Extended Data Fig. 7: Immunopeptidomics analysis identifies tryptophan-associated aberrant peptides.

a, Scheme summarizing the filtration steps that led to the detection of HLA-bound aberrant peptides, containing W-associated frameshift sequences (derived from 16 samples: 4 NT, 4 +IFNγ, 4 mTpr, 4 metastases from patient 55); immunopeptidomics spectra were searched against W-specific frameshifted human proteome. MS-identified peptides were filtered according to described steps, including HLA binding prediction, spectral quality and source gene expression validation in transcriptome or translatome datasets. Following the above steps, 28 aberrant peptides were detected either in MD55 metastasis or treated MD55A3 samples, and not in the untreated cells. b, Predicted hydrophobicity index (HI) and observed retention time (RT) of canonical peptides (black) and aberrant peptides at two filtration steps described in a, step 3 (blue) and step 7 (red). The observed correlation between the RT versus HI, (using the SSRCalc tool^31,70) supports the identification of the aberrant peptides presented in a. c, Schematic representation of aberrant peptides KCNK6 and PCNX2 detected via immunopeptidomics, derived from a −1 and +1 frameshift at a Trp codon (marked in red), respectively. The in-frame and out of frame amino acid sequences are indicated above (black) and below (red/green) the mRNA sequence, respectively. d, Venn diagram including the 28 aberrant peptides detected following the filtration steps presented in a (were not identified in the untreated MD55A3 cells), grouped according to the samples they were detected in. e, Mean LFQ normalized intensities. Canonical and aberrant peptides that exhibit above threefold change in their intensities, are marked in black and blue/red, respectively. f, g, Similarities between the aberrant (n = 28) and canonical (n = 6,304) bound peptides. f, Andromeda score. g, Binding affinity prediction with NetMHCpan showing a similar binding distribution of the canonical and aberrant peptides. The aberrant peptides are highlighted in red. Dashed line mark strong binders rank cutoff (<0.5). h, A list of eight out of the thirteen −1 and +1 out-of-frame and trans-frame peptides found in the metastasis samples of patient 55. The colours of the peptide sequences represent in-frame (black), +1 frame (green) and −1 frame (red).