However, identifying immunopeptides from mass spectrometry (MS) data remains challenging, with a large portion of measured spectra that are left unannotated. In an immunopeptidomics experiment, the MHC-peptide complexes are purified and the immunopeptides are eluted (Fig. ). Unlike conventional tryptic peptides, the generation of immunopeptides lacks well-defined protease specificity rules, necessitating that every possible protein subsequence within human leukocyte antigen (HLA) class-specific length constraints has to be considered during spectrum annotation. Consequently, the search space significantly expands, resulting in increased false positive spectrum annotation rates and reduced peptide identification sensitivity . A compounding factor hereof is that the search space in immunopeptidomics is often even further extended beyond the canonical human proteome, by incorporating somatic mutations , pathogen genomes , novel or unannotated open reading frames (nuORFs) , and post-translational modifications (PTMs) . Additionally, the non-tryptic nature of immunopeptides results in unique spectral characteristics, such as strong internal ion series and neutral losses . Because many immunopeptides do not contain basic lysine or arginine residues at the C-terminus, they are often singly charged , in contrast to multiply charged peptides encountered during bottom-up proteomics, making their MS analysis more intricate due to poor ionization and incomplete fragmentation coverage . Moreover, immunopeptides are often present at low abundances, which can also cause changes in fragment ion intensities .