Dissecting the source and contribution of non-canonical translation to the proteome and MHC class I immunopeptidome.

Combining RNA-sequencing, ribosome profiling and mass spectrometry, we elucidate the contribution of non-canonical translation to the proteome and MHC class I immunopeptidome. Recent advances in sequencing ribosome protected RNA fragments (Ribo-seq/ ribosome profiling) and mass spectrometry (MS) analyses reveal that many proteins originate from translating non-canonical open reading frames (ORF). The short length and low abundance of non-canonical proteins makes their detection challenging. Here, we leverage the remarkable feature of antigen processing and presentation machinery to sample the (hidden) proteome , by virtue of MHC class I molecules binding and extending the life-span of antigenic peptides, the majority of which derive from defective ribosomal products (DRiPs). We describe a novel proteogenomic approach to identify non-canonical translation products present in whole cell extracts and/or in the immunopeptidome. Our findings demonstrate new features of the non-canonical translatome and their critical contribution to tumor immunosurveillance.