Project Summary/Abstract The long-term goal of this project is to develop of a rapid, inexpensive, and sensitive screening diagnostic based on an innovative broad proteomic approach to improve our ability to diagnose infectious disease, monitor changes in the microbiome, measure the state of the host immune system, and identify disease specific biomarkers. Specifically, the initiative will provide proof of principle for a method to achieve simultaneous multi-pathogen detection and deep proteomic microbiome characterization. We will work to detect the presence of several hundred pathogens while also identifying thousands of microbes at clinically relevant levels. The short amino acid sequences or peptides from the proteins will provide markers that range from the virus variant level, such as SARS- CoV-2 variants of concern, to the family level, such as coronaviridae. In developing this diagnostic, a key challenge is to maximize the sensitivity of peptide marker detection while analyzing for many clinically relevant viruses and bacteria. We will therefore compare the performance of (a) a data-dependent acquisition method that searches a custom database and (b) a data-independent acquisition method that can identify thousands of proteins. The proposed study will begin with the analysis of approximately twenty model pathogens before proceeding to the more ambitious analysis of more than 230 oro-respiratory samples. We will assess the primary factors that determine the performance of each method and use our milestones to select one approach for further development. In the end, this research will demonstrate the feasibility of a broad protein-based screening diagnostic that can detect numerous pathogens and provide microbiome profiling to ultimately improve the diagnosis of disease and assessment of human health. The project team includes researchers with diverse expertise from Penn Engineering, Microbiology, and Penn Medicine.