The soluble HLA repertoire captures neoantigenic, cryptic, and tumour-associated peptides in melanoma

Melanoma is a highly immunogenic malignancy in which T cell recognition of HLA-presented peptides plays a central role in tumour control. While immunopeptidomics has enabled detailed characterisation of melanoma antigen repertoires, its clinical application is limited by restricted access to tumour tissue. Soluble HLA (sHLA) complexes in circulation present a potential minimally invasive alternative. However, the extent to which circulating HLA repertoires reflect tumour-derived antigen presentation remains unclear. Here, we applied an optimised SAPrIm workflow coupled with Orbitrap Astral mass spectrometry to analyse the immunopeptidome of matched tumour tissue and plasma samples from five melanoma patients collected at the time of tumour resection. We identified 6,590-17,690 non-redundant HLA-I peptides from tumour tissue (5-25 mg input) and 5,213-10,089 non-redundant HLA peptides from plasma-derived sHLA complexes (200-571 μL input within the volume of a routine biobank aliquot). Plasma-derived sHLA ligandomes retained key features of membrane-bound tumour HLA-I presentation, including dominant 9-mer peptide length distributions and conserved HLA-binding characteristics. Peptide recovery was strongly concordant between compartments following normalisation (Pearson r = 0.92), indicating that ligandome depth and recovery were primarily driven by intrinsic biological variation rather than sample input. Tumour-associated antigens were consistently detected in circulating sHLA peptides. Cryptic peptides derived from "peptideins" were preserved in circulation and comprised approximately 1% of the total repertoire. Somatic patient-specific variant calling revealed a candidate neo-peptide is also preserved and presented in sHLA. To assess tumour specificity and biomarker potential, identified HLA peptides were mapped to their corresponding source proteins and compared against healthy sHLA and benign tissue reference datasets. This analysis identified 247 candidate melanoma-associated source proteins not detected in healthy plasma or benign tissue reference datasets. Among these, 9 source proteins were recurrently detected across at least 4 of 5 patients, representing priority candidates for further biomarker validation. In this proof-of-concept study of five matched patients, these findings indicate that circulating sHLA ligandomes preserve core features of tumour antigen presentation and provide proof-of-concept support for plasma immunopeptidomics as a minimally invasive approach for antigen discovery and biomarker hypothesis generation.