Targeted in situ cross-linking for determination of structure and interactions of SARS-CoV-2 proteins in the cellular context

Discovery of host-pathogen intra-cellular interactions is critical for identification of mechanisms of infection and discovery. Affinity purification based on Mass Spectrometry (AP-MS) approaches can identify the host-pathogen protein-protein interaction network that includes hundreds of cellular proteins. For example, over 330 interactions were identified for SARS-CoV-2 major proteins. However, identification of the critical anchoring interactions directly induced by the viral proteins is still challenging. Here we developed a complete workflow of in-situ XL-MS followed by AP-MS, and employ it to investigate SARS-CoV2 protein inside the host cell. We focused on three Sars-Cov-2 proteins for which the structural information is either missing or incomplete: NSP1, NSP2, and the Nucleocapsid protein (N protein). AP-MS revealed that several human proteins were co-eluting with NSP1 and NSP2 while protein N eluted alone.