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The yield and productivity of biogas plants depend on the degradation performance of their microbiomes. The spatial separation of the anaerobic digestion (AD) process into a separate hydrolysis and a main fermenter should improve cultivation conditions of the microorganisms involved in the degradation of complex substrates like lignocellulosic biomass (LCB) and, thus, the performance of anaerobic digesters. However, relatively little is known about such two-stage processes. Here, we investigated the process performance of a two-stage agricultural AD over one year, focusing on chemical and technical process parameters and metagenome-centric metaproteomics. Technical and chemical parameters indicated stable operation of the main fermenter but varying conditions for the open hydrolysis fermenter. Matching this, the microbiome in the hydrolysis fermenter has a higher dynamic than in the main fermenter. Metaproteomics-based microbiome analysis revealed a partial separation between early and common steps in carbohydrate degradation and primary fermentation in the hydrolysis fermenter but complex carbohydrate degradation, secondary fermentation, and methanogenesis in the main fermenter. Detailed metagenomics and metaproteomics characterization of the single metagenome-assembled genomes showed that the species focus on specific substrate niches and do not utilize their full genetic potential to degrade, for example, LCB. Overall, it seems that a separation of AD in a hydrolysis and a main fermenter does not improve the cleavage of complex substrates but significantly improves the overall process performance. In contrast, the remaining methanogenic activity in the hydrolysis fermenter may cause methane losses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Metaproteomics represents a promising and fast method to analyze the taxonomic and functional composition of biogas plant microbiomes. However, metaproteomics sample preparation and bioinformatics analysis is still challenging due to the sample complexity and contaminants. In this chapter, a tailored workflow including sampling, phenol extraction in a ball mill, amido black protein quantification, FASP digestion, LC-MS/MS measurement as well as bioinformatics and biostatistical data evaluation are here described for the metaproteomics advancements applied to biogas plant samples., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The increasing amount and complexity of clinical data require an appropriate way of storing and analyzing those data. Traditional approaches use a tabular structure (relational databases) for storing data and thereby complicate storing and retrieving interlinked data from the clinical domain. Graph databases provide a great solution for this by storing data in a graph as nodes (vertices) that are connected by edges (links). The underlying graph structure can be used for the subsequent data analysis (graph learning). Graph learning consists of two parts: graph representation learning and graph analytics. Graph representation learning aims to reduce high-dimensional input graphs to low-dimensional representations. Then, graph analytics uses the obtained representations for analytical tasks like visualization, classification, link prediction and clustering which can be used to solve domain-specific problems. In this survey, we review current state-of-the-art graph database management systems, graph learning algorithms and a variety of graph applications in the clinical domain. Furthermore, we provide a comprehensive use case for a clearer understanding of complex graph learning algorithms. Graphical abstract., (© The Author(s) 2023. Published by Oxford University Press.)

Introduction: Investigating the taxonomic and functional composition of human microbiomes can aid in the understanding of disease etiologies, diagnosis, and therapy monitoring for several diseases, including inflammatory bowel disease or obesity. One method for microbiome monitoring is metaproteomics, which assesses human and microbial proteins and thus enables the study of host-microbiome interactions. This advantage led to increased interest in metaproteome analyses and significant developments to introduce this method into a clinical context., Areas Covered: This review summarizes the recent progress from a technical side and an application-related point of view., Expert Opinion: Numerous publications imply the massive potential of metaproteomics to impact human health care. However, the key challenges of standardization and validation of experimental and bioinformatic workflows and accurate quantification methods must be overcome.

High-calorie diets lead to hepatic steatosis and to the development of non-alcoholic fatty liver disease (NAFLD), which can evolve over many years into the inflammatory form of non-alcoholic steatohepatitis (NASH), posing a risk for the development of hepatocellular carcinoma (HCC). Due to diet and liver alteration, the axis between liver and gut is disturbed, resulting in gut microbiome alterations. Consequently, detecting these gut microbiome alterations represents a promising strategy for early NASH and HCC detection. We analyzed medical parameters and the fecal metaproteome of 19 healthy controls, 32 NASH patients, and 29 HCC patients, targeting the discovery of diagnostic biomarkers. Here, NASH and HCC resulted in increased inflammation status and shifts within the composition of the gut microbiome. An increased abundance of kielin/chordin, E3 ubiquitin ligase, and nucleophosmin 1 represented valuable fecal biomarkers, indicating disease-related changes in the liver. Although a single biomarker failed to separate NASH and HCC, machine learning-based classification algorithms provided an 86% accuracy in distinguishing between controls, NASH, and HCC. Fecal metaproteomics enables early detection of NASH and HCC by providing single biomarkers and machine learning-based metaprotein panels.

In metaproteomics, the study of the collective proteome of microbial communities, the protein inference problem is more challenging than in single-species proteomics. Indeed, a peptide sequence can be present not only in multiple proteins or protein isoforms of the same species, but also in homologous proteins from closely related species. To assign the taxonomy and functions of the microbial species, specialized tools have been developed, such as Prophane. This tool, however, is not directly compatible with post-processing tools such as Percolator. In this manuscript we therefore present Pout2Prot , which takes Percolator Output (.pout) files from multiple experiments and creates protein group and protein subgroup output files (.tsv) that can be used directly with Prophane. We investigated different grouping strategies and compared existing protein grouping tools to develop an advanced protein grouping algorithm that offers a variety of different approaches, allows grouping for multiple files, and uses a weighted spectral count for protein (sub)groups to reflect abundance. Pout2Prot is available as a web application at https://pout2prot.ugent.be and is installable via pip as a standalone command line tool and reusable software library. All code is open source under the Apache License 2.0 and is available at https://github.com/compomics/pout2prot.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments., (© 2021. The Author(s).)

Taxonomic and functional characterization of microbial communities from diverse environments such as the human gut or biogas plants by multi-omics methods plays an ever more important role. Researchers assign all identified genes, transcripts, or proteins to biological pathways to better understand the function of single species and microbial communities. However, due to the versality of microbial metabolism and a still-increasing number of newly biological pathways, linkage to standard pathway maps such as the KEGG central carbon metabolism is often problematic. We successfully implemented and validated a new user-friendly, stand-alone web application, the MPA_Pathway_Tool. It consists of two parts, called 'Pathway-Creator' and 'Pathway-Calculator'. The 'Pathway-Creator' enables an easy set-up of user-defined pathways with specific taxonomic constraints. The 'Pathway-Calculator' automatically maps microbial community data from multiple measurements on selected pathways and visualizes the results. The MPA_Pathway_Tool is implemented in Java and ReactJS.

Purpose: To retrospectively evaluate the diagnostic performance of a 1-min contrast-enhanced 3D-FLASH pulse sequence for detecting intracranial enhancing lesions compared to standard contrast-enhanced 3D-MPRAGE pulse sequence., Methods: Contrast-enhanced 3D-FLASH (acquisition time 49 s) and contrast-enhanced 3D-MPRAGE (4 min 35 s) pulse sequences were performed consecutively in 110 inpatient/emergency department 3T MRI brain examinations and analyzed by two independent neuroradiologist readers. For each sequence, the readers recorded (1) number of enhancing intracranial lesions; (2) intracranial susceptibility artifact (presence or absence; mm depth of intracranial signal loss); and (3) motion artifact (none, mild, moderate, severe). Inter and intra-reader agreement and reader accuracy relative to a reference standard were determined, and sequence comparison with respect to susceptibility and motion artifacts was performed., Results: There was substantial intra-reader, inter-sequence agreement [reader 1, κ = 0.70 (95% CI: [0.60, 0.81]); reader 2, κ = 0.70 (95% CI: [0.59, 0.82])] and substantial intra-sequence, inter-reader agreement [3D-MPRAGE assessment, κ = 0.76 (95% CI: [0.66, 0.86]); 3D-FLASH assessment, κ = 0.86 (95% CI: [0.77, 0.94]) for detection of intracranial enhancing lesions. For both readers, the diagnostic accuracy of 3D-FLASH and 3D-MPRAGE was similar (3D-MPRAGE: 86.4 and 88.1%; 3D-FLASH: 88.2 and 84.5%), with no inter-sequence diagnostic accuracy discordancy between the sequences for either reader. 3D-FLASH was associated with less susceptibility artifact (p < 0.001 both readers) and less motion artifact (p < 0.001 both readers)., Conclusion: On 3T brain MRI in the inpatient and emergency department setting, 1-min 3D-FLASH pulse sequence achieved comparable diagnostic performance to 4.5 min 3D-MPRAGE pulse sequence for detecting enhancing intracranial lesions, with reduced susceptibility and motion artifacts.

Gut microbiota-mediated inflammation promotes obesity-associated low-grade inflammation, which represents a hallmark of metabolic syndrome. To investigate if lifestyle-induced weight loss (WL) may modulate the gut microbiome composition and its interaction with the host on a functional level, we analyzed the fecal metaproteome of 33 individuals with metabolic syndrome in a longitudinal study before and after lifestyle-induced WL in a well-defined cohort. The 6-month WL intervention resulted in reduced BMI (-13.7%), improved insulin sensitivity (HOMA-IR, -46.1%), and reduced levels of circulating hsCRP (-39.9%), indicating metabolic syndrome reversal. The metaprotein spectra revealed a decrease of human proteins associated with gut inflammation. Taxonomic analysis revealed only minor changes in the bacterial composition with an increase of the families Desulfovibrionaceae, Leptospiraceae, Syntrophomonadaceae, Thermotogaceae and Verrucomicrobiaceae. Yet we detected an increased abundance of microbial metaprotein spectra that suggest an enhanced hydrolysis of complex carbohydrates. Hence, lifestyle-induced WL was associated with reduced gut inflammation and functional changes of human and microbial enzymes for carbohydrate hydrolysis while the taxonomic composition of the gut microbiome remained almost stable. The metaproteomics workflow has proven to be a suitable method for monitoring inflammatory changes in the fecal metaproteome.

We lack a predictive understanding of the environmental drivers determining the structure and function of archaeal communities as well as the proteome associated with these important soil organisms. Here, we characterized the structure (by 16S rRNA gene sequencing) and function (by metaproteomics) of archaea from 32 soil samples across terrestrial ecosystems with contrasting climate and vegetation types. Our multi-"omics" approach unveiled that genes from Nitrosophaerales and Thermoplasmata dominated soils collected from four continents, and that archaea comprise 2.3 ± 0.3% of microbial proteins in these soils. Aridity positively correlated with the proportion of Nitrosophaerales genes and the number of archaeal proteins. The interaction of climate x vegetation shaped the functional profile of the archaeal community. Our study provides novel insights into the structure and function of soil archaea across climates, and highlights that these communities may be influenced by increasing global aridity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Metaproteomics, the study of the collective protein composition of multi-organism systems, provides deep insights into the biodiversity of microbial communities and the complex functional interplay between microbes and their hosts or environment. Thus, metaproteomics has become an indispensable tool in various fields such as microbiology and related medical applications. The computational challenges in the analysis of corresponding datasets differ from those of pure-culture proteomics, e.g., due to the higher complexity of the samples and the larger reference databases demanding specific computing pipelines. Corresponding data analyses usually consist of numerous manual steps that must be closely synchronized. With MetaProteomeAnalyzer and Prophane, we have established two open-source software solutions specifically developed and optimized for metaproteomics. Among other features, peptide-spectrum matching is improved by combining different search engines and, compared to similar tools, metaproteome annotation benefits from the most comprehensive set of available databases (such as NCBI, UniProt, EggNOG, PFAM, and CAZy). The workflow described in this protocol combines both tools and leads the user through the entire data analysis process, including protein database creation, database search, protein grouping and annotation, and results visualization. To the best of our knowledge, this protocol presents the most comprehensive, detailed and flexible guide to metaproteomics data analysis to date. While beginners are provided with robust, easy-to-use, state-of-the-art data analysis in a reasonable time (a few hours, depending on, among other factors, the protein database size and the number of identified peptides and inferred proteins), advanced users benefit from the flexibility and adaptability of the workflow.

In anaerobic digestion plants (ADs), homogenization of the feed, fermenter content and microbial communities is crucial for efficient and robust biogas production. However, mixing also requires a significant amount of energy. For an 850 m 3 agricultural AD equipped with eight sampling ports, we investigated whether different feeding and stirring regimes enable a sufficient homogenization of the microbial community using metaproteomics and terminal restriction fragment length polymorphism (TRFLP) analysis. Systematic comparison of samples taken at the top and the bottom as well as at the rim and the center of the AD using scatter plots and students t-test revealed only a small number of differences in metaproteins, taxonomies and biological processes. Obviously, the applied stirring and feeding conditions were sufficient to largely homogenize the content of the AD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Although metaproteomics, the study of the collective proteome of microbial communities, has become increasingly powerful and popular over the past few years, the field has lagged behind on the availability of user-friendly, end-to-end pipelines for data analysis. We therefore describe the connection from two commonly used metaproteomics data processing tools in the field, MetaProteomeAnalyzer and PeptideShaker, to Unipept for downstream analysis. Through these connections, direct end-to-end pipelines are built from database searching to taxonomic and functional annotation.

The investigation of microbial proteins by mass spectrometry (metaproteomics) is a key technology for simultaneously assessing the taxonomic composition and the functionality of microbial communities in medical, environmental, and biotechnological applications. We present an improved metaproteomics workflow using an updated sample preparation and a new version of the MetaProteomeAnalyzer software for data analysis. High resolution by multidimensional separation (GeLC, MudPIT) was sacrificed to aim at fast analysis of a broad range of different samples in less than 24 h. The improved workflow generated at least two times as many protein identifications than our previous workflow, and a drastic increase of taxonomic and functional annotations. Improvements of all aspects of the workflow, particularly the speed, are first steps toward potential routine clinical diagnostics (i.e., fecal samples) and analysis of technical and environmental samples. The MetaProteomeAnalyzer is provided to the scientific community as a central remote server solution at www.mpa.ovgu.de.

Metaproteomics represent an important tool for the taxonomic and functional investigation of microbial communities in humans, environment, and technical applications. Due to the high complexity of the microbial communities, protein, and peptide fractionation is applied to improve the characterization of taxonomic and functional composition of microbial communities. In order to target scientific questions regarding taxonomic and functional composition adequately, a tradeoff between the number of fractions analyzed and the required depth of information has to be found. Two samples of a biogas plant were analyzed by either single LC-MS/MS measurement (1D) or LC-MS/MS measurements of fractions obtained after SDS-PAGE (2D) separation. Fractionation with SDS-PAGE increased the number of identified spectra by 273%, the number of peptides by 95%, and the number of metaproteins by 59%. Rarefaction plots of species and metaproteins against identified spectra showed that 2D separation was sufficient to identify most microbial families but not all metaproteins. More reliable quantitative comparison could be achieved with 2D. 1D separation enabled high-throughput analysis of samples, however, depth in functional descriptions and reliability of quantification were lost. Nevertheless, the proteotyping of multiple samples was still possible. 2D separations provided more reliable quantitative data combined with a deeper insight into the taxonomic and functional composition of the microbial communities. Regarding taxonomic and functional composition, metaproteomics based on 2D is just the tip of an iceberg., (© 2018 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

In nature microorganisms live in complex microbial communities. Comprehensive taxonomic and functional knowledge about microbial communities supports medical and technical application such as fecal diagnostics as well as operation of biogas plants or waste water treatment plants. Furthermore, microbial communities are crucial for the global carbon and nitrogen cycle in soil and in the ocean. Among the methods available for investigation of microbial communities, metaproteomics can approximate the activity of microorganisms by investigating the protein content of a sample. Although metaproteomics is a very powerful method, issues within the bioinformatic evaluation impede its success. In particular, construction of databases for protein identification, grouping of redundant proteins as well as taxonomic and functional annotation pose big challenges. Furthermore, growing amounts of data within a metaproteomics study require dedicated algorithms and software. This review summarizes recent metaproteomics software and addresses the introduced issues in detail., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)