Fig. 3: uORF/uoORF-derived HLA-presented peptides exhibit comparable characteristics to annotated peptides.

a Schematic overview of the uORF/uoORF database creation using Riboseq and PRICE prediction, followed by the identification of non-canonical HLA peptides through liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based immunopeptidomics. b Number of peptides mapping to the annotated proteome (left panel) and uORF/uoORF-derived proteome (right panel) of proliferating and mitotically arrested U-2 OS cells. The distribution of predicted binding to HLA alleles in U-2 OS cells is shown. Data from biologically independent experiments (n = 3) c Percentage of eluted ligand (EL) peptides predicted by NetMHCpan-4.1 plotted against predicted binding affinity for peptides derived from the annotated proteome (small dots) and uORF/uoORF-derived peptides (large dots) in proliferating and mitotically arrested U-2 OS cells. Predicted binding to HLA alleles in U-2 OS cells is shown. Peptides are categorized as strong binders (%EL rank 0–0.5), weak binders (%EL rank 0.5–2), or non-binders (%EL rank 2–100). Data from biologically independent experiments (n = 3) d Length distribution of detected peptides mapping to the annotated proteome (left panel) and uORF/uoORF-derived peptides (right panel) in proliferating and mitotically arrested U-2 OS cells. The proportion of predicted binding to U-2 OS HLA alleles is shown. Data from biologically independent experiments (n = 3) e Peptide motif plots of unique peptides from the annotated proteome (6624) and unique peptides derived from uORFs/uoORFs (58), confidently identified as binding to the U-2 OS allele HLA-B44:02. f Observed retention time (RT) plotted against predicted RT indices for peptides from the annotated proteome (black) and uORF/uoORF-derived proteome (red) across all HLA alleles in proliferating and mitotically arrested U-2 OS cells. R² represents the Pearson correlation coefficient. Data from biologically independent experiments (n = 3).