Tabatabaei Dakhili, S. Y.; Hinse, O.; Foster, L.; Bromme, D.

Cells generate peptides absent from reference proteomes, including products of reverse proteolysis and gene fusions. These are an increasingly pursued class of targets in autoimmune disease, cancer vaccines and T-cell therapies. The sequence is itself the discovery, yet a convincing spectral match does not establish it, because an ordinary genome-encoded peptide of the same mass can explain the spectrum equally well. Independent benchmarking has identified this ambiguity as a dominant source of error in reported non-canonical identifications. Here, we introduce TransPepMS, which adjudicates each proposed assignment and returns a verdict of retained, unresolved, or rejected. For example, on adjudicating 1,154 reported proteasome-spliced immunopeptides, only 53 survived same-mass competition, whereas 91 of 99 insulin-spike hybrids were retained and all 46 planted canonical peptides were rejected, showing that TransPepMS discriminates genuine non-canonical peptides rather than merely deflating claims. TransPepMS provides peptide validation before they guide biological interpretation and immune-target selection.