
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.09.612016
preprint
1
Article
High-quality peptide evidence for annotating non-canonical open reading frames as human proteins
Deutsch Eric W http://orcid.org/0000-0001-8732-0928

Kok Leron W http://orcid.org/0009-0008-0841-2313

Mudge Jonathan M http://orcid.org/0000-0003-4789-7495

Ruiz-Orera Jorge http://orcid.org/0000-0002-8317-0034

Fierro-Monti Ivo http://orcid.org/0000-0002-5460-2117

Sun Zhi http://orcid.org/0000-0003-3324-6851

Abelin Jennifer G http://orcid.org/0000-0001-5262-7241

Alba M Mar http://orcid.org/0000-0002-7963-7375

Aspden Julie L http://orcid.org/0000-0002-8537-6204

Bazzini Ariel A http://orcid.org/0000-0002-2251-5174

Bruford Elspeth A http://orcid.org/0000-0002-8380-5247

Brunet Marie A http://orcid.org/0000-0001-5973-3522

Calviello Lorenzo http://orcid.org/0000-0002-5600-0988

Carr Steven A http://orcid.org/0000-0002-7203-4299

Carvunis Anne-Ruxandra http://orcid.org/0000-0002-6474-6413

Chothani Sonia http://orcid.org/0000-0002-1010-7069

Clauwaert Jim http://orcid.org/0000-0002-5876-1406

Dean Kellie http://orcid.org/0000-0002-3213-8181

Faridi Pouya http://orcid.org/0000-0002-2712-3356

Frankish Adam http://orcid.org/0000-0002-4333-628X

Hubner Norbert http://orcid.org/0000-0002-1218-6223

Ingolia Nicholas T http://orcid.org/0000-0002-3395-1545

Magrane Michele http://orcid.org/0000-0003-3544-996X

Martin Maria Jesus http://orcid.org/0000-0001-5454-2815

Martinez Thomas F http://orcid.org/0000-0002-4011-8164

Menschaert Gerben http://orcid.org/0000-0002-7575-2085

Ohler Uwe http://orcid.org/0000-0002-0881-3116

Orchard Sandra http://orcid.org/0000-0002-8878-3972

Rackham Owen http://orcid.org/0000-0002-4390-0872

Roucou Xavier http://orcid.org/0000-0001-9370-5584

Slavoff Sarah A http://orcid.org/0000-0002-4443-2070

Valen Eivind http://orcid.org/0000-0003-1840-6108

Wacholder Aaron http://orcid.org/0000-0001-8739-0029

Weissman Jonathan S http://orcid.org/0000-0003-2445-670X

Wu Wei http://orcid.org/0000-0002-1092-603X

Xie Zhi http://orcid.org/0000-0002-5589-4836

Choudhary Jyoti http://orcid.org/0000-0003-0881-5477

Bassani-Sternberg Michal http://orcid.org/0000-0002-1934-954X

Vizcaíno Juan Antonio http://orcid.org/0000-0002-3905-4335

Ternette Nicola http://orcid.org/0000-0002-9283-0743

Moritz Robert L http://orcid.org/0000-0002-3216-9447

Prensner John R http://orcid.org/0000-0002-7024-636X

van Heesch Sebastiaan http://orcid.org/0000-0001-9593-1980

09 9 2024
2024.09.09.612016https://creativecommons.org/licenses/by-nd/4.0/ This work is licensed under a Creative Commons Attribution-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, and only so long as attribution is given to the creator. The license allows for commercial use.
http://biorxiv.org/lookup/doi/10.1101/2024.09.09.612016
nihpp-2024.09.09.612016.pdf
Abstract

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.
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