Proteogenomics-Guided Evaluation of RNA-Seq Assembly and Protein Database Construction for Emergent Model Organisms.

Proteogenomics is gaining momentum as, today, genomics, transcriptomics, and proteomics can be readily performed on any new species. This approach allows key alterations to molecular pathways to be identified when comparing conditions. For animals and plants, RNA-seq-informed proteomics is the most popular means of interpreting tandem mass spectrometry spectra acquired for species for which the genome has not yet been sequenced. It relies on high-performance de novo RNA-seq assembly and optimized translation strategies. Here, several pre-treatments for Illumina RNA-seq reads before assembly are explored to translate the resulting contigs into useful polypeptide sequences. Experimental transcriptomics and proteomics datasets acquired for individual Gammarus fossarum freshwater crustaceans are used, the most relevant procedure is defined by the ratio of MS/MS spectra assigned to peptide sequences. Removing reads with a mean quality score of less than 17-which represents a single probable nucleotide error on 150-bp reads-prior to assembly, increases the proteomics outcome. The best translation using Transdecoder is achieved with a minimal open reading frame length of 50 amino acids and systematic selection of ORFs longer than 900 nucleotides. Using these parameters, transcriptome assembly and translation informed by proteomics pave the way to further improvements in proteogenomics.

Shotgun proteomics datasets acquired on Gammarus pulex animals sampled from the wild.

This data article associated with the manuscript "Comparative proteomics in the wild: accounting for intrapopulation variability improves describing proteome response in a Gammarus pulex field population exposed to cadmium" refers to the shotgun proteomics analysis performed on 40 Gammarus pulex animals sampled from the wild. Proteins were extracted, digested with trypsin, and the resulting peptides were identified by tandem mass spectrometry. Here, we present the list of proteins from males and the list of proteins from females that are differentially detected between the Brameloup and the Pollon populations. Data are available via ProteomeXchange with identifiers PXD013656 and PXD013712, respectively.

De novo transcriptomes of 14 gammarid individuals for proteogenomic analysis of seven taxonomic groups.

Gammarids are amphipods found worldwide distributed in fresh and marine waters. They play an important role in aquatic ecosystems and are well established sentinel species in ecotoxicology. In this study, we sequenced the transcriptomes of a male individual and a female individual for seven different taxonomic groups belonging to the two genera Gammarus and Echinogammarus: Gammarus fossarum A, G. fossarum B, G. fossarum C, Gammarus wautieri, Gammarus pulex, Echinogammarus berilloni, and Echinogammarus marinus. These taxa were chosen to explore the molecular diversity of transcribed genes of genotyped individuals from these groups. Transcriptomes were de novo assembled and annotated. High-quality assembly was confirmed by BUSCO comparison against the Arthropod dataset. The 14 RNA-Seq-derived protein sequence databases proposed here will be a significant resource for proteogenomics studies of these ecotoxicologically relevant non-model organisms. These transcriptomes represent reliable reference sequences for whole-transcriptome and proteome studies on other gammarids, for primer design to clone specific genes or monitor their specific expression, and for analyses of molecular differences between gammarid species.

Comparative proteomics in the wild: Accounting for intrapopulation variability improves describing proteome response in a Gammarus pulex field population exposed to cadmium.

High-throughput proteomics can be performed on animal sentinels for discovering key molecular biomarkers signing the physiological response and adaptation of organisms. Ecotoxicoproteomics is today amenable by means of proteogenomics to small arthropods such as Gammarids which are well known sentinels of aquatic environments. Here, we analysed two regional Gammarus pulex populations to characterize the potential proteome divergence induced in one site by natural bioavailable mono-metallic contamination (cadmium) compared to a non-contaminated site. Two RNAseq-derived protein sequence databases were established previously on male and female individuals sampled from the reference site. Here, individual proteomes were acquired on 10 male and 10 female paired organisms sampled from each site. Proteins involved in protein lipidation, carbohydrate metabolism, proteolysis, innate immunity, oxidative stress response and lipid transport were found more abundant in animals exposed to cadmium, while hemocyanins were found in lower abundance. The intrapopulation proteome variability of long-term exposed G. pulex was inflated relatively to the non-contaminated population. These results show that, while remaining a challenge for such organisms with not yet sequenced genomes, taking into account intrapopulation variability is important to better define the molecular players induced by toxic stress in a comparative field proteomics approach.

Fine-scale haplotype mapping of MUT, AACS, SLC6A15 and PRKCA genes indicates association with insulin resistance of metabolic syndrome and relationship with branched chain amino acid metabolism or regulation.

Branched chain amino acids (BCAA) are essential elements of the human diet, which display increased plasma levels in obesity and regained particular interest as potential biomarkers for development of diabetes. To define determinants of insulin resistance (IR) we investigated 73 genes involved in BCAA metabolism or regulation by fine-scale haplotype mapping in two European populations with metabolic syndrome. French and Romanians (n = 465) were genotyped for SNPs (Affymetrix) and enriched by imputation (BEAGLE 4.1) at 1000 genome project density. Initial association hits detected by sliding window were refined (HAPLOVIEW 3.1 and PHASE 2.1) and correlated to homeostasis model assessment (HOMAIR) index, in vivo insulin sensitivity (SI) and BCAA plasma levels (ANOVA). Four genomic regions were associated with IR located downstream of MUT, AACS, SLC6A15 and PRKCA genes (P between 9.3 and 3.7 x 10-5). Inferred haplotypes up to 13 SNPs length were associated with IR (e.g. MUT gene with P < 4.9 x 10-5; Bonferroni 1.3 x 10-3) and synergistic to HOMAIR. SNPs in the same regions were also associated with one order of magnitude lower P values (e.g. rs20167284 in the MUT gene with P < 1.27 x 10-4) and replicated in Mediterranean samples (n = 832). In French, influential haplotypes (OR > 2.0) were correlated with in vivo insulin sensitivity (1/SI) except for SLC6A15 gene. Association of these genes with BCAA levels was variable, but influential haplotypes confirmed implication of MUT from BCAA metabolism as well as a role of regulatory genes (AACS and PRKCA) and suggested potential changes in transcriptional activity. These data drive attention towards new regulatory regions involved in IR in relation with BCAA and show the ability of haplotypes in phased DNA to detect signals complimentary to SNPs, which may be useful in designing genetic markers for clinical applications in ethnic populations.

Ecotoxicoproteomics: A decade of progress in our understanding of anthropogenic impact on the environment.

Anthropogenic pollutants are found worldwide. Their fate and effects on human and ecosystem health must be appropriately monitored. Today, ecotoxicology is focused on the development of new methods to assess the impact of pollutant toxicity on living organisms and ecosystems. In situ biomonitoring often uses sentinel animals for which, ideally, molecular biomarkers have been defined thanks to which environmental quality can be assessed. In this context, high-throughput proteomics methods offer an attractive approach to study the early molecular responses of organisms to environmental stressors. This approach can be used to identify toxicity pathways, to quantify more precisely novel biomarkers, and to draw the possible adverse outcome pathways. In this review, we discuss the major advances in ecotoxicoproteomics made over the last decade and present the current state of knowledge, emphasizing the technological and conceptual advancements that allowed major breakthroughs in this field, which aims to "make our planet great again". SIGNIFICANCE: Ecotoxicoproteomics is a protein-centric methodology that is useful for ecotoxicology and could have future applications as part of chemical risk assessment and environmental monitoring. Ecotoxicology employing non-model sentinel organisms with highly divergent phylogenetic backgrounds aims to preserve the functioning of ecosystems and the overall range of biological species supporting them. The classical proteomics workflow involves protein identification, functional annotation, and extrapolation of toxicity across species. Thus, it is essential to develop multi-omics approaches in order to unravel molecular information and construct the most suitable databases for protein identification and pathway analysis in non-model species. Current instrumentation and available software allow relevant combined transcriptomic/proteomic studies to be performed for almost any species. This review summarizes these approaches and illustrates how they can be implemented in ecotoxicology for routine biomonitoring.

Branched-Chain Amino Acid Database Integrated in MEDIPAD Software as a Tool for Nutritional Investigation of Mediterranean Populations.

Branched-chained amino acids (BCAA) are essential dietary components for humans and can act as potential biomarkers for diabetes development. To efficiently estimate dietary intake, we developed a BCAA database for 1331 food items found in the French Centre d'Information sur la Qualité des Aliments (CIQUAL) food table by compiling BCAA content from international tables, published measurements, or by food similarity as well as by calculating 267 items from Greek, Turkish, Romanian, and Moroccan mixed dishes. The database embedded in MEDIPAD software capable of registering 24 h of dietary recalls (24HDR) with clinical and genetic data was evaluated based on archived 24HDR of the Saint Pierre Institute (France) from 2957 subjects, which indicated a BCAA content up to 4.2 g/100 g of food and differences among normal weight and obese subjects across BCAA quartiles. We also evaluated the database of 119 interviews of Romanians, Turkish and Albanians in Greece (27⁻65 years) during the MEDIGENE program, which indicated mean BCAA intake of 13.84 and 12.91 g/day in males and females, respectively, comparable to other studies. The MEDIPAD is user-friendly, multilingual, and secure software and with the BCAA database is suitable for conducting nutritional assessment in the Mediterranean area with particular facilities for food administration.