Poster Presentation 27th Annual Lorne Proteomics Symposium 2022

The role of modifiers of the antigen presentation pathway on spliced peptide presentation in melanoma (#104)

Ritchlynn Mr Aranha 1 , Anthony Prof Purcell 1 , Nathan Dr Croft 1 , Pouya Dr Faridi 1
  1. Monash University, Clayton, VIC, Australia

The antigen presentation pathway is a complex pathway involving many proteases and aminopeptidases that shape the repertoire of peptides displayed by the cell surface (immunopeptidome). Any aberrations to this pathway can cause drastic changes to the cells immunopeptidome. Some of these aberrations are caused by the endoplasmic reticulum aminopeptidase (ERAP) that trims HLA-bound peptides at their N-terminus, while interferon-γ induces the immunoproteasome, thus leading to the presentation of a modified immunopeptidome. This immunopeptidome consists of both linear and spliced peptides. Splicing has been attributed to proteasomal-catalysed processes, but it is unknown what effect other aminopeptidases play. Studies have also shown that inhibition of ERAP leads to an increase in the presentation of immunogenic peptides. In this study, we used patient-derived melanoma cell lines treated in the presence or absence of an ERAP1 inhibitor or interferon-γ to investigate the impact on the immunopeptidome and identify potential novel peptide targets for T-cell immunotherapy. We used both data-dependent (DDA) and data-independent acquisition (DIA) mass spectrometry with PEAKS Studio software and bioinformatics algorithms to identify and quantify linear and spliced peptides. A large spectral library of ~120,000 peptides was generated, and this was used to interrogate DIA data, allowing quantification of ~5000-11000 unique peptides per sample. Spliced peptides made up between 23-34% of the immunopeptidome of cells, with no significant changes in the proportion of spliced peptides presented between different treatments. Although there was no significant change in the proportion of peptides, interferon-γ and ERAP1 inhibition remodelled the immunopeptidome of the melanoma cells and led to significant increases in the presentation of peptides derived from melanoma-associated antigens thus creating potentially novel linear and spliced targets for T-cell immunotherapy.