Detection of human unannotated microproteins by mass spectrometry-based proteomics: a community assessment

Wacholder, A.; Deutsch, E. W.; Kok, L. W.; van Dinter, J. T.; Lee, J.; Wright, J. C.; Leblanc, S.; Jayatissa, A. H.; Jiang, K.; Arefiev, I.; Cao, K.; Bourassa, F.; Trifiro, F.-A.; Bassani-Sternberg, M.; Baranov, P. V.; Bogaert, A.; Chothani, S.; Fierro-Monti, I.; Fijalkowska, D.; Gevaert, K.; Hubner, N.; Mudge, J. M.; Ruiz-Orera, J.; Schulz, J.; Vizcaino, J. A.; Prensner, J. R.; Brunet, M. A.; Martinez, T. F.; Slavoff, S. A.; Roucou, X.; Chaudhary, J. S.; van Heesch, S.; Moritz, R. L.; Carvunis, A.-R.

doi:10.1101/2025.02.19.639069

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bioRxiv · 10.1101/2025.02.19.639069

Detection of human unannotated microproteins by mass spectrometry-based proteomics: a community assessment

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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.

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Wacholder, A., Deutsch, E. W., Kok, L. W., van Dinter, J. T., Lee, J., Wright, J. C., Leblanc, S., Jayatissa, A. H., Jiang, K., Arefiev, I., Cao, K., Bourassa, F., Trifiro, F.-A., Bassani-Sternberg, M., Baranov, P. V., Bogaert, A., Chothani, S., Fierro-Monti, I., Fijalkowska, D., Gevaert, K., Hubner, N., Mudge, J. M., Ruiz-Orera, J., Schulz, J., Vizcaino, J. A., Prensner, J. R., Brunet, M. A., Martinez, T. F., Slavoff, S. A., Roucou, X., Chaudhary, J. S., van Heesch, S., Moritz, R. L., Carvunis, A.-R.. 2025-02-23. Detection of human unannotated microproteins by mass spectrometry-based proteomics: a community assessment. https://doi.org/10.1101/2025.02.19.639069

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