Community benchmarking and evaluation of human unannotated microprotein detection by mass spectrometry based proteomics
Thousands of short open reading frames (sORFs) are translated outside of annotated coding sequences. Recent studies have pioneered searching for sORF-encoded microproteins in mass spectrometry (MS)-based proteomics and peptidomics datasets. Here, we assessed literature-reported MS-based identifications of unannotated human proteins. We find that studies vary by three orders of magnitude in the number of unannotated proteins they report. Of nearly 10,000 reported sORF-encoded peptides, 96% were unique to a single study, and 12% mapped to annotated proteins or proteoforms. Manual curation of a benchmark dataset of 406 manually evaluated spectra from 204 sORF-encoded proteins revealed large variation in peptide-spectrum match (PSM) quality between studies, with immunopeptidomics studies generally reporting higher quality PSMs than conventional enzymatic digests of whole cell lysates. We estimate that 65% of predicted sORF-encoded protein detections in immunopeptidomics studies were supported by high-quality PSMs versus 7.8% in non-immunopeptidomics datasets. Our work stresses the need for standardized protocols and analysis workflows to guide future advancements in microprotein detection by MS towards uncovering how many human microproteins exist.
Authors
- Kevin Jiang (ORCID: https://orcid.org/0000-0003-3258-4445)
- Jyoti S. Choudhary (ORCID: https://orcid.org/0000-0003-0881-5477)
- Sébastien Leblanc (ORCID: https://orcid.org/0000-0003-2599-6716)
- Sonia Chothani (ORCID: https://orcid.org/0000-0002-1010-7069)
- Thomas F. Martínez (ORCID: https://orcid.org/0000-0002-4011-8164)
- Ivo Fierro-Monti
- Xavier ROUCOU
- Jonathan M. Mudge (ORCID: https://orcid.org/0000-0003-4789-7495)
- Jorge Ruiz‐Orera (ORCID: https://orcid.org/0000-0002-8317-0034)
- Sarah A. Slavoff (ORCID: https://orcid.org/0000-0002-4443-2070)
- Anne‐Ruxandra Carvunis (ORCID: https://orcid.org/0000-0002-6474-6413)
- Juan Antonio Vizcaíno (ORCID: https://orcid.org/0000-0002-3905-4335)
- Jip T. van Dinter (ORCID: https://orcid.org/0000-0002-3749-1531)
- James C. Wright (ORCID: https://orcid.org/0000-0001-6950-4328)
- Jiwon Lee (ORCID: https://orcid.org/0009-0009-2250-3315)
- Marie A. Brunet (ORCID: https://orcid.org/0000-0001-5973-3522)
- Michal Bassani‐Sternberg (ORCID: https://orcid.org/0000-0002-1934-954X)
- Sebastiaan van Heesch (ORCID: https://orcid.org/0000-0001-9593-1980)
- Leron W. Kok (ORCID: https://orcid.org/0009-0008-0841-2313)
- Ihor Arefiev
- Francis Bourassa (ORCID: https://orcid.org/0009-0001-9012-7245)
- Félix-Antoine Trifiro
- Ayodya H Jayatissa (ORCID: https://orcid.org/0009-0002-7063-6365)
- Kevin Cao
- Jana Schulz
- Eric W. Deutsch
- Annelies Bogaert
- Aaron Wacholder
- Pavel V. Baranov
- Robert L. Moritz
- John R. Prensner
- Kris Gevaert
Institutions
- European Bioinformatics Institute (GB)
- Helmholtz Association of German Research Centres (DE)
- Université de Sherbrooke (CA)
- Institute of Cancer Research (GB)
- University of Pittsburgh (US)
- University of California, Irvine (US)
- Max Delbrück Center (DE)
- Heidelberg University (DE)
- University College Cork (IE)
- University of Michigan (US)
- Cancer Research UK (GB)
- Centre Hospitalier Universitaire de Sherbrooke (CA)
- Centre Hospitalier Universitaire Vaudois (CH)
- Institute for Systems Biology (US)
- Ghent University (BE)
- Yale University (US)
- Health and Social Services Centre University Institute of Geriatrics of Sherbrooke (CA)
- Granta Design (United Kingdom) (GB)
- Oncode Institute (NL)
- Ludwig Cancer Research (CH)
- Systems Biology Institute (JP)
- Duke-NUS Medical School (SG)
- Princess Máxima Center (NL)
- German Centre for Cardiovascular Research (DE)
- VIB-UGent Center for Medical Biotechnology (BE)
- Charité - Universitätsmedizin Berlin (DE)
- Heidelberg University (US)
Publication Details
- Journal
- Nature Communications
- Published
- 2026-01-21
- DOI
- https://doi.org/10.1038/s41467-025-68002-x
- Citations
- 3
- Primary Topic
- Advanced Proteomics Techniques and Applications
- Type
- article
- Field-Weighted Citation Impact
- 48.78
Funders
- National Science Foundation
- Damon Runyon Cancer Research Foundation
- CureSearch for Children's Cancer
- Ludwig Institute for Cancer Research
- Wellcome Trust
- Deutsches Zentrum für Herz-Kreislaufforschung
- Morgan Adams Foundation
- European Molecular Biology Laboratory
- Hope Foundation
- Curing Kids Cancer
- British Heart Foundation
- European Commission
- Deutsche Forschungsgemeinschaft
- Fondation Leducq
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
- Fonds Wetenschappelijk Onderzoek
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek
- Vlaamse regering
- Swiss Cancer Research Foundation
- Swiss Bridge Foundation
- Stichting Villa Joep
- National Institutes of Health
- Canadian Institutes of Health Research
- Fonds de Recherche du Québec - Santé
- National Human Genome Research Institute
- National Cancer Institute
- Hyundai Hope On Wheels