Most non-canonical proteins uniquely populate the proteome or immunopeptidome

Translation events that were previously thought to result in the production of non-biologically relevant proteins are now being reconsidered as increasing evidence shows that some non-canonical proteins may play an important role in immunity and disease. As our understanding of the flexibility of translation increases, so do the challenges with predicting what potential translation products may need to be considered when using databases to interpret LC-MS/MS data. Further, since many of these non-canonical proteins are low in abundance and degrade easily, finding enriched populations is key for optimising analysis workflows. In a recent paper from Ruiz Cuevas and others, these proteins were found to be overrepresented in the immunopeptidome from multiple B cell lymphomas, with high representation of novel isoforms and proteins encoded by putative non-coding regions or frameshifted canonical genes. Their proteogenomic strategy that utilized Ribo-seq and RNA sequencing (RNA-seq) identified translation events to produce a sample-specific database for MS analysis. The paper examines the unique features of the non-canonical translatome and is an important read for anyone interested cell surface MHC-I-associated peptides (MAPs) and how they may influence tumor immunosurveillance.

How was PEAKS used?

PEAKS Studio X was used to complete the database (DB) search on LC-MS/MS data obtained from peptides collected from cancer cells. The authors used their customized sample-specific databases in the DB workflow parameter settings to focus the search space to relevant peptides. They also completed the DB + PTM search workflow with a standard reference DB to check for improperly assigned post-translationally modified canonical peptides within their results.

Ruiz Cuevas MV, Hardy MP, Hollý J, Bonneil É, Durette C, Courcelles M, Lanoix J, Côté C, Staudt LM, Lemieux S, Thibault P, Perreault C, Yewdell JW. Most non-canonical proteins uniquely populate the proteome or immunopeptidome. Cell Rep. 2021 Mar 9;34(10):108815. doi:10.1016/j.celrep.2021.108815. PMID: 33691108; PMCID: PMC8040094.


Combining RNA sequencing, ribosome profiling, and mass spectrometry, we elucidate the contribution of non-canonical translation to the proteome and major histocompatibility complex (MHC) class I immunopeptidome. Remarkably, of 14,498 proteins identified in three human B cell lymphomas, 2,503 are non-canonical proteins. Of these, 28% are novel isoforms and 72% are cryptic proteins encoded by ostensibly non-coding 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 hinders downstream translation of genes involved in transcription, translation, and antiviral responses. Novel protein isoforms show strong enrichment for signaling pathways deregulated 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.