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| Item Type: | Article |
|---|---|
| Title: | Community benchmarking and evaluation of human unannotated microprotein detection by mass spectrometry based proteomics |
| Creators Name: | Wacholder, Aaron, Deutsch, Eric W., Kok, Leron W., van Dinter, Jip T., Lee, Jiwon, Wright, James C., Leblanc, Sebastien, Jayatissa, Ayodya H., Jiang, Kevin, Arefiev, Ihor, Cao, Kevin, Bourassa, Francis, Trifiro, Felix-Antoine, Bassani-Sternberg, Michal, Baranov, Pavel V., Bogaert, Annelies, Chothani, Sonia, Fierro-Monti, Ivo, Fijalkowska, Daria, Gevaert, Kris, Hubner, Norbert, Mudge, Jonathan M., Ruiz-Orera, Jorge, Schulz, Jana, Vizcaíno, Juan Antonio, Prensner, John R., Brunet, Marie A., Martinez, Thomas F., Slavoff, Sarah A., Roucou, Xavier, Choudhary, Jyoti S., van Heesch, Sebastiaan, Moritz, Robert L. and Carvunis, Anne-Ruxandra |
| 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 nonimmunopeptidomics 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. |
| Source: | Nature Communications |
| ISSN: | 2041-1723 |
| Publisher: | Nature Publishing Group |
| Date: | 21 January 2026 |
| Official Publication: | https://doi.org/10.1038/s41467-025-68002-x |
| PubMed: | View item in PubMed |
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