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

Mol Cell Proteomics. 2018 Dec;17(12):2347-2357. doi: 10.1074/mcp.RA118.000877. Epub 2018 Aug 31.


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.

Keywords: Bioinformatics searching; De novo sequencing; Human Leukocyte Antigen; Immunology; Immunopeptidomics; Mass Spectrometry; Peptidomics; Proteasome-spliced peptides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigen Presentation / physiology
  • Cell Line, Tumor
  • Computational Biology / methods*
  • Exome
  • Exome Sequencing
  • HLA Antigens / metabolism*
  • Humans
  • Ligands
  • Peptides / genetics*
  • Peptides / metabolism*
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Splicing*
  • Proteome
  • Signal Transduction
  • Tandem Mass Spectrometry


  • HLA Antigens
  • Ligands
  • Peptides
  • Proteome
  • Proteasome Endopeptidase Complex