# The Metaproteomics Initiative: a coordinated approach for propelling the functional characterization of microbiomes

Tim van den Bossche, Magnus Ø. Arntzen, Dörte Becher, Dirk Benndorf, Vincent G H Eijsink, Céline Henry, Pratik D Jagtap, Nico Jehmlich, Catherine Juste, Benoit J Kunath, et al.

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Tim van den Bossche, Magnus Ø. Arntzen, Dörte Becher, Dirk Benndorf, Vincent G H Eijsink, et al. The Metaproteomics Initiative: a coordinated approach for propelling the functional characterization of microbiomes. Microbiome, 2021, 9 (1), pp.243. 10.1186/s40168-021-01176-w. hal-04127775

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# The Metaproteomics Initiative: a coordinated approach for propelling the functional characterization of microbiomes

Tim Van Den Bossche¹,² ID, Magnus Ø. Arntzen³ ID, Dörte Becher⁴ ID, Dirk Benndorf⁵,⁶,⁷ ID, Vincent G. H. Eijsink³ ID, Céline Henry⁸ ID, Pratik D. Jagtap⁹ ID, Nico Jehmlich¹⁰ ID, Catherine Juste⁸ ID, Benoit J. Kunath¹¹ ID, Bart Mesuere¹,¹² ID, Thilo Muth¹³ ID, Phillip B. Pope³,¹⁴ ID, Jana Seifert¹⁵,¹⁶ ID, Alessandro Tanca¹⁷ ID, Sergio Uzzau¹⁷ ID, Paul Wilmes¹¹ ID, Robert L. Hettich<fnref n="18" />*† ID and Jean Armengaud¹⁹† ID

## Abstract

Through connecting genomic and metabolic information, metaproteomics is an essential approach for understanding how microbiomes function in space and time. The international metaproteomics community is delighted to announce the launch of the Metaproteomics Initiative (www.metaproteomics.org), the goal of which is to promote dissemination of metaproteomics fundamentals, advancements, and applications through collaborative networking in microbiome research. The Initiative aims to be the central information hub and open meeting place where new-comers and experts interact to communicate, standardize, and accelerate experimental and bioinformatic methodologies in this field. We invite the entire microbiome community to join and discuss potential synergies at the interfaces with other disciplines, and to collectively promote innovative approaches to gain deeper insights into microbiome functions and dynamics.

**Keywords:** Microbiome, Metaproteomics, Multi-omics, Education, Interactions, Networking

## Background

Microbial communities are major drivers of biogeochemical cycles, vital components of the biosphere including plants and animals, and key factors in human health. High-throughput genome sequencing technologies have revealed the immense diversity of microbes and the complexity of the ecological interactions within microbiomes [1]. In the past, deciphering the functioning of these complex microbial systems has been a daunting challenge, but is now becoming more approachable due to recent technological advances that allow diving

much deeper into the functional biomolecular machinery. Metaproteomics is one such emerging approach that is beginning to dramatically advance our understanding of microbiome functioning through analyzing the spatiotemporal expression of microbial genes and dynamics within a microbial consortium.

Proteins are the workhorses of metabolism, homeostasis, cell division, transport of nutrients, cell-cell communication, protein synthesis, and the construction of cellular and extracellular structures. While the proteome defines the set of proteins in a single organism, the metaproteome comprises the complete suite of proteins from the variety of microorganisms present in a sample or ecosystem. As a result, a myriad of proteins, some of which may share relatively similar sequences, should be identified and quantified to acquire phenotypically relevant knowledge of the

<footnote>*Correspondence: hettichrl@ornl.gov</footnote>

<footnote>†Robert L. Hettich and Jean Armengaud are shared last authors</footnote>

<footnote n="18">¹⁸ Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
functioning of microbiomes [2]. Importantly, metapro-
teomic information complements the genetic poten-
tial and gene expression information obtained through
metagenomics and metatranscriptomics, respectively,
by identifying the temporal and spatial abundance of
metabolic enzymes and other proteins. Metaproteom-
ics is also highly complementary to metabolomics in
explaining the phenotype of biological systems. Impor-
tantly, due to sequence variation among metabolic
enzymes, metaproteomics data provides insight into
“which organism is doing what” in a microbiome eco-
system and enables identification of specific members
of the microbiome that are responsible for a change in
abundance of a given metabolite. Thus, metaproteom-
ics is pivotally positioned amongst other multi-omics
approaches to enable direct genotype-phenotype link-
ages. In particular, it facilitates the connection between
nucleotide information and metabolic outputs. Fur-
thermore, the identification of structural proteins,
biofilm protein components, and other extracellular
enzymes sets metaproteomics apart from the other
omics layers, resulting in unique insights into micro-
biome functioning. Thereby, it provides robust data
for unraveling microbial networks within microbiomes
and contributes unique information to fully under-
stand the interactions with their environment [3, 4].
Metaproteomics can capture data across all domains
of life, which is crucial for example to understand host-
associated ecosystems in which proteins and peptides
represent essential molecular currencies of exchange.
Hence, metaproteomics “snapshots” at the host-symbi-
ont interfaces are unique in mapping out microbial and
host functions that are reciprocally related. Improve-
ments in sample preparation, high-resolution peptide/
protein separation and analysis, and refined strategies
for data interpretation have propelled metaproteomics
dramatically forward in the past decade [4–7]. Indeed,
metaproteomics has already helped answer a number of
research questions that otherwise would have remained
elusive, primarily by functional assessment of micro-
bial communities based on the identification and quan-
tification of taxon-specific peptides [8]. For example,
whether polyphenols accumulated in soils are micro-
bially unavailable under anoxia was challenged with
metaproteomics on laboratory-controlled reactors [9];
unusual pathways for carbon and energy use were high-
lighted in *Olavius algarvensis*, a gutless marine worm
relying on its bacterial symbionts to cope with nutri-
ent-poor environments [10]; and fecal metaproteomics
of patients with metabolic syndrome revealed the level
of gut inflammation and the subtle changes in bacterial
composition and their metabolism [11].

**Introduction of the metaproteome initiative**

While metagenomic and metatranscriptomic approaches share a common technical measurement platform that is fairly uniform and standardized, the experimental protocol for metaproteomics is much more broad and under active development. This presents challenges for assessing and comparing results and capabilities. In addition, metaproteomics currently benefits greatly by the sequencing, assembly, and annotation of metagenomes for accurate interpretation of the data. Thus, to propel metaproteomics to an unprecedented level of microbiome functional analyses requires an expanded and concerted stage of development where all possible synergies between analytical scientists, protein biochemists, bioinformaticians, statisticians, microbiologists, and other multi-omics specialists should be exploited. The current positive momentum of metaproteomics is particularly ripe for such a shift in gear. Importantly, the microbiome research community needs knowledge dissemination via improved educational resources so that community members are informed of the possibilities metaproteomics can offer and have an understanding of the realistic status of the field. Ideally, the microbiome and metaproteomics community should have easier access to the most recent updates of methodologies through specialized open platforms equipped with state-of-the-art instrumentation and strong expertise in data analysis. The full impact of active research to further improve metaproteomics in terms of methodology and data analysis will only be achieved if we improve its standards, educate the whole community, and foster new conceptual advances, thus facilitating the applicability of metaproteomics to solve a wide range of key biological questions. Clearly, metaproteomics has now matured sufficiently to be a viable component of experimental design in current and future microbiome research projects.

A growing community of researchers using metapro-
teomics had the opportunity to meet at several inter-
national symposia (Magdeburg, Germany in February
2016, Alghero, Italy, in June 2017, Leipzig, Germany in
December 2018, an online edition in June 2021, and Lux-
embourg City, Luxembourg in September 2021) and
launched specific actions such as training sessions and
interlaboratory comparisons. Over the past year, mem-
bers of this community have regularly discussed a variety
of subjects, such as the need for a place where newcomers
and experts can discuss and initiate new projects, the
desire to forge metaproteomics as a critical methodology
of choice for functional analyses, and the need to estab-
lish and disseminate the best options and standards for
this methodology. This has enabled the development of
a roadmap in order to assess the possibilities of tackling
more complex projects, such as meta-omics projects and
spatio-temporal studies of hundreds of differing conditions involving a large number of samples and replicates to be analyzed at increasing depth and throughput.

This international community, which at the time of writing brings together over 120 members from 48 research groups from 15 countries, has proposed a new initiative under the aegis of the European Proteomics Association (EuPA, https://eupa.org), the federation of European national proteomics societies, with a clear mission and vision statement: “the Metaproteomics Initiative promotes dissemination of metaproteomics fundamentals, advancements, and applications through collaborative networking in microbiome research. We aim to be the central information hub and open meeting place where newcomers and experts interact to communicate, standardize, and accelerate experimental and bioinformatic methodologies in this field”. This will be achieved initially through a dedicated website (https://metaproteomics.org) and Twitter account (@MetaP_Init), presentations and other educational resources, an online communication channel, inter-laboratory comparisons, and regular symposia. We are delighted to announce that as of February 2021 the Metaproteomics Initiative was officially launched and supported by EuPA. We are confident that this Initiative will benefit from an open structure, facilitating exchanges and interactions with other disciplines, and favouring additional synergies at the interfaces with other omics, data science, and microbiology. Through this commentary, we invite the entire microbiome community to join this Initiative and promote collective innovative and holistic approaches to understand how microbiomes function.

The formulation and execution of a community-driven, multi-lab comparison of established metaproteomic workflows, the “Critical Assessment of MetaProteome Investigation” (CAMPI) study, is the first tangible action of the Metaproteomics Initiative [12]. CAMPI has compared sample preparation, mass spectrometry analysis, and bioinformatics workflows for data analysis. This benchmarking effort has led to findings which have highlighted the importance of each step of the analytical pipeline. Such a benchmarking effort calls for discussion not only on the needs of standardized experimental procedures and data treatment workflows, but also specific guidelines in data reporting and data sharing. The CAMPI effort has also paved the way for subsequent inter-laboratory assays where advanced methodological questions will be tackled by the strength and multiple expertises of the whole community. In this regard, it is clear that the definition of appropriate standardized biological samples or benchmark datasets presents important tasks for the community.

Among the other major activities already initiated by the Metaproteomics Initiative are (i) an inaugural online symposium in June 2021, (ii) its promotion among the microbiome community by means of active communication channels, (iii) gathering tutorials and lectures on metaproteomics for offering simple and free access to high-standard educational resources, and (iv) establishment of a consensual framework to stimulate, initiate and coordinate open working groups conducting projects with defined objectives and outputs. To achieve its objectives, the Metaproteomics Initiative will favor the organization of training courses for newcomers and advanced users, further promote its vision in regions currently under-represented, regularly organize new CAMPI editions open to all volunteers, propose brainstorming sessions and hackathons for solving specific bottlenecks, arrange specific round-tables for improving guidelines, and recommending metrics for high-quality results, and establish a platform where scientists with key scientific questions can meet metaproteomics specialists for smooth and efficient collaboration.

As pointed out above, the benefits of a united and stronger metaproteomics community and its successful insertion at the crossroads of many disciplines are huge. Although under the EuPA umbrella, the Metaproteomics Initiative is not limited to a given geographical area and welcomes all positive ideas and feedback. As the puzzling complexity of microbiome samples has been an important driver of methodological improvements in meta-omic sciences, open challenges in metaproteomics are expected to further stimulate significant advancements in mass spectrometry-based proteome analysis and proteome bioinformatics [12, 13]. Due to the myriad of possible applications, metaproteomic approaches are increasingly used and the field is becoming attractive to young scientists. Indeed, a new generation of scientists trained in one or more of the interrelated areas of meta-omics, data science, modeling, and microbiology is emerging. Engaging, inspiring, and educating early-career researchers are undoubtedly a rewarding prospect for the microbiome community.

## Conclusions

Ambitious long-term plans such as the Human Genome Project, the European Green Deal, and the Human Brain Project have pushed the boundaries of science. The multidisciplinary giant Tara Oceans project aimed at deciphering the complexity of ocean life by means of planetary exploration sampling and huge metagenomics efforts is an emblematic model for environmental microbiology [14]. The International Human Microbiome Standards Project is also a pivotal project for improving data quality and optimizing omics comparability in the
human microbiome field. Such long-range frameworks
offer challenging objectives for many researchers, cut-
ting-edge research infrastructure to advance methodolo-
gies and knowledge, and a visibility that is attractive for
stakeholders, media, and the public. The alliance of the
Metaproteomics Initiative and other microbiome disci-
plines could become the cornerstone of such a long-term,
cutting-edge project on the comprehensive molecular
analysis of microbiome ecosystem functioning and adap-
tation. Through its unique positioning, we are confident
that the Metaproteomics Initiative will contribute to
the best possible returns on investment for microbiome
research.

**Supplementary Information**

The online version contains supplementary material available at https://
doi.org/10.1186/s40168-021-01176-w.Acknowledgements
None.

**Authors' contributions**

T.V.D.B, R.L.H, and J.A coordinated development and construction of the manuscript, with input from all co-authors. All co-authors contributed edits and input throughout the manuscript construction, and all have read and approved the final version.

Funding

We would like to thank the European Proteomics Association (EuPA) and the European national proteomics societies for their support, as well as the early supporters and members of this Initiative. TVDB would like to acknowledge the Research Foundation-Flanders (FWO) [SB Grant 1S90918N]. PW acknowl- edges the European Research Council (ERC-CoG 863664).

Availability of data and materials

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

All authors declare that they have no competing interests.

Author details

Received: 16 June 2021 Accepted: 10 October 2021

Published online: 20 December 2021

**References**

1. Berg G, Rybakova D, Fischer D, Cernava T, Vergès M-CC, Charles T, et al. Microbiome definition re-visited: old concepts and new challenges. Microbiome. 2020;8:103.

2. Heintz-Buschart A, Wilmes P. Human gut microbiome: function matters. Trends Microbiol. 2018;26:563-74.

3. Zhang X, Li L, Butcher J, Stintzi A, Figeys D. Advancing functional and translational microbiome research using meta-omics approaches. Microbiome. 2019;7:154.

4. Schiebenhoefer H, Van Den Bossche T, Fuchs S, Renard BY, Muth T, Martens L. Challenges and promise at the interface of metaproteomics and genomics: an overview of recent progress in metaproteogenomic data analysis. Expert Rev Proteomics. 2019;16:375-90.

5. Karaduta O, Dvanajscak Z, Zybailov B. Metaproteomics-an advantageous option in studies of host-microbiota interaction. Microorganisms. 2021;9 Available from: https://doi.org/10.3390/microorganisms9050980.

6. Saito MA, Bertrand EM, Duffy ME, Gaylord DA, Held NA, Hervey WJ 4th, et al. Progress and challenges in ocean metaproteomics and proposed best practices for data sharing. J Proteome Res. 2019;18:1461-76.

7. Issa Isaac N, Philippe D, Nicholas A, Raoult D, Eric C. Metaproteomics of the human gut microbiota: Challenges and contributions to other OMICS. Clin Mass Spectrom. Elsevier BV. 2019;14:18-30.

8. Kleiner M. Metaproteomics: much more than measuring gene expression in microbial communities. mSystems. 2019;4 Available from: https://doi.org/10.1128/mSystems.00115-19.

9. McGivern BB, Tfaily MM, Borton MA, Kosina SM, Daly RA, Nicora CD, et al. Decrypting bacterial polyphenol metabolism in an anoxic wetland soil. Nat Commun. 2021;12:2466.

10. Kleiner M, Wentrup C, Lott C, Teeling H, Wetzel S, Young J, et al. Metaproteomics of a gutless marine worm and its symbiotic microbial community reveal unusual pathways for carbon and energy use. Proc Natl Acad Sci U S A. 2012;109:E1173-82.

11. Biemann R, Buß E, Benndorf D, Lehmann T, Schallert K, Püttker S, et al. Fecal metaproteomics reveals reduced gut inflammation and changed microbial metabolism following lifestyle-induced weight loss. Biomolecules. 2021;11 Available from: https://doi.org/10.3390/biom11050726.

12. Van Den Bossche T, Kunath BJ, Schallert K, Schäpe SS, Abraham PE, Armengaud J, et al. Critical assessment of metaproteome investigation (CAMPI): A multi-lab comparison of established workflows. bioRxiv. 2021; Available from: http://biorxiv.org/lookup/doi/10.1101/2021.03.05.433915.

13. Wilmes P, Heintz-Buschart A, Bond PL. A decade of metaproteomics: where we stand and what the future holds. Proteomics. 2015;15:3409-17.

14. Sunagawa S, Acinas SG, Bork P, Bowler C, Tara Oceans Coordinators, Eveil-lard D, et al. Tara Oceans: towards global ocean ecosystems biology. Nat Rev Microbiol. 2020;18:428-45.

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<footnote n="1">¹VIB-UGent Center for Medical Biotechnology, VIB, 9000 Ghent, Belgium.
²Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium. ³Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, 1432 Ås, Norway. ⁴Institute for Microbiology, Department for Microbial Proteomics, University of Greifswald, 17498 Greifswald, Germany. ⁵Bioprocess Engineering, Otto von Guericke University, 39106 Magdeburg, Germany.
⁶Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany. ⁷Microbiology, Anhalt University of Applied Sciences, 06354 Köthen, Germany. ⁸Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France. ⁹Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.</footnote>

<footnote n="10">¹⁰Helmholtz-Centre for Environmental Research GmbH-UFZ, Department of Molecular Systems Biology, Permoserstrasse 15, 04318 Leipzig, Germany.</footnote>

<footnote n="11">¹¹Luxembourg Centre for Systems Biomedicine and Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.</footnote>

<footnote n="12">¹²Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Ghent, Belgium. ¹³Section eScience (S.3), Federal Institute for Materials Research and Testing, Berlin, Germany. ¹⁴Faculty of Biosciences, NMBU - Norwegian University of Life Sciences, 1432 Ås, Norway. ¹⁵HoL-MiR - Hohenheim Center for Livestock Microbiome Research, University of Hohenheim, Leonore-Blosser-Reisen-Weg 3, 70599 Stuttgart, Germany.</footnote>

<footnote n="16">¹⁶Institute of Animal Science, University of Hohenheim, Emil-Wolff-Str. 6-10, 70599 Stuttgart, Germany. ¹⁷Center for Research and Education on the Microbiota, Department of Biomedical Sciences, University of Sassari, Sassari, Italy.</footnote>

<footnote n="18">¹⁸Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.</footnote>

<footnote n="19">¹⁹Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technolo-
gies pour la Santé (DMTS), SPI, 30200 Bagnols-sur-Cèze, France.</footnote>