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

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Item Type:	Preprint
Title:	Detection of human unannotated microproteins by mass spectrometry-based proteomics: a community assessment
Creators Name:	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. and Carvunis, A.R.
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.
Source:	bioRxiv
Publisher:	Cold Spring Harbor Laboratory Press
Article Number:	2025.02.19.639069v2
Date:	26 February 2025
Official Publication:	https://doi.org/10.1101/2025.02.19.639069
PubMed:	View item in PubMed

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