TransPepMS: A Tool to Verify Peptides Absent from Reference Proteomes

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.