What Can Ribo-Seq, Immunopeptidomics, and Proteomics Tell Us About the Noncanonical Proteome?

Ribosome profiling (Ribo-Seq) has proven transformative for our understanding of the human genome and proteome by illuminating thousands of noncanonical sites of ribosome translation outside the currently annotated coding sequences (CDSs). A conservative estimate suggests that at least 7000 noncanonical ORFs are translated, which, at first glance, has the potential to expand the number of human protein CDSs by 30%, from ∼19,500 annotated CDSs to over 26,000 annotated CDSs. Yet, additional scrutiny of these ORFs has raised numerous questions about what fraction of them truly produce a protein product and what fraction of those can be understood as proteins according to conventional understanding of the term. Adding further complication is the fact that published estimates of noncanonical ORFs vary widely by around 30-fold, from several thousand to several hundred thousand. The summation of this research has left the genomics and proteomics communities both excited by the prospect of new coding regions in the human genome but searching for guidance on how to proceed. Here, we discuss the current state of noncanonical ORF research, databases, and interpretation, focusing on how to assess whether a given ORF can be said to be “protein coding.” [Display omitted] •Ribo-seq paired with proteomics-based methods optimally detects noncanonical ORFs.•Data quality and analytical pipelines impact the output of a Ribo-seq experiment.•Noncanonical ORF catalogs variably report both high- and low-stringency nominations.•A framework for standardized noncanonical ORF evidence will advance the field. The human genome encodes thousands of noncanonical ORFs along with protein-coding genes. As a nascent field, many questions about them remain: How many exist? Do they encode proteins? What evidence is needed for their verification? Central to these debates has been the advent of ribosome profiling (Ribo-Seq) to discern genome-wide ribosome occupancy and immunopeptidomics to detect peptides presented by major histocompatibility complex molecules. This article synthesizes the current state of noncanonical ORF research and proposes standards for their future investigation and reporting.