Estimating the Contribution of Proteasomal Spliced Peptides to the HLA-I Ligandome

It has been reported that about 30% of the HLA-I ligands are produced by proteasomal splicing of two noncontiguous fragments of a parental protein. We report that the identification of many of those spliced peptides is ambiguous. With an alternative workflow, based on de novo sequencing and subsequent verification with multiple search tools, we estimate that the upper bound for the proportion of cis-spliced peptides is 2–6%. Nevertheless, the true contribution of spliced peptides to the ligandome may be much smaller. [Display omitted] Highlights •Reported proteasomal spliced HLA peptides do not fit the consensus binding motifs.•Their MS/MS spectrum matches suggest that many of them are ambiguous.•Our workflow is based on de novo sequencing, alignment, and multiple search tools.•The upper bound proportion of cis-spliced peptides is 2–6% and likely much smaller. 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 false discovery rate (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.