Are There Indeed Spliced Peptides in the Immunopeptidome?

The claims that a large fraction of the immunopeptidome is composed of spliced major histocompatibility complex (MHC) peptides have stirred significant excitement and raised controversy. Here, I suggest that there are likely no spliced peptides in the immunopeptidome, and if they exist at all, they are extremely rare. I base this claim on both biochemical and bioinformatics considerations. First, as a reactant in normal proteolytic reactions, water will compete with transpeptidation, which has been suggested as the mechanism of peptide splicing. The high mobility and abundance of water in aqueous solutions renders transpeptidation very inefficient and therefore unlikely to occur. Second, new studies have refuted the bioinformatics assignments to spliced peptides of most of the immunopeptidome MS data, suggesting that the correct assignments are likely other canonical, noncanonical, and post-translationally modified peptides. Therefore, I call for rigorous experimental methodology using heavy stable isotope peptides spiking into the immunoaffinity-purified mixtures of natural MHC peptides and analysis by the highly reliable targeted MS, to claim that MHC peptides are indeed spliced.