Updated project metadata. Spliced peptides are short protein fragments spliced together in the proteasome by peptide bond formation. True estimation of the contribution of proteasome-spliced peptides (PSPs) to the global Human Leukocyte Antigen (HLA) ligandome is critical. A recent study suggested that PSPs contribute up to 30% of the HLA ligandome. We performed a thorough reanalysis of the reported results using multiple computational tools and various validation steps and concluded that only a fraction of the proposed PSPs passes the quality filters. To better estimate the actual number of PSPs, we present an alternative workflow. We performed de-novo sequencing of the HLA-peptide spectra and discarded all de-novo sequences found in the UniProt database. We checked whether the remaining de-novo sequences could match spliced peptides from human proteins. The spliced sequences were appended to the UniProt fasta file, which was searched by two search tools at a FDR of 1%. We find that 2-6% of the HLA ligandome could be explained as spliced protein fragments. The majority of these potential PSPs have good peptide-spectrum match properties and are predicted to bind the respective HLA molecules. However, it remains to be shown how many of these potential PSPs actually originate from proteasomal splicing events.