Fig. 2: Immunopeptidomics unravels DAC + SB939-induced novel ORFs-derived HLA ligands.
a Number of ORFs predicted from DAC + SB939-induced transcripts stratified by their host transcript classification. b Frequency distribution of the number and (c) length of the predicted ORFs. d Volcano plot of differential protein abundance analyses, comparing DAC + SB939 vs DMSO treated NCI-H1299 cells measured by MS using predicted ORFs from DAC + SB939-induced transcripts as reference. e MS-identified HLA class I ligands from DAC + SB939 vs DMSO treated cells (n = 3 biological replicates; mean values ± SD; p-value was calculated using an unpaired two-tailed t test). f Overlap analysis of MS-identified HLA ligands from DAC + SB939 vs DMSO treated cells from all identified ligands (left) or novel ORF-exclusive ligands (right). g Peptide length distribution of HLA ligands exclusively assigned to novel ORFs compared to all identified canonical HLA ligands. h Ranked intensity values of MS acquired data derived from the combined immunopeptidomes of DAC + SB939 and DMSO treated cells (n = 3). Positions of novel ORF-derived HLA ligands are projected on the curve. i Comparative profiling of ORF-derived HLA ligands (n = 112) based on HLA-restricted presentation frequency in immunopeptidomes of DAC + SB939 or DMSO treated cells. Frequencies of positive immunopeptidomes for the respective HLA ligands (x-axis) are indicated on the y-axis. The box highlights treatment-induced neopeptides (t-neopeptides; n = 15) with presentation in 100% of the biological replicates. The corresponding calculated spectral correlation coefficients (R2) of the fragment spectra comparison are indicated below. j Fragment spectra comparison (m/z on the x-axis) of the experimentally eluted DAC + SB939-induced novel ORF-derived HLA class I-presented ligand LLLSYRYIY (PA*32) extracted from the DAC + SB939 treated cells (identification) to the synthetic peptide (validation, mirrored on the x-axis) with the calculated R2. Identified b- and y-ions are marked in red and blue, respectively. k Calculated R2 of the fragment spectra comparison of experimentally eluted and synthetic peptides plotted against the number of samples with ligand identification (n = 6). The band indicates the median, and the box indicates the first and third quartiles.
