top-down MS.
Main efforts in the project have focussed on assessing soft X-rays at FLASH and FLASH II free-electron lasers and PETRA III synchrotron for efficient fragmentation and dissociation of standard proteins and protein complexes. The results show potential for the methodology especially in combination with conformational separation in ion mobility.
The main biological question of SPOCk’S MS is how coronaviral replication/transcription complexes assemble and function and thereby facilitate replication of coronaviruses (CoVs). In a first publication (Krichel et al 2020), polyprotein processing of the regulatory region nsp7-10 through the viral protease in SARS-CoV-1 was monitored by native MS assessing processing order and efficiency of the three cleavage sites. Crucially, complex formation of released nsp7 and nsp8 could be observed simultaneously showing a heterotetramer, of which the topology was deduced. The complex was examined further in a follow-up (Krichel et al. 2021). The study revealed two distinct assembly pathways for nsp7+8 complexes resulting in alternate stoichiometry and topology in different CoVs. Processing was then also compared for nsp7-11 (the full intermediate observed in cells) in four different CoVs, which were also included in the complexation study. While processing order is largely conserved, subtle differences exist and are also reflected upon assembly of released subunits to nsp16. Crucially, we could deduce rate constants for all four cleavage sites in parallel (Schamoni-Kast et al. 2024 bioRxiv).
Importantly, in light of the SARS-CoV-2 pandemic, efforts on the biological aspects of the project were intensified. Instead of SARS-CoV-1, we switched to production and analysis of SARS-CoV-2 proteins and then also compared to various human CoVs to deduce general principles. Moreover, we assisted in screening antiviral targeting the main viral protease and further mass spectrometric investigations of this emergent virus (Günther et al 2021). Additionally, we performed investigations on Lassa virus, which revealed insights into viral assembly (Sänger et al 2023).
