The immunopeptidome comprises the suite of human leukocyte antigen (HLA)-bound peptides that are presented at the cell surface for recognition by patrolling T cells, which is key to both antibody and cytotoxic responses associated with protection against pathogens and cancer. HLA molecules may present peptides with post-translational modifications (PTMs), contributing to the diversity of possible T-cell targets. While glycosylation is an important and ubiquitous PTM within the cell, its presence within the immunopeptidome remains underexplored. We have shown that the HLA class II repertoire is populated extensively by trimmed N-glycans; however, the situation for HLA class I remains unclear, largely due to the lower proportion of glycopeptides presented by these molecules and the technical challenges associated with the annotation and identification of their mass spectra.
A major analytical challenge posed by class I-bound glycoimmunopeptides is that glycans are modifying the already diverse repertoire of immunopeptides, resulting in a vast and complex search space. We sought to streamline and improve analysis of glycoimmunopeptidome data through in silico enrichment of putative glycosylated spectra, filtering for signature oxonium ions liberated following collision-induced dissociation LC-MS/MS. We also developed an in-house algorithm to generate dataset-specific glycan lists to serve as a pseudo-glycomics database. Benchmarking this approach using Byonic, a leading search engine for glycopeptides, we found that search times for immunopeptide data were reduced from days to hours. We found that HLA class I immunopeptidome data contains a small yet significant fraction (~0.5-10%) of glycopeptide-derived spectra that conform to expected HLA class I binding motifs. These glycopeptides are decorated by a suite of N- and O-linked glycans of variable proportions dependent upon the presenting HLA allotype.
These tools improve our ability to search immunopeptidomic datasets for glycopeptides, which is an important step towards comprehensive characterisation of glycoimmunopeptides and enriching understanding of the glycosylated immunopeptidome.