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Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides that derive from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass-spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single cell transcriptomics, ribosome profiling, and a combination of two MS/MS search tools. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides and of an immunogenic peptide from a downstream reading frame in the melanoma stem cell marker gene ABCB5. It holds great promise for the discovery of novel cancer antigens for cancer immunotherapy.

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