Characterization of Proteasome-Generated Spliced Peptides Detected by Mass Spectrometry
peer-reviewed · The Journal of Immunology · 2022
| Date | 2022-06-15 |
| Type | peer-reviewed |
| Venue | The Journal of Immunology |
| Publisher | Oxford University Press (OUP) |
| Contribution | downstream-application |
| DOI | 10.4049/jimmunol.2100717 |
| Citations (OpenAlex) | 5 |
Abstract
CD8 + T cells recognize peptides displayed by HLA class I molecules and monitor intracellular peptide pools. It is known that the proteasome splices two short peptide fragments. Recent studies using mass spectrometry (MS) and bioinformatics analysis have suggested that proteasome-generated spliced peptides (PSPs) may account for a substantial proportion of HLA class I ligands. However, the authenticity of the PSPs identified using bioinformatics approaches remain ambiguous. In this study, we employed MS-based de novo sequencing to directly capture cryptic HLA ligands that were not templated in the genome. We identified two PSPs originating from the same protein in a human colorectal cancer line with microsatellite instability. Healthy donor-derived CD8 + T cells readily responded to the two PSPs, showing their natural HLA presentation and antigenicity. Experiments using minigene constructs demonstrated proteasome-dependent processing of two PSPs generated by standard and reverse cis splicing, respectively. Our results suggest a broader diversity of HLA class I Ag repertoires generated by proteasomal splicing, supporting the advantage of MS-based approaches for the comprehensive identification of PSPs.
Methods and tools
- Proteasome-spliced HLA ligand discovery: De novo sequencing of HLA class I ligands from a colorectal cancer line found genome-untemplated proteasome-spliced peptides that CD8+ T cells recognise.
Methods it uses
- PEAKS: Commercial DP-based de novo