High-resolution multi-omics enhances prediction and detection of smORF-encoded proteins in the human gut microbiome

Megan E. Davin; Júlia Ortís Sunyer; Luis F. Delgado; Steven L. Tavis; Tuesday Lowndes; Zainab Zafar; Jordan Caussin; Rashi Halder; Oskar Hickl; Cédric C. Laczny; Etienne Hanslian; Daniela A. Koppold; Anika Rajput-Khokhar; Nico Steckhan; Sebastian Schade; Jochen Schneider; Brit Mollenhauer; Andreas Michalsen; Patrick May; Robert L. Hettich; Paul Wilmes

doi:10.1038/s41467-026-72762-5

High-resolution multi-omics enhances prediction and detection of smORF-encoded proteins in the human gut microbiome

Megan E. Davin
, Júlia Ortís Sunyer
, Luis F. Delgado
, Steven L. Tavis
, Tuesday Lowndes
, Zainab Zafar
, Jordan Caussin
, Rashi Halder
, Oskar Hickl
, Cédric C. Laczny
, Etienne Hanslian
, Daniela A. Koppold
, Anika Rajput-Khokhar
, Nico Steckhan
, Sebastian Schade
, Jochen Schneider
, Brit Mollenhauer
, Andreas Michalsen
, Patrick May
, Robert L. Hettich

Paul Wilmes

Bioanalytical Mass Spectrometry

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

Small open reading frames (smORFs), which encode proteins under 100 amino acids, represent an underexplored dimension of the human gut microbiome, despite growing evidence of their essential biological roles. Due to small size and poor annotation, smORFs are typically excluded from metagenomic/metaproteomic analyses. Here, we present a high-resolution multi-omic workflow that integrates smORF prediction into metaproteome searches and enables ultra-deep detection of smORF-encoded proteins (SEPs), without experimental size-based enrichment, utilizing state-of-the-art mass spectrometry instrumentation. Applied to human gut microbiomes, this approach resulted in the largest number of detected SEPs to date, allowing identification of over 25,000 SEPs in the metaproteome, alongside the measurements of the larger proteins. Our multi-omics integrative strategy is critical for advancing human metaproteome research. It also provides a generalizable strategy for comprehensive SEP discovery across diverse microbial ecosystems greatly expanding the previously hidden proteomic landscape.

Original language	English
Article number	6296
Journal	Nature Communications
Volume	17
Issue number	1
DOIs	https://doi.org/10.1038/s41467-026-72762-5
State	Published - Dec 2026

Funding

We thank the staff of the Luxembourg Center for Systems Biomedicine (LCSB), particularly the sequencing platform, for running the sequencing analysis. The bioinformatics analyses presented in this paper were carried out using the HPC facilities of the University of Luxembourg68. We also thank Richard J. Giannone from the bioanalytical mass spectrometry group at Oak Ridge National Laboratory for technical insight and internal review of this project. Senior authors P.W. and R.L.H. disclose support for the research of this work to all co-authors from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program [grant agreement No. 863664] and P.W. further acknowledges support from the Luxembourg National Research Fund [grant number C23/BM/18091896/Infectome]. Co-first authors M.E.D. and J.O.S. disclose individual support, with M.E.D. supported by the National Science Foundation Graduate Research Fellowship Program and J.O.S. supported by a Pélican Grant from the Fondation de Luxembourg. P.W. further discloses individual support from a Fulbright Research Scholarship from the Commission for Educational Exchange between the United States, Belgium, and Luxembourg.

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Davin, M. E., Ortís Sunyer, J., Delgado, L. F., Tavis, S. L., Lowndes, T., Zafar, Z., Caussin, J., Halder, R., Hickl, O., Laczny, C. C., Hanslian, E., Koppold, D. A., Rajput-Khokhar, A., Steckhan, N., Schade, S., Schneider, J., Mollenhauer, B., Michalsen, A., May, P., ... Wilmes, P. (2026). High-resolution multi-omics enhances prediction and detection of smORF-encoded proteins in the human gut microbiome. Nature Communications, 17(1), Article 6296. https://doi.org/10.1038/s41467-026-72762-5

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title = "High-resolution multi-omics enhances prediction and detection of smORF-encoded proteins in the human gut microbiome",

abstract = "Small open reading frames (smORFs), which encode proteins under 100 amino acids, represent an underexplored dimension of the human gut microbiome, despite growing evidence of their essential biological roles. Due to small size and poor annotation, smORFs are typically excluded from metagenomic/metaproteomic analyses. Here, we present a high-resolution multi-omic workflow that integrates smORF prediction into metaproteome searches and enables ultra-deep detection of smORF-encoded proteins (SEPs), without experimental size-based enrichment, utilizing state-of-the-art mass spectrometry instrumentation. Applied to human gut microbiomes, this approach resulted in the largest number of detected SEPs to date, allowing identification of over 25,000 SEPs in the metaproteome, alongside the measurements of the larger proteins. Our multi-omics integrative strategy is critical for advancing human metaproteome research. It also provides a generalizable strategy for comprehensive SEP discovery across diverse microbial ecosystems greatly expanding the previously hidden proteomic landscape.",

author = "Davin, \{Megan E.\} and \{Ort{\'i}s Sunyer\}, J{\'u}lia and Delgado, \{Luis F.\} and Tavis, \{Steven L.\} and Tuesday Lowndes and Zainab Zafar and Jordan Caussin and Rashi Halder and Oskar Hickl and Laczny, \{C{\'e}dric C.\} and Etienne Hanslian and Koppold, \{Daniela A.\} and Anika Rajput-Khokhar and Nico Steckhan and Sebastian Schade and Jochen Schneider and Brit Mollenhauer and Andreas Michalsen and Patrick May and Hettich, \{Robert L.\} and Paul Wilmes",

note = "Publisher Copyright: {\textcopyright} The Author(s) 2026.",

year = "2026",

month = dec,

doi = "10.1038/s41467-026-72762-5",

language = "English",

volume = "17",

journal = "Nature Communications",

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Davin, ME, Ortís Sunyer, J, Delgado, LF, Tavis, SL, Lowndes, T, Zafar, Z, Caussin, J, Halder, R, Hickl, O, Laczny, CC, Hanslian, E, Koppold, DA, Rajput-Khokhar, A, Steckhan, N, Schade, S, Schneider, J, Mollenhauer, B, Michalsen, A, May, P, Hettich, RL & Wilmes, P 2026, 'High-resolution multi-omics enhances prediction and detection of smORF-encoded proteins in the human gut microbiome', Nature Communications, vol. 17, no. 1, 6296. https://doi.org/10.1038/s41467-026-72762-5

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T1 - High-resolution multi-omics enhances prediction and detection of smORF-encoded proteins in the human gut microbiome

AU - Davin, Megan E.

AU - Ortís Sunyer, Júlia

AU - Delgado, Luis F.

AU - Tavis, Steven L.

AU - Lowndes, Tuesday

AU - Zafar, Zainab

AU - Caussin, Jordan

AU - Halder, Rashi

AU - Hickl, Oskar

AU - Laczny, Cédric C.

AU - Hanslian, Etienne

AU - Koppold, Daniela A.

AU - Rajput-Khokhar, Anika

AU - Steckhan, Nico

AU - Schade, Sebastian

AU - Schneider, Jochen

AU - Mollenhauer, Brit

AU - Michalsen, Andreas

AU - May, Patrick

AU - Hettich, Robert L.

AU - Wilmes, Paul

PY - 2026/12

Y1 - 2026/12

N2 - Small open reading frames (smORFs), which encode proteins under 100 amino acids, represent an underexplored dimension of the human gut microbiome, despite growing evidence of their essential biological roles. Due to small size and poor annotation, smORFs are typically excluded from metagenomic/metaproteomic analyses. Here, we present a high-resolution multi-omic workflow that integrates smORF prediction into metaproteome searches and enables ultra-deep detection of smORF-encoded proteins (SEPs), without experimental size-based enrichment, utilizing state-of-the-art mass spectrometry instrumentation. Applied to human gut microbiomes, this approach resulted in the largest number of detected SEPs to date, allowing identification of over 25,000 SEPs in the metaproteome, alongside the measurements of the larger proteins. Our multi-omics integrative strategy is critical for advancing human metaproteome research. It also provides a generalizable strategy for comprehensive SEP discovery across diverse microbial ecosystems greatly expanding the previously hidden proteomic landscape.

AB - Small open reading frames (smORFs), which encode proteins under 100 amino acids, represent an underexplored dimension of the human gut microbiome, despite growing evidence of their essential biological roles. Due to small size and poor annotation, smORFs are typically excluded from metagenomic/metaproteomic analyses. Here, we present a high-resolution multi-omic workflow that integrates smORF prediction into metaproteome searches and enables ultra-deep detection of smORF-encoded proteins (SEPs), without experimental size-based enrichment, utilizing state-of-the-art mass spectrometry instrumentation. Applied to human gut microbiomes, this approach resulted in the largest number of detected SEPs to date, allowing identification of over 25,000 SEPs in the metaproteome, alongside the measurements of the larger proteins. Our multi-omics integrative strategy is critical for advancing human metaproteome research. It also provides a generalizable strategy for comprehensive SEP discovery across diverse microbial ecosystems greatly expanding the previously hidden proteomic landscape.

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DO - 10.1038/s41467-026-72762-5

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VL - 17

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 6296

ER -

High-resolution multi-omics enhances prediction and detection of smORF-encoded proteins in the human gut microbiome

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