Updated project metadata. Proteasomes are large multi-subunit enzymes that act as the main producers of antigenic peptides presented at the cell surface to CD8+ T cells. They can simply cut proteins or ligate non-contiguous peptide fragments thereby generating novel sequences, i.e. spliced peptides via a process called proteasome-catalysed peptide splicing (PCPS). In order to benchmark methods for spliced peptide identification, we here perform in vitro digestions of TSN2 and TSN89 synthetic polypeptide substrates by K562-derived 20S proteasome followed by LC-MS/MS measurements using Orbitrap Fusion Lumos mass spectrometer. Michele Mishto, Head of the research group Molecular Immunology at King’s College London and the Francis Crick Institute, London (UK). Email: michele.mishto@kcl.ac.uk