PROJECT SUMMARY Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the viral pneumonia outbreak of coronavirus disease 2019 (COVID-19) causing widespread morbidity and mortality. Main protease (Mpro) encoded by SARS-CoV-2 processes the viral polyproteins and facilitates viral replication. Once activated, this enzyme can evade the host innate immune responses by cleaving host proteins. The host protein substrates of SARS-CoV-2 Mpro are not well characterized. The major challenges with the direct detection of host protein targets is their low abundance and technical limitations with the specificity and sensitivity of the current methods. Thus, this proposal is designed to develop a more specific and sensitive approach termed N-terminomics to discover unique protein fragments that are generated by Mpro during SARS CoV-2 infection. The Specific Aims of this proposal are: 1) Development and validation of novel probes to identify protease fragments generated by SARS-CoV-2 Mpro in cell lysate and 2) Identify and quantify the endogenous substrates of SARS-CoV-2 Mpro in infected mammalian cells. The rationale for the proposed research is that its success would facilitate a greater understanding of which host pathways are altered by this SARS-CoV-2 to cause a severe disease condition. Moreover, host targets of MPro can be used to develop treatment regimens to treat severe cases if COVID-19. The expected outcome of this research is that our approach will be more sensitive and readily adaptable for the host substrate profiling of any pathogenic proteases and it will aid in mapping cellular pathways which are hijacked by pathogens to invade the host system.