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| Item Type: | Article |
|---|---|
| Title: | Community benchmarking and evaluation of human unannotated microprotein detection by mass spectrometry based proteomics |
| Creators: |
Wacholder, Aaron  ORCID: https://orcid.org/0000-0001-8739-0029, Deutsch, Eric W. ORCID: https://orcid.org/0000-0001-8732-0928, Kok, Leron W. ORCID: https://orcid.org/0009-0008-0841-2313, van Dinter, Jip T. ORCID: https://orcid.org/0000-0002-3749-1531, Lee, Jiwon ORCID: https://orcid.org/0000-0002-4079-7494, Wright, James C. ORCID: https://orcid.org/0000-0001-6950-4328, Leblanc, Sebastien, Jayatissa, Ayodya H., Jiang, Kevin ORCID: https://orcid.org/0000-0003-3258-4445, Arefiev, Ihor, Cao, Kevin, Bourassa, Francis ORCID: https://orcid.org/0009-0001-9012-7245, Trifiro, Felix-Antoine, Bassani-Sternberg, Michal ORCID: https://orcid.org/0000-0002-1934-954X, Baranov, Pavel V., Bogaert, Annelies, Chothani, Sonia ORCID: https://orcid.org/0000-0002-1010-7069, Fierro-Monti, Ivo ORCID: https://orcid.org/0000-0002-5460-2117, Fijalkowska, Daria, Gevaert, Kris ORCID: https://orcid.org/0000-0002-4237-0283, Hubner, Norbert ORCID: https://orcid.org/0000-0002-1218-6223, Mudge, Jonathan M. ORCID: https://orcid.org/0000-0003-4789-7495, Ruiz-Orera, Jorge ORCID: https://orcid.org/0000-0002-8317-0034, Schulz, Jana, Vizcaíno, Juan Antonio ORCID: https://orcid.org/0000-0002-3905-4335, Prensner, John R. ORCID: https://orcid.org/0000-0002-7024-636X, Brunet, Marie A. ORCID: https://orcid.org/0000-0001-5973-3522, Martinez, Thomas F. ORCID: https://orcid.org/0000-0002-4011-8164, Slavoff, Sarah A. ORCID: https://orcid.org/0000-0002-4443-2070, Roucou, Xavier ORCID: https://orcid.org/0000-0001-9370-5584, Choudhary, Jyoti S. ORCID: https://orcid.org/0000-0003-0881-5477, van Heesch, Sebastiaan ORCID: https://orcid.org/0000-0001-9593-1980, Moritz, Robert L. ORCID: https://orcid.org/0000-0002-3216-9447 and Carvunis, Anne-Ruxandra ORCID: https://orcid.org/0000-0002-6474-6413
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| Abstract: | 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. |
| Keywords: | Benchmarking, Mass Spectrometry, Molecular Sequence Annotation, Open Reading Frames, Peptides, Protein Databases, Proteins, Proteomics |
| Source: | Nature Communications |
| ISSN: | 2041-1723 |
| Publisher: | Nature Publishing Group |
| Volume: | 17 |
| Number: | 1 |
| Page Range: | 1241 |
| Date: | 2 February 2026 |
| Additional Information: | Erratum in: Nat Commun 17(1):4882. |
| Official Publication: | https://doi.org/10.1038/s41467-025-68002-x |
| PubMed: | View item in PubMed |
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