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At least 19 recordsLinked to original sources
PubMed2005

Metaproteomic analysis of Chesapeake Bay microbial communities.

BACKGROUND: Natural microbial communities are extremely complex and dynamic systems in terms of their population structure and functions. However, little is known about the in situ functions of the microbial communities. RESULTS: This study describes the application of proteomic approaches (metaproteomics) to observe expressed protein profiles of natural microbial communities (metaproteomes). The technique was validated using a constructed community and subsequently used to analyze Chesapeake Bay microbial community (0.2 to 3.0 microm) metaproteomes. Chesapeake Bay metaproteomes contained proteins from pI 4-8 with apparent molecular masses between 10-80 kDa. Replicated middle Bay metaproteomes shared approximately 92% of all detected spots, but only shared 30% and 70% of common protein spots with upper and lower Bay metaproteomes. MALDI-TOF analysis of highly expressed proteins produced no significant matches to known proteins. Three Chesapeake Bay proteins were tentatively identified by LC-MS/MS sequencing coupled with MS-BLAST searching. The proteins identified were of marine microbial origin and correlated with abundant Chesapeake Bay microbial lineages, Bacteroides and alpha-proteobacteria. CONCLUSION: Our results represent the first metaproteomic study of aquatic microbial assemblages and demonstrate the potential of metaproteomic approaches to link metagenomic data, taxonomic diversity, functional diversity and biological processes in natural environments.

Journal Article↗
PubMed2026

A streamlined workflow for high throughput metaproteomic analysis of the rumen microbiome.

Metaproteomics can provide direct functional insights into complex microbial communities, yet its application in rumen research remains limited due to labor-intensive and low-throughput sample preparation workflows before the MS analysis. This work aimed to develop and characterize a streamlined, high throughput metaproteomic workflow optimized for rumen samples. Key steps, including microbial cell extraction, cell lysis, protein digestion, and LC-MS/MS acquisition, were systematically assessed and optimized to reduce hands-on time while maintaining deep proteome coverage. The optimized workflow integrates a minimized cell extraction protocol using 0.5 g starting material and in-solution tryptic digestion. Application of the final workflow to 72 samples from in vitro fermentation revealed that biological variability between inocula dominated technical variability, which remained moderate (median CV of 21-24% across batches). Overall, the optimized workflow supports robust taxonomic and functional characterization of the rumen microbiome with improved scalability. These advances provide a foundation for applying metaproteomics to larger experimental designs, including nutritional trials and cohort studies, thereby enabling broader functional interrogation of rumen microbial ecosystems. SIGNIFICANCE: This study addresses current limitations in the application of metaproteomics to rumen microbiome research by developing a streamlined and scalable sample preparation workflow. By optimizing key steps and reducing sample input while maintaining reproducibility and proteome coverage, this work enables more efficient processing of larger sample sets. These advances support the broader use of metaproteomics in rumen studies and facilitate functional investigations relevant to animal nutrition and sustainable livestock production.

Animals↗
PubMed2025

Functional metaproteomics for enzyme discovery.

Discovery of microbial biocatalysts traditionally relied on activity screening of isolated bacterial strains. However, since most microorganisms cannot be cultivated in the lab, such an approach leaves the majority of the microbial enzyme diversity untapped. Metagenomic approaches, in which the DNA from a microbial community is directly isolated and then used either for the creation of an expression library or for sequencing and metagenome annotation have alleviated this shortcoming to an extent, but have their own limitations: the generation of large expression libraries is time-consuming and their screening is costly, while metagenome annotation can infer biocatalytic function only from prior knowledge. We have thus developed a functional metaproteomic approach, which combines the immediacy of traditional activity screening with the comprehensiveness of a meta-omics approach. Briefly, the whole metaproteome of an environmental sample is separated on a 2-D gel, biocatalytically active proteins are visualized in-gel through zymography, and those candidate biocatalysts are then identified through mass spectrometry, searching against a metagenome-derived database obtained from the very same environmental sample. Here we explain the process in detail, with a focus on esterases, and give guidelines on how to develop a functional metaproteomic workflow for enzyme discovery.

Proteomics↗
PubMed2026

Pilot metaproteomic profiling reveals bacterial diversity and potential medical and veterinary relevance of tick microbiomes in northern Algeria.

Ticks are major ectoparasites and vectors of pathogens affecting humans, livestock, and wildlife. They harbor diverse microbial communities that may influence tick biology and interactions with microorganisms; however, functional information on tick-associated microbiomes remains limited, particularly in North Africa. In this pilot study, we applied a metaproteomic approach based on high-resolution tandem mass spectrometry to characterize bacterial communities associated with three tick species collected in Algeria: Rhipicephalus sanguineus sensu lato, Hyalomma aegyptium, and Hyalomma dromedarii. Peptide spectra were assigned to taxa using a two-step database search strategy based on NCBInr, and bacterial composition and relative abundance were compared across tick species and sampling locations. A total of 40 bacterial genera belonging to 32 families and four phyla were identified. Microbiome composition differed significantly between tick genera and collection locations, suggesting an influence of species-specific and geographical factors on microbial community structure. Dominant genera included Streptomyces, Bacillus, Clostridium, Escherichia, Flavobacterium, Paenibacillus, and Providencia. Peptides related to Coxiella spp. were frequently detected, consistent with previous reports of Coxiella-like endosymbionts in ticks. This pilot study provides a first metaproteomic characterization of tick-associated communities in Algeria. The results reveal species- and location-associated differences in microbial composition and highlight the potential of metaproteomics for exploring tick-associated microbiomes in North Africa.

Animals↗
PubMed2024

metaExpertPro: A Computational Workflow for Metaproteomics Spectral Library Construction and Data-Independent Acquisition Mass Spectrometry Data Analysis.

Analysis of large-scale data-independent acquisition mass spectrometry metaproteomics data remains a computational challenge. Here, we present a computational pipeline called metaExpertPro for metaproteomics data analysis. This pipeline encompasses spectral library generation using data-dependent acquisition MS, protein identification and quantification using data-independent acquisition mass spectrometry, functional and taxonomic annotation, as well as quantitative matrix generation for both microbiota and hosts. By integrating FragPipe and DIA-NN, metaExpertPro offers compatibility with both Orbitrap and timsTOF MS instruments. To evaluate the depth and accuracy of identification and quantification, we conducted extensive assessments using human fecal samples and benchmark tests. Performance tests conducted on human fecal samples indicated that metaExpertPro quantified an average of 45,000 peptides in a 60-min diaPASEF injection. Notably, metaExpertPro outperformed three existing software tools by characterizing a higher number of peptides and proteins. Importantly, metaExpertPro maintained a low factual false discovery rate of approximately 5% for protein groups across four benchmark tests. Applying a filter of five peptides per genus, metaExpertPro achieved relatively high accuracy (F-score = 0.67-0.90) in genus diversity and showed a high correlation (rSpearman = 0.73-0.82) between the measured and true genus relative abundance in benchmark tests. Additionally, the quantitative results at the protein, taxonomy, and function levels exhibited high reproducibility and consistency across the commonly adopted public human gut microbial protein databases IGC and UHGP. In a metaproteomic analysis of dyslipidemia patients, metaExpertPro revealed characteristic alterations in microbial functions and potential interactions between the microbiota and the host.

Proteomics↗
PubMed2026

The need for standardization and improved open (meta)data practices in metaproteomics.

Metaproteomics enables functional insight into microbial communities by identifying and quantifying proteins in complex samples. Yet, heterogeneous analytical workflows and the lack of standardization across experimental and bioinformatics stages hinder reproducibility and comparability, limiting integration with other omics data. We here present a community-developed reporting checklist tailored to the specific needs of metaproteomics. We also outline current efforts to enable structured and interoperable metadata capture, drawing on standards from proteomics and microbiome research wherever possible. By promoting transparent reporting and advancing metadata practices, our recommendations aim to align metaproteomics more closely with FAIR principles and support reproducible and interoperable research practices. Video Abstract.

Proteomics↗
PubMed2006

Towards exposure of elusive metabolic mixed-culture processes: the application of metaproteomic analyses to activated sludge.

Protein expression is a direct reflection of specific microbial activities in any ecosystem. In order to assess protein expression in mixed microbial communities, the feasibility of applying proteomic techniques to activated sludge samples has recently been demonstrated. We report the application of metaproteomics to two activated sludges from a laboratory-scale sequencing batch reactor with dissimilar phosphorus removal performances. Fluorescence in situ hybridization (FISH) revealed that the sludge with good enhanced biological phosphorus removal performance (EBPR) was dominated by Betaproteobacteria (65% of EUBMIX binding cells) and gave positive signals for the Rhodocyclus-type PAO specific probe (59%). The non-EBPR sludge was dominated by tetrad-forming Alphaproteobacteria (75%). With regard to the proteomic investigation, 630 individual protein spots were matched across the replicate groups of the anaerobic and aerobic phases of the EBPR sludge with 9.4% of all spots being statistically different between the two phases. The non-EBPR metaproteomic maps exhibited 590 matched spots with 14.7% statistical differences between the two phases. Overall, the non-EBPR sludge expressed around 30% more significant differences than the EBPR sludge. The comparison of protein expression in the two sludges showed that their metaproteomes were substantially different and this was reflected in their microbial community structures and metabolic transformations.

Base Sequence↗
PubMed2025

Metaproteomic Analysis to Assess the Impact of Storage Media on Human Gut Microbiome in Fecal Samples.

The human gut microbiome is a diverse community of microorganisms residing in the gastrointestinal tract. The storage condition of fecal samples may impact the taxonomic and protein compositions of microbiomes in these samples. Here, we performed a mass spectrometry-based metaproteomic study to assess the impact of storage media on human gut microbiome in fecal samples. We evaluated FDA-authorized OMNIgene·GUT (OG), phosphate-buffered saline (PBS), and RNALater (RNAL) buffers and identified 38,185 microbial peptides corresponding to 7348 microbial proteins, which matched 16 phyla, 20 classes, 50 orders, 104 families, 332 genera, and 453 species. We found a high similarity among the fecal microbiomes preserved in OG, PBS, and RNAL in terms of the identification of proteins, taxa, and functional annotations. Both alpha and beta diversity suggested the high similarity among samples stored in the three media. Nonetheless, we also found some notable differences among buffers regarding the abundances of a few taxon groups. A partial human proteome (over 400 proteins) was identified in the fecal samples, with most of these proteins associated with the membrane and extracellular regions. The findings indicate the similarity among microbiomes in the fecal samples stored in OG, PBS, and RNAL regarding proteome profile, taxa, and functional capacity. SUMMARY: This study thoroughly analyzed and compared the metaproteomes of fecal samples preserved at -80°C in PBS, RNALater, and OMNIgene·GUT Dx buffers, offering novel insights into the effectiveness of these buffers in maintaining the stability and composition of the human gut microbiome. We found a high similarity in the identification and quantification of proteins, taxa, and functional annotations across the three buffers, with notable quantitative differences highlighting subtle yet important variations in preservation efficacy. The unique datasets and findings could offer valuable revelations into the impact of fecal sample preservation on translational and clinical analyses of the human gut microbiome.

Humans↗
PubMed2026

Metaproteomic profiling reveals viral proteins and associated host proteomic alterations in glioblastoma.

Glioblastoma (GB) is a WHO grade 4 brain cancer with dismal prognosis, yet its aetiology remains poorly defined. Although viral involvement has been proposed, findings across studies remain inconsistent, reflecting inherent limitations of individual technologies and cohort size. Here we applied metaproteomic profiling to a publicly available GB proteome dataset (12 control, 21 adjacent, 159 tumour) and an independent cohort of 81 samples (37 control, 44 tumour) to detect viral proteins in tumour and controls tissues. Across cohorts, we detected viral proteins from diverse species, with human herpesviruses (HHV-1, 2, and 8) more frequently detected in GB tumours compared with control tissues. Analysis of the host tumour proteome revealed differential abundance of proteins related to transcriptional regulation, RNA processing, protein translation, immune responses, and mitochondrial-associated metabolism. Correlation analysis identified associations between viral and human proteins, with several linked to biological processes previously implicated in DNA virus-host interactions. Further stratification of tumour by HHV-1 status showed consistent alterations in proteins associated with mitochondrial-associated metabolism, protein turnover, and cell adhesion/signalling.In summary, this study demonstrates the feasibility of metaproteomics for detecting viral components in archival GB tissues. Using this approach, we observed differences in viral protein landscape across cohorts and identified associations between viral presence and host proteomic features, providing a protein-level framework for future studies of virus-host interactions in GB.

Humans↗
PubMed2006

Metaproteomics: studying functional gene expression in microbial ecosystems.

The recent availability of extensive metagenomic sequences from various environmental microbial communities has extended the postgenomic era to the field of environmental microbiology. Although still restricted to a small number of studies, metaproteomic investigations have revealed interesting aspects of functional gene expression within microbial habitats that contain limited microbial diversity. These studies highlight the potential of proteomics for the study of microbial consortia. However, the application of proteomic investigations to complex microbial assemblages such as seawater and soil still presents considerable challenges. Nonetheless, metaproteomics will enhance the understanding of the microbial world and link microbial community composition to function.

Computational Biology↗
PubMed2006

Metaproteomics approach to study the functionality of the microbiota in the human infant gastrointestinal tract.

A metaproteomics approach comprising two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry) was applied to the largely uncultured infant fecal microbiota for the first time. The fecal microbial metaproteome profiles changed over time, and one protein spot contained a peptide sequence that showed high similarity to those of bifidobacterial transaldolases.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2026

Integrated metagenomic and metaproteomic insights into current-carrying-coil magnetic field enhanced synergistic methanogenic system and antibiotic resistance gene reduction in cow manure anaerobic digestion.

Anaerobic digestion (AD) is a sustainable strategy for valorizing cow manure (CM). However, the high ammonia (NH3) concentration and low biodegradability of CM limit hydrolysis and methane production. This study investigated the application of a current-carrying-coil-based magnetic field (CCC-MF) to AD of CM. The CCC-MF digesters showed higher soluble chemical oxygen demand and attained 16.59 % higher ammonium nitrogen reduction, contributing to a 37.50 % higher average methane yield than the control. Further, CCC-MF digesters showed higher enzyme activities (alkaline protease + 30 %, acetate kinase + 22 % and hydrazine dehydrogenase + 26 %) and increased microbial metabolic indices (dehydrogenase activity + 17 % and electron transport system activity + 10 %) than the control. Metagenomics analysis revealed that abundances of the bacterial genera Mesotoga, Aminobacterium, Xiashengella, unclassified Candidatus Cloacimonadota, Advenella, Pseudomonas, and Comamonas increased, whereas the acetoclastic methanogen Methanothrix decreased by 2.58 %, accompanied by 2.07- and 1.64-fold increases in hydrogenotrophic methanogens Methanospirillum and Methanobacterium, respectively, in CCC-MF digesters. The abundance of nitrogen dissimilation and assimilation genes NirK, NorB, NarB, NapA, nmo, and GLT1 were enhanced by 1.14, 1.04, 2.30, 1.32, 1.17, and 1.29-fold in CCC-MF digesters compared to the control. Moreover, metaproteomics revealed higher up-regulated differentially expressed proteins in NH3 reduction-related amino acid metabolism pathways in CCC-MF digester compared to control. Additionally, reduced abundances of bacitracin, polymyxin, sulfonamide, and multidrug antibiotic resistance (MAR) gene types were observed in the CCC-MF digesters. The findings suggest that applying CCC-MF may be associated with higher methane production and ammonium reduction, potentially linked to a more favorable synergistic methanogenic system and nitrogen transformation pathways.

Manure↗
PubMed2006

Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform.

Bacteriophages are the most abundant forms of life in the biosphere and carry genomes characterized by high genetic diversity and mosaic architectures. The complete sequences of 30 mycobacteriophage genomes show them collectively to encode 101 tRNAs, three tmRNAs, and 3,357 proteins belonging to 1,536 "phamilies" of related sequences, and a statistical analysis predicts that these represent approximately 50% of the total number of phamilies in the mycobacteriophage population. These phamilies contain 2.19 proteins on average; more than half (774) of them contain just a single protein sequence. Only six phamilies have representatives in more than half of the 30 genomes, and only three-encoding tape-measure proteins, lysins, and minor tail proteins-are present in all 30 phages, although these phamilies are themselves highly modular, such that no single amino acid sequence element is present in all 30 mycobacteriophage genomes. Of the 1,536 phamilies, only 230 (15%) have amino acid sequence similarity to previously reported proteins, reflecting the enormous genetic diversity of the entire phage population. The abundance and diversity of phages, the simplicity of phage isolation, and the relatively small size of phage genomes support bacteriophage isolation and comparative genomic analysis as a highly suitable platform for discovery-based education.

DNA, Viral↗
PubMed2025

Large Quantities of Bacterial DNA and Protein in Common Dietary Protein Source Used in Microbiome Studies.

Diet has been shown to greatly impact the intestinal microbiota. To understand the role of individual dietary components, defined diets with purified components are frequently used in diet-microbiota studies. Defined diets frequently use purified casein as the protein source. Previous work indicated that casein contains microbial DNA potentially impacting results of microbiome studies. Other diet-based microbially derived molecules that may impact microbiome measurements, such as proteins detected by metaproteomics, have not been determined for casein. Additionally, other protein sources used in microbiome studies have not been characterized for their microbial content. We used metagenomics and metaproteomics to identify and quantify microbial DNA and protein in a casein-based defined diet to better understand potential impacts on metagenomic and metaproteomic microbiome studies. We further tested six additional defined diets with purified protein sources with an integrated metagenomic-metaproteomic approach and found that contaminating microbial protein is unique to casein within the tested set as microbial protein was not identified in diets with other protein sources. We also illustrate the contribution of diet-derived microbial protein in diet-microbiota studies by metaproteomic analysis of stool samples from germ-free mice (GF) and mice with a conventional microbiota (CV) following consumption of diets with casein and non-casein protein. This study highlights a potentially confounding factor in diet-microbiota studies that must be considered through evaluation of the diet itself within a given study.

Animals↗
PubMed2006

Protein extraction and fingerprinting optimization of bacterial communities in natural environment.

Recent development in molecular approaches allows access to genetic structure and diversity of indigenous microbial communities. In contrast, the functional analysis of microorganisms in their environment is still hampered by methodological limitations. Analysis of total proteins expressed at the whole community level (metaproteome) has been proposed to characterize the functional structure of microbial communities in their environment. However, developments are still required to perform such analysis. Our aim was to optimize methods to extract and characterize metaproteome of indigenous microbial communities. Experiments were first conducted in monoxenic bacterial cultures, and various methods were examined to define a procedure of protein extraction ensuring an efficient recovery regardless of the taxonomic affiliation of the cells. These developments were next applied to characterize the metaproteome from indigenous bacterial communities in freshwater samples. Bacterial cells were recovered from water using a high-speed density gradient centrifugation method before protein extraction and fingerprinting. The reactivity and sensitivity of this metaproteomic approach were tested by analyzing the variations of protein fingerprints according to perturbations (cadmium or mercury contamination). The genetic structure of the corresponding communities was also characterized by automated ribosomal spacer analysis (ARISA) DNA fingerprinting. Both protein and DNA fingerprints were statistically analyzed. Results obtained showed that the method developed for protein recovery and fingerprinting was efficient, sensitive, and reproducible. Both the functional and genetic structures of the freshwater bacterial community were complex and varied with perturbations. These variations occurred at both population and protein expression levels and were specific to the perturbation applied.

Bacteria↗
PubMed2025

Consumption of traditional Sardinian fermented milk promotes changes in the rat gut microbiota composition and functions.

BACKGROUND: Fermented milk products are part of the staple diet for many Mediterranean populations. Most of these traditional foods are enriched with lactobacilli and other lactic acid bacteria, as well as with metabolites resulting from lactose fermentation. Currently, there is very little scientific knowledge on how dietary supplementation with fermented milk affects the composition of the gut microbiota and its metabolic activities. RESULTS: We integrated 16 S rRNA gene-based taxonomic profiling with metaproteomics-based functional analysis to investigate gut microbiota changes in rats exposed to an 8-week dietary supplementation with casu axedu, a traditional fermented milk produced within rural communities in Sardinia (Italy). Several microbial taxa showed a significantly increased abundance at the end of the dietary treatment, including Phascolarctobacterium, Prevotella, Blautia glucerasea, and Lactococcus lactis, while Bacteroides dorei and Helicobacter rodentium were decreased compared to the control rats. Metaproteomic analysis highlighted a striking reshaping of the Prevotella proteome in agreement with its blooming in casu axedu-fed animals, suggesting an increase of the glycolytic activity through the Embden-Meyerhof-Parnas pathway over the Entner-Doudoroff pathway. Moreover, an increased production of enzymes involved in succinate biosynthesis was observed, which in turn significantly boosted the abundance of Phascolarctobacterium and its production of propionate. Fermented milk consumption also promoted microbial synthesis of branched chain essential amino acids L-valine and L-leucine. Finally, metaproteomic data indicated a reduction of bacterial virulence factors and host inflammatory markers, suggesting that the consumption of casu axedu can have beneficial effects on the gut mucosa health. CONCLUSIONS: Our integrated multi-omics approach reveals that dietary supplementation with the traditional Sardinian fermented milk, casu axedu, induces significant shifts in the rat gut microbiota composition and function, characterized by the enrichment of beneficial taxa and metabolic pathways associated with improved gut health and reduced inflammation.

Animals↗
PubMed2026

MegaPX: fast and space-efficient peptide assignment method using IBF-based multi-indexing.

MOTIVATION: A central problem for metaproteomic analysis is the often-unknown taxonomic composition of the analyzed microbiomes. Using a database search, the standard approach requires prior knowledge of which proteins and taxa to include in the protein reference database or to use tailored metagenome-derived databases, which are expensive and error-prone in their generation. A possible strategy to circumvent this database search issue is de novo sequencing, where peptide sequences are directly identified from mass spectra. However, these sequences must still be mapped back to potentially extensive databases. Here, alignment-based approaches enable robust and precise results, with the potential drawback of high memory usage and long run times. RESULTS: We present MegaPX, a software for rapidly classifying de novo peptide sequences against large protein databases. MegaPX implemented as a C++-based tool, uses an alignment-free, k-mer approach as a taxonomic classification method with the possibility of generating mutated reference databases for error-tolerant searching. It uses various algorithms, including interleaved Bloom filters, to efficiently compute approximate membership queries, ensuring fast processing times while querying and indexing large databases in a multi-indexing fashion. We demonstrate the potential of MegaPX by analyzing different samples, including metaproteomics, against extensive reference databases, highlighting its use as a fast screening tool.

Software↗
PubMed2004

The application of two-dimensional polyacrylamide gel electrophoresis and downstream analyses to a mixed community of prokaryotic microorganisms.

Summary In the post-genomic era, the focus of numerous researchers has moved to studying the functional products of gene expression. In microbiology, these "omic" approaches have largely been limited to pure cultures of microorganisms. Consequently, they do not provide information on gene expression in a complex mixture of microorganisms as found in the environment. Our method enabled the successful extraction and purification of the entire proteome from a laboratory-scale activated sludge system optimized for enhanced biological phosphorus removal, its separation by two-dimensional polyacrylamide gel electrophoresis and the mapping of this metaproteome. Highly expressed protein spots were excised and identified using quadrupole time-of-flight mass spectrometry with de novo peptide sequencing. The proteins isolated were putatively identified as an outer membrane protein (porin), an acetyl coenzyme A acetyltransferase and a protein component of an ABC-type branched-chain amino acid transport system. These proteins possibly stem from the dominant and uncultured Rhodocyclus-type polyphosphate-accumulating organism in the activated sludge. We propose the term "metaproteomics" for the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time.

Acetyl-CoA C-Acyltransferase↗