Fig. 1: A proteogenomics approach for the robust identification of noncHLAp.

a A schematic of the entire workflow is shown, where tissue samples or tumor cell lines were obtained from patients, and exome, RNA- and Ribo-Seq were performed to provide a framework to assess the non-canonical antigen repertoire. HLAp were immunoaffinity-purified from cancer cell lines and matched tumor/healthy lung tissues and then analyzed by MS. Immunopeptidomics spectra were then searched against RNA- and Ribo-Seq-based personalized protein sequence databases that contain non-canonical polypeptide sequences. MS-identified noncHLAIp were validated by targeted MS-based PRM and tested for immunogenicity using autologous T cells or PBMCs. b The percentage of predicted HLA binders of length 8–14 mer peptides with a MixMHCpred p-value ≤ 0.05 was used to evaluate the accuracy of the identified HLAIp by MaxQuant at 1% FDR as a function of database size (blue line). The percentage of predicted binders obtained for each condition is shown for each bar for the melanoma cell line 0D5P. c Different protein sequence databases combining whole-exome sequencing and inferences from RNA-Seq and Ribo-Seq data were utilized. NewAnce was implemented by retaining the PSM intersection of the two MS search tools MaxQuant and Comet, and applying group-specific FDR calculations for protHLAp and noncHLAp. Source data are provided as a Source Data file.