Summary
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 maximally 2-4% 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.
- Abbreviations
- APPM
- Antigen processing and presentation machinery
- HLA
- Human Leukocyte Antigen
- HLA-Ip
- HLA class I binding peptides
- FDR
- False discovery rate; FP: false positive
- AA
- amino acid
- LC
- liquid chromatography
- MS
- Mass spectrometry
- MS/MS
- Tandem mass spectrometry
- PSPs
- Proteasome-spliced peptides
- DeNovo_spliced
- Spliced peptides identified by de-novo
- DeNovo_non-spliced
- Non-spliced peptides not in UniProt identified by de-novo
- LM_spliced
- Spliced peptides identified by Liepe et al.
- LM_UniProt
- UniProt peptides identified by Liepe et al.
- PSM
- Peptide spectrum match