Beta-cells produce hybrid insulin peptides (HIPs) by linking insulin fragments to other peptides through peptide bonds. HIPs have unique amino acid sequences and are targeted by autoreactive T cells in type 1 diabetes (T1D). Individuals with recent-onset T1D have significantly higher levels of HIP-reactive T cells in their blood compared to non-diabetic control subjects. HIP-reactive T cells have also been found in the residual pancreatic islets of deceased T1D organ donors. In non-obese diabetic (NOD) mice, a major T1D animal model, several CD4 T cell clones that trigger diabetes have been shown to target HIPs. Through mass spectrometry, a subgroup of HIPs containing N-terminal amine groups of various peptides linked to aspartic acid residues of insulin C-peptide has been detected in NOD islets. Our research reveals that these HIPs form spontaneously in beta-cells via an aspartic anhydride intermediate mechanism. This process leads to the creation of a regular HIP with a standard peptide bond and a HIP-isomer (isoHIP) with an isopeptide bond linked to the carboxylic acid side-chain of the aspartic acid residue. Our mass spectrometric analyses confirmed the presence of both HIP isomers in murine islets, thereby validating the occurrence of this new reaction mechanism in beta-cells. The spontaneous formation of neoepitopes through the development of new peptide bonds within cells may contribute to the pathogenesis of T1D and other autoimmune diseases.