Most Non-Canonical Proteins Uniquely Populate the Proteome or 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. Remarkably, of 14,498 proteins identified in three human B cell lymphomas, 2,503 are unannotated. Of these, 28% are new isoforms, and 72% are cryptic proteins encoded by ostensibly noncoding regions (60%) or frameshifted canonical genes (12%). Cryptic proteins are translated as efficiently as canonical proteins, have more predicted disordered residues and lower stability and critically, generate MHC-I peptides 5-fold more efficiently per translation event. Translating 5’ “untranslated” regions selectively hindered downstream translation of genes involved in transcription, translation and anti-viral responses. Novel protein isoforms showed strong enrichment for signaling pathways dysregulated in cancer. Only a small fraction of cryptic proteins detected in the proteome contribute to the MHC-I immunopeptidome, demonstrating the high preferential access of cryptic defective ribosomal products to the class I pathway.