The impact of sequence database choice on metaproteomic results in gut microbiota studies

Background: Elucidating the role of gut microbiota in physiological and pathological processes has recently emerged as a key research aim in life sciences. In this respect, metaproteomics, the study of the whole protein complement of a microbial community, can provide a unique contribution by revealing which functions are actually being expressed by specific microbial taxa. However, its wide application to gut microbiota research has been hindered by challenges in data analysis, especially related to the choice of the proper sequence databases for protein identification. Results: Here, we present a systematic investigation of variables concerning database construction and annotation and evaluate their impact on human and mouse gut metaproteomic results. We found that both publicly available and experimental metagenomic databases lead to the identification of unique peptide assortments, suggesting parallel database searches as a mean to gain more complete information. In particular, the contribution of experimental metagenomic databases was revealed to be mandatory when dealing with mouse samples. Moreover, the use of a "merged" database, containing all metagenomic sequences from the population under study, was found to be generally preferable over the use of sample-matched databases. We also observed that taxonomic and functional results are strongly database-dependent, in particular when analyzing the mouse gut microbiota. As a striking example, the Firmicutes/Bacteroidetes ratio varied up to tenfold depending on the database used. Finally, assembling reads into longer contigs provided significant advantages in terms of functional annotation yields. Conclusions: This study contributes to identify host- and database-specific biases which need to be taken into account in a metaproteomic experiment, providing meaningful insights on how to design gut microbiota studies and to perform metaproteomic data analysis. In particular, the use of multiple databases and annotation tools has to be encouraged, even though this requires appropriate bioinformatic resources

Defining the core proteome of the chloroplast envelope membranes

High-throughput protein localization studies require multiple strategies. Mass spectrometric analysis of defined cellular fractions is one of the complementary approaches to a diverse array of cell biological methods. In recent years, the protein content of different cellular (sub-)compartments was approached. Despite of all the efforts made, the analysis of membrane fractions remains difficult, in that the dissection of the proteomes of the envelope membranes of chloroplasts or mitochondria is often not reliable because sample purity is not always warranted. Moreover, proteomic studies are often restricted to single (model) species, and therefore limited in respect to differential individual evolution. In this study we analyzed the chloroplast envelope proteomes of different plant species, namely, the individual proteomes of inner and outer envelope (OE) membrane of Pisum sativum and the mixed envelope proteomes of Arabidopsis thaliana and Medicago sativa. The analysis of all three species yielded 341 identified proteins in total, 247 of them being unique. 39 proteins were genuine envelope proteins found in at least two species. Based on this and previous envelope studies we defined the core envelope proteome of chloroplasts. Comparing the general overlap of the available six independent studies (including ours) revealed only a number of 27 envelope proteins. Depending on the stringency of applied selection criteria we found 231 envelope proteins, while less stringent criteria increases this number to 649 putative envelope proteins. Based on the latter we provide a map of the outer and inner envelope core proteome, which includes many yet uncharacterized proteins predicted to be involved in transport, signaling, and response. Furthermore, a foundation for the functional characterization of yet unidentified functions of the inner and OE for further analyses is provided

Interplay between R513 methylation and S516 phosphorylation of the cardiac voltage-gated sodium channel

Arginine methylation is a novel post-translational modification within the voltage-gated ion channel superfamily, including the cardiac sodium channel, Naᵥ1.5. We show that Naᵥ1.5 R513 methylation decreases S516 phosphorylation rate by 4 orders of magnitude, the first evidence of protein kinase A inhibition by arginine methylation. Reciprocally, S516 phosphorylation blocks R513 methylation. Naᵥ1.5 p.G514C, associated to cardiac conduction disease, abrogates R513 methylation, while leaving S516 phosphorylation rate unchanged. This is the first report of methylation–phosphorylation cross-talk of a cardiac ion channel

Mass Spectrometry in the Elucidation of the Glycoproteome of Bacterial Pathogens

Presently some three hundred post-translational modifications are known to occur in bacteria in vivo. Many of these modifications play critical roles in the regulation of proteins and control key biological processes. One of the most predominant modifications, N- and O-glycosylations are now known to be present in bacteria (and archaea) although they were long believed to be limited to eukaryotes. In a number of human pathogens these glycans have been found attached to the surfaces of pilin, flagellin and other surface and secreted proteins where it has been demonstrated that they play a role in the virulence of these bacteria. Mass spectrometry characterization of these glycosylation events has been the enabling key technology for these findings. This review will look at the use of mass spectrometry as a key technology for the detection and mapping of these modifications within microorganisms, with particular reference to the human pathogens, Campylobacter jejuni and Mycobacterium tuberculosis. The overall aim of this review will be to give a basic understanding of the current ‘state-of-the-art’ of the key techniques, principles and technologies, including bioinformatics tools, involved in the analysis of the glycosylation modifications

Development of antiseptic adaptation and cross-adapatation in selected oral pathogens in vitro

There is evidence that pathogenic bacteria can adapt to antiseptics upon repeated exposure. More alarming is the concomitant increase in antibiotic resistance that has been described for some pathogens. Unfortunately, effects of adaptation and cross-adaptation are hardly known for oral pathogens, which are very frequently exposed to antiseptics. Therefore, this study aimed to determine the in vitro increase in minimum inhibitory concentrations (MICs) in oral pathogens after repeated exposure to chlorhexidine or cetylpyridinium chloride, to examine if (cross-)adaptation to antiseptics/antibiotics occurs, if (cross-)adaptation is reversible and what the potential underlying mechanisms are. When the pathogens were exposed to antiseptics, their MICs significantly increased. This increase was in general at least partially conserved after regrowth without antiseptics. Some of the adapted species also showed cross-adaptation, as shown by increased MICs of antibiotics and the other antiseptic. In most antiseptic-adapted bacteria, cell-surface hydrophobicity was increased and mass-spectrometry analysis revealed changes in expression of proteins involved in a wide range of functional domains. These in vitro data shows the adaptation and cross-adaptation of oral pathogens to antiseptics and antibiotics. This was related to changes in cell surface hydrophobicity and in expression of proteins involved in membrane transport, virulence, oxidative stress protection and metabolism

ProteoClade: A taxonomic toolkit for multi-species and metaproteomic analysis

We present ProteoClade, a Python toolkit that performs taxa-specific peptide assignment, protein inference, and quantitation for multi-species proteomics experiments. ProteoClade scales to hundreds of millions of protein sequences, requires minimal computational resources, and is open source, multi-platform, and accessible to non-programmers. We demonstrate its utility for processing quantitative proteomic data derived from patient-derived xenografts and its speed and scalability enable a novel de novo proteomic workflow for complex microbiota samples

Gene induction during differentiation of human monocytes into dendritic cells: an integrated study at the RNA and protein levels

Changes in gene expression occurring during differentiation of human monocytes into dendritic cells were studied at the RNA and protein levels. These studies showed the induction of several gene classes corresponding to various biological functions. These functions encompass antigen processing and presentation, cytoskeleton, cell signalling and signal transduction, but also an increase in mitochondrial function and in the protein synthesis machinery, including some, but not all, chaperones. These changes put in perspective the events occurring during this differentiation process. On a more technical point, it appears that the studies carried out at the RNA and protein levels are highly complementary.Comment: website publisher: http://www.springerlink.com/content/ha0d2c351qhjhjdm

In-depth proteomics identifies a role for autophagy in controlling reactive oxygen species mediated endothelial permeability

Endothelial cells (ECs) form the inner layer of blood vessels and physically separate the blood from the surrounding tissue. To support tissues with nutrients and oxygen, the endothelial monolayer is semipermeable. When EC permeability is altered, blood vessels are not functional, and this is associated with disease. A comprehensive knowledge of the mechanisms regulating EC permeability is key in developing strategies to target this mechanism in pathologies. Here we have used an in vitro model of human umbilical vein endothelial cells mimicking the formation of a physiologically permeable vessel and performed time-resolved in-depth molecular profiling using stable isotope labeling by amino acids in cell culture mass spectrometry (MS)-proteomics. Autophagy is induced when ECs are assembled into a physiologically permeable monolayer. By using siRNA and drug treatment to block autophagy in combination with functional assays and MS proteomics, we show that ECs require autophagy flux to maintain intracellular reactive oxygen species levels, and this is required to maintain the physiological permeability of the cells

Exploitation of proteomics strategies in protein structure-function studies

Mass spectrometry plays a central role in structural proteomics, particularly in highly intensive structural genomics projects. This review paper reports some examples taken from recent work from the authors' laboratory and is aimed at showing that modem proteomics strategies are instrumental in the integration of structural genomic projects in fields such as: (i) protein-protein interactions, (ii) protein-DNA interactions, (iii) protein-ligand interactions, and (iv) protein-folding intermediates