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Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory bowel diseases (IBD) of the gastrointestinal tract. This study used non-invasive LC-MS/MS to find disease specific microbial and human proteins which might be used later for an easier diagnosis. Therefore, 17 healthy controls, 11 CD patients and 14 UC patients but also 13 Irritable Bowel Disease (IBS) patients, 8 Colon Adenoma (CA) patients, and 8 Gastric Carcinoma (GCA) patients were investigated. The proteins were extracted from the fecal samples with liquid phenol in a ball mill. Subsequently, the proteins were digested tryptically to peptides and analyzed by an Orbitrap LC-MS/MS. For protein identification and interpretation of taxonomic and functional results, the MetaProteomeAnalyzer software was used. Cluster analysis and non-parametric test (analysis of similarities) separated healthy controls from patients with CD and UC as well as from patients with GCA. Among others, CD and UC correlated with an increase of neutrophil extracellular traps and immune globulins G (IgG). In addition, a decrease of human IgA and the transcriptional regulatory protein RprY from Bacillus fragilis was found for CD and UC. A specific marker in feces for CD was an increased amount of the human enzyme sucrose-isomaltase. Crohn's Disease and Ulcerative Colitis are chronic inflammatory diseases of the gastrointestinal tract, whose diagnosis required comprehensive medical examinations including colonoscopy. The impact of the microbial communities in the gut on the pathogenesis of these diseases is poorly understood. Therefore, this study investigated the impact of gut microbiome on these diseases by a metaproteome approach, revealing several disease specific marker proteins. Overall, this indicated that fecal metaproteomics has the potential to be useful as non-invasive tool for a better and easier diagnosis of both diseases. Unlabelled Image • Fecal metaproteome analyses separated healthy controls from CD patients, UC patients and GCA patients. • Increase of NETs and IgG and decrease of IgA and transcriptional regulatory protein RprY from B. fragilis while CD and UC. • Identification of potential marker metaproteins for CD, UC, IBS and GCA, such as human enzyme sucrose-isomaltase for CD. [ABSTRACT FROM AUTHOR]

Complex microbial communities are the functional core of anaerobic digestion processes taking place in biogas plants (BGP). So far, however, a comprehensive characterization of the microbiomes involved in methane formation is technically challenging. As an alternative, enriched communities from laboratory-scale experiments can be investigated that have a reduced number of organisms and are easier to characterize by state of the art mass spectrometric-based (MS) metaproteomic workflows. Six parallel laboratory digesters were inoculated with sludge from a full-scale BGP to study the development of enriched microbial communities under defined conditions. During the first three month of cultivation, all reactors (R1-R6) were functionally comparable regarding biogas productions (375–625 NL L reactor volume −1 d −1 ), methane yields (50–60%), pH values (7.1–7.3), and volatile fatty acids (VFA, <5 mM). Nevertheless, a clear impact of the temperature (R3, R4) and ammonia (R5, R6) shifts was observed for the respective reactors. In both reactors operated under thermophilic regime, acetic and propionic acid (10–20 mM) began to accumulate. While R4 recovered quickly from acidification, the levels of VFA remained to be high in R3 resulting in low pH values of 6.5–6.9. The digesters R5 and R6 operated under the high ammonia regime (>1 gNH 3 L −1 ) showed an increase to pH 7.5–8.0, accumulation of acetate (>10 mM), and decreasing biogas production (<125 NL L reactor volume −1 d −1 ). Tandem MS (MS/MS)-based proteotyping allowed the identification of taxonomic abundances and biological processes. Although all reactors showed similar performances, proteotyping and terminal restriction fragment length polymorphisms (T-RFLP) fingerprinting revealed significant differences in the composition of individual microbial communities, indicating multiple steady-states. Furthermore, cellulolytic enzymes and cellulosomal proteins of Clostridium thermocellum were identified to be specific markers for the thermophilic reactors (R3, R4). Metaproteins found in R3 indicated hydrogenothrophic methanogenesis, whereas metaproteins of acetoclastic methanogenesis were identified in R4. This suggests not only an individual evolution of microbial communities even for the case that BGPs are started at the same initial conditions under well controlled environmental conditions, but also a high compositional variance of microbiomes under extreme conditions. [ABSTRACT FROM AUTHOR]

Abstract: Biogas production from energy crops and biodegradable waste is one of the major sources for renewable energies in Germany. Within a biogas plant (BGP) a complex microbial community converts biomass to biogas. Unfortunately, disturbances of the biogas process occur occasionally and cause economic losses of varying extent. Besides technical failures the microbial community itself is commonly assumed as a reason for process instability. To improve the performance and efficiency of BGP, a deeper knowledge of the composition and the metabolic state of the microbial community is required and biomarkers for monitoring of process deviations or even the prediction of process failures have to be identified. Previous work based on 2D-electrophoresis demonstrated that the analysis of the metaproteome is well suited to provide insights into the apparent metabolism of the microbial communities. Using SDS-PAGE with subsequent mass spectrometry, stable protein patterns were evaluated for a number of anaerobic digesters. Furthermore, it was shown that severe changes in process parameters such as acidification resulted in significant modifications of the metaproteome. Monitoring of changing protein patterns derived from anaerobic digesters, however, is still a challenge due to the high complexity of the metaproteome. In this study, different combinations of separation techniques to reduce the complexity of proteomic BGP samples were compared with respect to the subsequent identification of proteins by tandem mass spectrometry (MS/MS): (i) 1D: proteins were tryptically digested and the resulting peptides were separated by reversed phase chromatography prior to MS/MS. (ii) 2D: proteins were separated by GeLC-MS/MS according to proteins molecular weights before tryptic digestion, (iii) 3D: proteins were separated by gel-free fractionation using isoelectric focusing (IEF) conducted before GeLC-MS/MS. For this study, a comparison of two anaerobic digesters operated at mesophilic and at thermophilic conditions was conducted. The addition of further separation dimensions before protein identification increased the number of identified proteins. On the other hand additional fractionation steps increased the experimental work load and the time required for LC-MS/MS measurement. The high resolution of the 3D-approach enabled the detection of approximately 750 to 1650 proteins covering the main pathways of hydrolysis, acidogenesis, acetogenesis and methanogenesis. Methanosarcinales dominated in the mesophilic BGP, whereas Methanomicrobiales were highly abundant in the thermophilic BGP. Pathway analysis confirmed the taxonomic results and revealed that the acetoclastic methanogenesis occurred preferentially at mesophilic conditions, whereas exclusively hydrogenotrophic methanogenesis was detected in thermophilic BGP. However, for the identification of process biomarkers by comprehensive screening of BGP it will be indispensable to find a balance between the experimental efforts and analytical resolution. [Copyright &y& Elsevier]

Soil microbes, and the proteins they produce, are responsible for a myriad of soil processes which are integral to life on Earth, supporting soil fertility, nutrient fluxes, trace gas emissions, and plant production. However, how and why the composition of soil microbial proteins (the metaproteome) changes across wide gradients of vegetation, climatic and edaphic conditions remains largely undetermined. By applying high-resolution mass spectrometry to soil samples collected from four continents, we identified the most common proteins in soils, and investigated the primary environmental factors driving their distributions across climate and vegetation types. We found that soil proteins involved in carbohydrate metabolism, DNA repair, lipid metabolism, transcription regulation, tricarboxylic acid cycling, nitrogen (N) fixation and one-carbon metabolism dominate soils across a wide range of climates, vegetation types and edaphic conditions. Vegetation type and climate were important factors determining the community composition of the topsoil metaproteome. Moreover, we show that vegetation type, climate, and key edaphic proporties (mainly soil C fractions, pH and texture) influenced the proportion of important proteins involved in biogeochemical cycles and cellular processes. We also found that protein-based taxonomic information based on proteins has a greater resolution than 16S rRNA gene sequencing with regards to the ability to detect significant correlations with environmental variables. Together, our work identifies the dominant proteins produced by microbes living in a wide range of soils, and advances our understanding of how environmental changes can influence the structure and function of the topsoil metaproteome and the soil processes that they support. • Proteins are the catalyzers of microbial-mediated processes in soil. • We identified the most common metabolic and biogeochemical proteins across biomes. • Vegetation and climate determine the community composition of soil metaproteome. • Protein-based bacterial taxonomy correlated with environmental variables. • The proportion of N-fixation and C1-metabolism proteins was shaped by vegetation. [ABSTRACT FROM AUTHOR]