Abstract Type 1 diabetes (T1D) is caused by an autoimmune response that targets and destroys insulin-producing beta cells in the pancreas. Despite advancements in managing T1D, identifying specific antigens critical for T1D onset has been challenging. Recent research has uncovered hybrid insulin peptides (HIPs), which induce an autoimmune CD4 T cell response and may serve as specific autoantigens in T1D. HIPs are generated when a peptide fragment of proinsulin binds to a peptide fragment from other beta cell proteins. Presence of various HIPs has been validated through mass spectrometric analyses of human and murine islet samples. Several of those identified HIPs have a known CD4 T cell that specifically targets them in human and murine disease. We consider these HIPs as disease-relevant because we not only have mass spectrometric evidence for their existence, but also immunological evidence verifying their role in disease. In this proposal, we plan to study the role of disease-relevant HIPs formed by the enzyme Cathepsin D (CatD) in the progression of diabetes in non- obese diabetic (NOD) mice, a amahor animal model used for the study of T1D. We hypothesize that a mutation of an insulin leucine residue (targeted by CatD) can effectively prevent CatD-mediated HIP formation, resulting in a reduction of disease incidence in genetically modified mice compared to wildtype mice. We will assess HIP formation by analyzing mouse islet samples using mass spectrometry, confirm HIP content through T cell assays with HIP-reactive T cell clones, and monitor mice for disease incidence and immune cell infiltration of the pancreatic islets. The researchers will also investigate the origin of another subgroup of HIPs (identified in human islets) that do not form through a CatD-mediated process. For this we apply a proteomic strategy to probe human islet samples by mass spectrometry with the objective to identify the enzyme responsible for their formation. The results of these experiments will provide a better understanding of the role of HIPs in disease onset. Furthermore, the identification of additional HIP-forming processes in human islets may lead to the discovery of a new therapeutic target, that may allow us to selectively block formation of HIPs in beta-cells. The proposed studies also offer me the opportunity to expand my understanding of several fields including immunology, proteomics, and mass spectrometry. These combined skillsets will provide me with expertise needed to study numerous diseases and pathogenic process throughout my anticipated career as independent investigator.