Olivier Pible

Taking the shortcut for high-throughput shotgun proteomic analysis of bacteria.

Currently, proteomic tools are able to establish a complete list of the most abundant proteins present in a sample, providing the opportunity to study at high resolution the physiology of any bacteria for which the genome sequence is available. For a comprehensive list, proteins should be first resolved into fractions that are then proteolyzed by trypsin. The resulting peptide mixtures are analyzed by a high-throughput tandem mass spectrometer that records thousands of MS/MS spectra for each fraction. These spectra are then assigned to peptides, which are used as evidence of the existence of proteins. In addition to generating a list of protein identifications, this shortcut to proteomics uses the number of spectra recorded for each protein to quantify the observations. Here, we describe one of the most simple sample preparation methods for high-throughput proteomics of bacteria, as well as the subsequent data processing to extract quantitative information based on the spectral count approach.

Proteogenomic insights into salt tolerance by a halotolerant alpha-proteobacterium isolated from an Andean saline spring.

UNLABELLED Tistlia consotensis is a halotolerant Rhodospirillaceae that was isolated from a saline spring located in the Colombian Andes with a salt concentration close to seawater (4.5%w/vol). We cultivated this microorganism in three NaCl concentrations, i.e. optimal (0.5%), without (0.0%) and high (4.0%) salt concentration, and analyzed its cellular proteome. For assigning tandem mass spectrometry data, we first sequenced its genome and constructed a six reading frame ORF database from the draft sequence. We annotated only the genes whose products (872) were detected. We compared the quantitative proteome data sets recorded for the three different growth conditions. At low salinity general stress proteins (chaperons, proteases and proteins associated with oxidative stress protection), were detected in higher amounts, probably linked to difficulties for proper protein folding and metabolism. Proteogenomics and comparative genomics pointed at the CrgA transcriptional regulator as a key-factor for the proteome remodeling upon low osmolarity. In hyper-osmotic condition, T. consotensis produced in larger amounts proteins involved in the sensing of changes in salt concentration, as well as a wide panel of transport systems for the transport of organic compatible solutes such as glutamate. We have described here a straightforward procedure in making a new environmental isolate quickly amenable to proteomics. BIOLOGICAL SIGNIFICANCE The bacterium Tistlia consotensis was isolated from a saline spring in the Colombian Andes and represents an interesting environmental model to be compared with extremophiles or other moderate organisms. To explore the halotolerance molecular mechanisms of the bacterium T. consotensis, we developed an innovative proteogenomic strategy consisting of i) genome sequencing, ii) quick annotation of the genes whose products were detected by mass spectrometry, and iii) comparative proteomics of cells grown in three salt conditions. We highlighted in this manuscript how efficient such an approach can be compared to time-consuming genome annotation when pointing at the key proteins of a given biological question. We documented a large number of proteins found produced in greater amounts when cells are cultivated in either hypo-osmotic or hyper-osmotic conditions. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Proteomic Investigation of Male Gammarus fossarum, a Freshwater Crustacean, in Response to Endocrine Disruptors

While the decrease in human sperm count in response to pollutants is a worldwide concern, little attention is being devoted to its causes and occurrence in the biodiversity of the animal kingdom. Arthropoda is the most species-rich phyla, inhabiting all aquatic and terrestrial ecosystems. During evolution, key molecular players of the arthropod endocrine system have diverged from the vertebrate counterparts. Consequently, arthropods may have different sensitivities toward endocrine disrupting chemicals (EDCs). Here alteration of sperm quality in a crustacean, Gammarus fossarum, a popular organism in freshwater risk assessment, was investigated after laboratory exposure to various concentrations of three different xenobiotics: cadmium, methoxyfenozide, and pyriproxyfen. The integrity of the reproductive process was assessed by means of sperm-quality markers. For each substance, semiquantitative/relative proteomics based on spectral counting procedure was carried out on male gonads to observe the biological impact. The changes in a total of 871 proteins were monitored in response to toxic pressure. A drastic effect was observed on spermatozoon production, with a dose-response relationship. While exposure to EDCs leads to strong modulations of male-specific proteins in testis, no induction of female-specific proteins was noted. Also, a significant portion of orphans proved to be sensitive to toxic stress.

Improving the quality of genome, protein sequence, and taxonomy databases: A prerequisite for microbiome meta‐omics 2.0

High‐throughput shotgun metaproteomic approaches on environmental or medical microbiomes are producing huge amounts of tandem mass spectrometry data. These can be interpreted either with a general protein sequence database comprising tens of thousands of sequenced genomes or with a more customized database such as those obtained after metagenome sequencing of the DNA extracted from the same sample. However, not all entries in a nucleotide or protein sequence database are of equal quality and this can critically impact metaproteomic data interpretation. In this viewpoint article, we exemplify several key issues. First, either genome or transcriptome data interpretation due to inaccurate contig assembly and gene prediction may be erroneous, for its mitigation the metaproteogenomic strategies could have an interesting perspective. Errors in sample handling and taxonomical characterization may also be problematic. Cross‐contamination of genome sequences is also underestimated while frequent. As a consequence of these structural errors regarding protein sequences and additional problems due to homology‐based functional annotation of proteins, specific efforts for better interpretation of metaproteomic data are required. We propose the development of new bioinformatic pipelines devoted to detection and correction of errors and contaminations to improve the overall quality of sequence and taxonomy databases for metaproteomics.

Proteomics meets blue biotechnology: a wealth of novelties and opportunities.

Blue biotechnology, in which aquatic environments provide the inspiration for various products such as food additives, aquaculture, biosensors, green chemistry, bioenergy, and pharmaceuticals, holds enormous promise. Large-scale efforts to sequence aquatic genomes and metagenomes, as well as campaigns to isolate new organisms and culture-based screenings, are helping to push the boundaries of known organisms. Mass spectrometry-based proteomics can complement 16S gene sequencing in the effort to discover new organisms of potential relevance to blue biotechnology by facilitating the rapid screening of microbial isolates and by providing in depth profiles of the proteomes and metaproteomes of marine organisms, both model cultivable isolates and, more recently, exotic non-cultivable species and communities. Proteomics has already contributed to blue biotechnology by identifying aquatic proteins with potential applications to food fermentation, the textile industry, and biomedical drug development. In this review, we discuss historical developments in blue biotechnology, the current limitations to the known marine biosphere, and the ways in which mass spectrometry can expand that knowledge. We further speculate about directions that research in blue biotechnology will take given current and near-future technological advancements in mass spectrometry.

Clinical implications of recent advances in proteogenomics

ABSTRACT Proteogenomics, the alliance of proteomics, transcriptomics, genomics and bioinformatics, was first proposed for refining genome annotation using experimental data acquired on gene products. With high-throughput analysis of proteins made possible with next-generation tandem mass spectrometers, proteogenomics is greatly improving human genome annotation per se, and is helping to decrypt the numerous gene and protein modifications occurring during development, aging, illness and cancer progression. Further efforts are required to obtain a comprehensive picture of human genes, their products, functions, and drift over time or in reaction to microbiota and pathogen stimuli. This should be performed not only to obtain a general overview of the human population, but also to gain specific information at the individual level. This review focuses on the clinical implications of proteogenomics: novel biological insights into fundamental biology, better characterization of pathogens and parasites, discovery of novel diagnostic approaches for cancer, and personalized medicine.

Proteogenomic insights into the core-proteome of female reproductive tissues from crustacean amphipods.

As a result of the poor genome sequence coverage of crustacean amphipods, characterization of their evolutionary biology relies mostly on phenotypic traits. Here, we analyzed the proteome of ovaries from five amphipods, all from the Senticaudata suborder, with the objective to obtain insights into the core-proteome of female reproductive systems. These amphipods were from either the Gammarida infraorder: Gammarus fossarum, Gammarus pulex, Gammarus roeseli, or the Talitrida infraorder: Parhyale hawaiensis and Hyalella azteca. Ovaries from animals sampled at the end of their reproductive cycle were dissected. Their whole protein contents were extracted and their proteomes were recorded by high-throughput nanoLC-MS/MS with a high-resolution mass spectrometer. We interpreted tandem mass spectrometry data with the protein sequence resource from G. fossarum and P. hawaiensis, both recently established by RNA sequencing. The large molecular biodiversity within amphipods was assessed by the ratio of MS/MS spectra assigned for each sample, which tends to diverge rapidly along the taxonomic level considered. The core-proteome was defined as the proteins conserved along all samples, thus detectable by the homology-based proteomic assignment procedure. This specific subproteome may be further enriched in the future with the analysis of new species and update of the protein sequence resource.

Subcellular localization and interaction network of the mRNA decay activator Pat1 upon UV stress.

To identify nucleo‐cytoplasmic shuttle proteins that relocate to the nucleus upon UV stress, we selected 18 targets on the basis of their conservation amongst eukaryotes and their relatively poor functional description. Their relocation was assayed using quantitative nuclear relocation assay (QNR). We focused on Pat1, a component of the cytoplasmic foci called processing bodies (p‐bodies), because it had the strongest response to the stress. We verified that Pat1 accumulates in the nucleus after GFP tagging and fluorescence microscopy. Using tandem affinity purification coupled to a mass spectrometry shotgun detection and quantitation approach, we explored the dynamics of Pat1 protein–protein interaction network after UV stress. We have shown that Pat1 co‐purifies with Dhh1 specifically upon UV stress. We observed that the nuclear accumulation of Pat1 upon UV stress is abolished in a dhh1∆ strain. These data provide the first evidence that Dhh1 is required for Pat1 nuclear relocation after UV stress. Copyright

Data for comparative proteomics of ovaries from five non-model, crustacean amphipods.

Ovaries were taken from five sexually mature amphipods: Gammarus fossarum, Gammarus pulex, Gammarus roeseli, Hyallela azteca and Parhyale hawaiensis. The soluble proteome extracted from individual pair of ovaries from five biological replicates was trypsin digested and the resulting peptides were analyzed by high resolution tandem mass spectrometry. The spectra were assigned with four protein sequence databases with different specificities: a RNAseq-derived G. fossarum database; a RNAseq-derived P. hawaiensis database; both originating from ovaries transcriptome; the Daphnia pulex database derived from whole-genome sequencing and the NCBInr database. The best interpretation was obtained for most animals with the specific RNA-seq protein database previously established by means of RNAseq carried out on G. fossarum. Proteins identified in the five amphipod species allow defining the core-proteome of female reproductive tissues of the Senticaudata suborder. The data accompanying the manuscript describing the database searches and comparative analysis Trapp et al., 2015 [1] have been deposited to the ProteomeXchange with identifiers PXD002253 (G. fossarum), PXD002254 (G. pulex), PXD002255 (G. roeseli), PXD002256 (H. Azteca), and PXD002257 (P. hawaiensis).

Ovary and embryo proteogenomic dataset revealing diversity of vitellogenins in the crustacean Gammarus fossarum.

Ovaries and embryos from sexually mature Gammarus fossarum were sampled at different stages of the reproductive cycle. The soluble proteome was extracted for five biological replicates and samples were subjected to trypsin digestion. The resulting peptides were analyzed by high resolution tandem mass spectrometry with a LTQ-Orbitrap XL instrument. The MS/MS spectra were assigned with a previously described RNAseq-derived G. fossarum database. The proteins highlighted by proteogenomics were monitored and their abundance kinetics over the different stages revealed a large panel of vitellogenins. Criteria were i) accumulation during oogenesis, ii) decrease during embryogenesis, iii) classified as female-specific, and iv) sequence similarity and phylogenetic analysis. The data accompanying the manuscript describing the database searches and comparative analysis (“High-throughput proteome dynamics for discovery of key proteins in sentinel species: unsuspected vitellogenins diversity in the crustacean Gammarus fossarum” by Trapp et al. [1]) have been deposited to the ProteomeXchange via the PRIDE repository with identifiers PRIDE: PXD001002.