Proteomics Of Non-model Organisms Research Articles

Proteomics to study adaptations in marine organisms to environmental stress.

Comparisons of proteomic responses of closely related congeners and populations have shown which cellular processes are critical to adapt to environmental stress. For example, several proteomic species comparisons showed that increasing abundances of oxidative stress proteins indicate that reactive oxygen species (ROS) represent a ubiquitous signal and possible co-stressor of warm and cold temperature, acute hyposaline and low pH stress, possibly causing a shift from pro-oxidant NADH-producing to anti-oxidant NADPH-producing and -consuming metabolic pathways. Changes in cytoskeletal and actin-binding proteins in response to several stressors, including ROS, suggest that both are important structural and functional elements in responding to stress. Disruption of protein homeostasis, e.g., increased abundance of molecular chaperones, was severe in response to acute heat stress, inducing proteolysis, but was also observed in response to chronic heat and cold stress and was concentrated to the endoplasmic reticulum during hyposaline stress. Small GTPases affecting vesicle formation and transport, Ca(2+)-signaling and ion transport responded to salinity stress in species- and population-specific ways. Aerobic energy metabolism was in general down-regulated in response to temperature, hypoxia, hyposalinity and low pH stress, but other metabolic pathways were activated to respond to increased oxidative stress or to switch metabolic fuels. Thus, comparative proteomics is a powerful approach to identify functionally adaptive variation. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network.

Chickpea is grown over 10 million hectares of land worldwide, and global production hovers around 8.5 million metric tons annually. Despite its nutritional merits, it is often referred to as 'orphan' legume and has remained outside the realm of large-scale functional genomics studies. While current chickpea genome initiative has primarily focused on sequence information and functional annotation, proteomics analyses are limited. It is thus important to study the proteome of the cell organelle particularly the nucleus, which harbors most of the genetic information and gene expression machinery. Phosphorylation-dependent modulation of gene expression plays a vital role but the complex networks of phosphorylation are poorly understood. This inventory of nuclear phosphoproteins would provide valuable insights into the dynamic regulation of cellular phenotype through phosphorylation. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Proteomic-based comparison between populations of the Great Scallop, Pecten maximus

Comparing populations residing in contrasting environments is an efficient way to decipher how organisms modulate their physiology. Here we present the proteomic signatures of two populations in a non-model marine species, the great scallop Pecten maximus, living in the northern (Hordaland, Norway) and in the center (Brest, France) of this species' latitudinal distribution range. The results showed 38 protein spots significantly differentially accumulated in mantle tissues between the two populations. We could unambiguously identify 11 of the protein spots by Maldi TOF-TOF mass spectrometry. Eight proteins corresponded to different isoforms of actin, two were identified as filamin, another protein related to the cytoskeleton structure, and one was the protease elastase. Our results suggest that scallops from the two populations assayed may modulate their cytoskeleton structures through regulation of intracellular pools of actin and filamin isoforms to better adapt to their environment. Marine mollusks are non-model organisms that have been poorly studied at the proteomic level, and this article is the first studying the great scallop (P. maximus) at this level. Furthermore, it addresses population proteomics, a new promising field, especially in environmental sciences. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Transcriptome and proteomic analysis of mango (Mangifera indica Linn) fruits

Here we used Illumina RNA-seq technology for transcriptome sequencing of a mixed fruit sample from 'Zill' mango (Mangifera indica Linn) fruit pericarp and pulp during the development and ripening stages. RNA-seq generated 68,419,722 sequence reads that were assembled into 54,207 transcripts with a mean length of 858bp, including 26,413 clusters and 27,794 singletons. A total of 42,515(78.43%) transcripts were annotated using public protein databases, with a cut-off E-value above 10(-5), of which 35,198 and 14,619 transcripts were assigned to gene ontology terms and clusters of orthologous groups respectively. Functional annotation against the Kyoto Encyclopedia of Genes and Genomes database identified 23,741(43.79%) transcripts which were mapped to 128 pathways. These pathways revealed many previously unknown transcripts. We also applied mass spectrometry-based transcriptome data to characterize the proteome of ripe fruit. LC-MS/MS analysis of the mango fruit proteome was using tandem mass spectrometry (MS/MS) in an LTQ Orbitrap Velos (Thermo) coupled online to the HPLC. This approach enabled the identification of 7536 peptides that matched 2754 proteins. Our study provides a comprehensive sequence for a systemic view of transcriptome during mango fruit development and the most comprehensive fruit proteome to date, which are useful for further genomics research and proteomic studies. Our study provides a comprehensive sequence for a systemic view of both the transcriptome and proteome of mango fruit, and a valuable reference for further research on gene expression and protein identification. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Cotton proteomics for deciphering the mechanism of environment stress response and fiber development

Cotton fiber is considered as the backbone of the textile industry. The productivity of cotton crop is severely hampered by the occurrence of pathogens, pests, and various environmental factors. Nevertheless, cotton plant has developed sophisticated mechanisms to respond to environment stresses to avoid detrimental effects on its growth and development. Therefore, understanding the mechanisms of cotton fiber development and environment stress response is of considerable interest for designing agriculture breeding strategies to ensure sustainable productivity. The application of proteomics technologies to advance our knowledge in cotton fiber development and abiotic/biotic stress tolerance has increased dramatically in the last 5years as evidenced by the large amount of publications in this area. This review summarizes the work which has been reported for cotton proteomics and evaluates the findings in context of the approaches that are widely employed with the aim to generate novel insight useful for cotton improvement. Cotton (Gossypium spp.) is considered as the foremost commercially important fiber crop grown all over the world and is deemed as the backbone of the textile industry. Cotton is also an important source of edible oil seed and a nutrient-rich food crop as cottonseed contains high-quality protein and oil. The growth and productivity of cotton crop are often hampered by various biotic stress factors, such as insect pests and pathogens. In addition, cotton plants are frequently subjected to unavoidable environmental factors that cause abiotic stress, such as salt, heat and drought. Proteomic techniques provide one of the best options for understanding the gene function and phenotypic changes during cotton fiber development and stress response. This review first summarizes the work which has been reported for cotton proteomics about cotton fiber development and abiotic/biotic stress tolerance, and also evaluates the findings in context of the approaches that are widely employed during last 5years, with the aim to generate novel insight useful for cotton improvement. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

It's time to pop a cork on champagne's proteome!

Champagne is a world-renowned French sparkling wine, which undergoes many steps (fermentation, aging …) for its elaboration. Various compounds might evolve during this winemaking process and thus modify its final quality. Here, we report the first proteome analysis of two standard commercial Champagne wines, using the powerful Combinatorial Peptide Ligand Library (CPLL) technique. Indeed, wine proteins are present in small amounts but they are key compounds, likely to impact on both foam quality and aroma behavior. Forty-three unique gene products were retrieved in a single-varietal champagne and a blended champagne. Several proteins from Vitis vinifera together with seven yeast proteins were undoubtedly identified in these Champagne wines. The main advantage of CPLLs was the detection of low abundance proteins despite the absence of purification or pre-concentration step. It is an important fact to take into account, since Champagne wines generally contain a low amount of proteins (5-10mg/L) that implies to usually concentrate wine proteins before 1D or 2D electrophoresis. Most Champagne proteins are grape and yeast glycoproteins which are considered as good foam "promoters". Some of these proteins might also interact with wine aromas, and thus contribute to the overall quality of Champagne wines. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Comparative proteomics reveals proteins impacted by nitrogen deprivation in wild-type and high lipid-accumulating mutant strains of Tisochrysis lutea

Understanding microalgal lipid accumulation under nitrogen starvation is of major interest for biomass feedstock, food and biofuel production. Using a domesticated oleaginous algae Tisochrysis lutea, we performed the first comparative proteomic analysis on the wild type strain and a selected lipid over-accumulating mutant. 2-DE analysis was made on these strains cultured in two metabolic conditions, with and without nitrogen deprivation, which revealed significant differences in proteomes according to both strain and nitrogen availability. Mass spectrometry allowed us to identify 37 proteins that were differentially expressed between the two strains, and 17 proteins regulated by nitrogen starvation concomitantly with lipid accumulation. The proteins identified are known to be involved in various metabolic pathways including lipid, carbohydrate, amino acid, energy and pigment metabolisms, photosynthesis, protein translation, stress response and cell division. Four candidates were selected for possible implication in the over-accumulation of lipids during nitrogen starvation. These include the plastid beta-ketoacyl-ACP reductase protein, the coccolith scale associated protein and two glycoside hydrolases involved in biosynthesis of fatty acids, carbon homeostasis and carbohydrate catabolism, respectively. This proteomic study confirms the impact of nitrogen starvation on overall metabolism and provides new perspectives to study the lipid over-accumulation in the prymnesiophyte haptophyte T. lutea. This paper study consists of the first proteomic analysis on Tisochrysis lutea, a non-model marine microalga of interest for aquaculture and lipids production. Comparative proteomics revealed proteins putatively involved in the up-accumulation of neutral lipids in a mutant strain during nitrogen starvation. The results are of great importance for future works to improve lipid accumulation in microalgae of biotechnological interest for biofuel production. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Immunological profile of antivenoms: Preclinical analysis of the efficacy of a polyspecific antivenom through antivenomics and neutralization assays

Parenteral administration of animal-derived antivenoms constitutes the mainstay in the treatment of snakebite envenomings. Despite the fact that this therapy has been available for over a century, the detailed understanding of the neutralizing and immunoreactivity profiles of the majority of antivenoms is pending. Currently, a combination of preclinical neutralization tests and 'antivenomics', i.e. a proteomic-based assessment of antivenom immunoreactivity, provides a powerful analytical platform to investigate the preclinical efficacy of antivenoms. In this review, the studies performed on the polyvalent antivenom manufactured by Instituto Clodomiro Picado, Costa Rica, are summarized. This antivenom is prepared by immunizing horses with a mixture of the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys, and is used in Central America for the treatment of envenomings by viperid species. Overall, the antivenom shows a widespread pattern of immunological reactivity against homologous and heterologous venoms, which correlates with its ability to neutralize lethal, hemorrhagic, myotoxic, coagulant, defibrinogenating, phospholipase A2 and proteinase activities of viperid venoms. At the same time, antivenomics detected several venom components against which the antivenom shows only partial or negligible immunorecognition, such as low molecular mass vasoactive peptides, disintegrins, and some phospholipases A2, P-I metalloproteinases and serine proteinases. Such information can be used to design strategies for enhancing the antibody response of horses against poorly immunogenic, toxicologically-relevant venom components in order to further improve the efficacy of this antivenom. The timely parenteral administration of an appropriate antivenom remains, more than a century after the development of the first serum antivenimeux by Calmette and Phisalix and Bertrand, the only currently effective treatment for snakebite envenomings. A key technical issue in the generation of novel antivenoms is the design of optimized immunization venom mixtures that ensure that the resulting antidotes will be effective against the highest number of venoms from snakes of medical concern across the geographical range where they will be used. Antivenomics is a proteomics-based protocol developed to complement in vitro and in vivo standard preclinical tests in the qualitative and quantitative characterization of the immunological profile and the extent of cross-reactivity of antivenoms against homologous and heterologous venoms. Antivenomics is translational venomics. The combination of antivenomics and neutralization assays represents a powerful analytical platform to investigate the efficacy of antivenoms at the molecular and preclinical levels. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Venomous snakes of Costa Rica: Biological and medical implications of their venom proteomic profiles analyzed through the strategy of snake venomics

In spite of its small territory of ~50,000km(2), Costa Rica harbors a remarkably rich biodiversity. Its herpetofauna includes 138 species of snakes, of which sixteen pit vipers (family Viperidae, subfamily Crotalinae), five coral snakes (family Elapidae, subfamily Elapinae), and one sea snake (Family Elapidae, subfamily Hydrophiinae) pose potential hazards to human and animal health. In recent years, knowledge on the composition of snake venoms has expanded dramatically thanks to the development of increasingly fast and sensitive analytical techniques in mass spectrometry and separation science applied to protein characterization. Among several analytical strategies to determine the overall protein/peptide composition of snake venoms, the methodology known as 'snake venomics' has proven particularly well suited and informative, by providing not only a catalog of protein types/families present in a venom, but also a semi-quantitative estimation of their relative abundances. Through a collaborative research initiative between Instituto de Biomedicina de Valencia (IBV) and Instituto Clodomiro Picado (ICP), this strategy has been applied to the study of venoms of Costa Rican snakes, aiming to obtain a deeper knowledge on their composition, geographic and ontogenic variations, relationships to taxonomy, correlation with toxic activities, and discovery of novel components. The proteomic profiles of venoms from sixteen out of the 22 species within the Viperidae and Elapidae families found in Costa Rica have been reported so far, and an integrative view of these studies is hereby presented. In line with other venomic projects by research groups focusing on a wide variety of snakes around the world, these studies contribute to a deeper understanding of the biochemical basis for the diverse toxic profiles evolved by venomous snakes. In addition, these studies provide opportunities to identify novel molecules of potential pharmacological interest. Furthermore, the establishment of venom proteomic profiles offers a fundamental platform to assess the detailed immunorecognition of individual proteins/peptides by therapeutic or experimental antivenoms, an evolving methodology for which the term 'antivenomics' was coined (as described in an accompanying paper in this special issue). Venoms represent an adaptive trait and an example of both divergent and convergent evolution. A deep understanding of the composition of venoms and of the principles governing the evolution of venomous systems is of applied importance for exploring the enormous potential of venoms as sources of chemical and pharmacological novelty but also to fight the consequences of snakebite envenomings. Key to this is the identification of evolutionary and ecological trends at different taxonomical levels. However, the evolution of venomous species and their venoms do not always follow the same course, and the identification of structural and functional convergences and divergences among venoms is often unpredictable by a phylogenetic hypothesis. Snake venomics is a proteomic-centered strategy to deconstruct the complex molecular phenotypes the venom proteomes. The proteomic profiles of venoms from sixteen out of the 22 venomous species within the Viperidae and Elapidae families found in Costa Rica have been completed so far. An integrative view of their venom composition, including the identification of geographic and ontogenic variations, is hereby presented. Venom proteomic profiles offer a fundamental platform to assess the detailed immunorecognition of individual venom components by therapeutic or experimental antivenoms. This aspect is reviewed in the companion paper. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Improving the identification rate of data independent label-free quantitative proteomics experiments on non-model crops: A case study on apple fruit

Complex peptide extracts from non-model crops are troublesome for proper identification and quantification. To increase the identification rate of label free DIA experiments of Braeburn apple a new workflow was developed where a DDA database was constructed and linked to the DIA data. At a first level, parent masses found in DIA were searched in the DDA database based on their mass to charge ratio and retention time; at a second level, masses of fragmentation ions were compared for each of the linked spectrum. Following this workflow, a tenfold increase of peptides was identified from a single DIA run. As proof of principle, the designed workflow was applied to determine the changes during a storage experiment, achieving a two-fold identification increase in the number of significant peptides. The corresponding protein families were divided into nine clusters, representing different time profiles of changes in abundances during storage. Up-regulated protein families already show a glimpse of important pathways affecting aging during long-term storage, such as ethylene synthesis, and responses to abiotic stresses and their influence on the central metabolism. Proteomics research on non-model crops causes additional difficulties in identifying the peptides present in, often complex, samples. This work proposes a new workflow to retrieve more identifications from a set of quantitative data, based on linking DIA and DDA data at two consecutive levels. As proof of principle, a storage experiment on Braeburn apple resulted in twice as much identified storage related peptides. Important proteins involved in central metabolism and stress are significantly up-regulated after long term storage. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Surface analysis of Dicrocoelium dendriticum. The molecular characterization of exosomes reveals the presence of miRNAs

With the aim of characterizing the molecules involved in the interaction of Dicrocoelium dendriticum adults and the host, we have performed proteomic analyses of the external surface of the parasite using the currently available datasets including the transcriptome of the related species Echinostoma caproni. We have identified 182 parasite proteins on the outermost surface of D. dendriticum. The presence of exosome-like vesicles in the ESP of D. dendriticum and their components has also been characterized. Using proteomic approaches, we have characterized 84 proteins in these vesicles. Interestingly, we have detected miRNA in D. dendriticum exosomes, thus representing the first report of miRNA in helminth exosomes. In order to identify potential targets for intervention against parasitic helminths, we have analyzed the surface of the parasitic helminth Dicrocoelium dendriticum. Along with the proteomic analyses of the outermost layer of the parasite, our work describes the molecular characterization of the exosomes of D. dendriticum. Our proteomic data confirm the improvement of protein identification from "non-model organisms" like helminths, when using different search engines against a combination of available databases. In addition, this work represents the first report of miRNAs in parasitic helminth exosomes. These vesicles can pack specific proteins and RNAs providing stability and resistance to RNAse digestion in body fluids, and provide a way to regulate host-parasite interplay. The present data should provide a solid foundation for the development of novel methods to control this non-model organism and related parasites. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Improving the quality of protein identification in non-model species. Characterization of Quercus ilex seed and Pinus radiata needle proteomes by using SEQUEST and custom databases

Nowadays the most used pipeline for protein identification consists in the comparison of the MS/MS spectra to reference databases. Search algorithms compare obtained spectra to an in silico digestion of a sequence database to find exact matches. In this context, the database has a paramount importance and will determine in a great deal the number of identifications and its quality, being this especially relevant for non-model plant species. Using a single Viridiplantae database (NCBI, UniProt) and TAIR is not the best choice for non-model species since they are underrepresented in databases resulting in poor identification rates. We demonstrate how it is possible to improve the rate and quality of identifications in two orphan species, Quercus ilex and Pinus radiata, by using SEQUEST and a combination of public (Viridiplantae NCBI, UniProt) and a custom-built specific database which contained 593,294 and 455,096 peptide sequences (Quercus and Pinus, respectively). These databases were built after gathering and processing (trimming, contiging, 6-frame translation) publicly available RNA sequences, mostly ESTs and NGS reads. A total of 149 and 1533 proteins were identified from Quercus seeds and Pinus needles, representing a 3.1- or 1.5-fold increase in the number of protein identifications and scores compared to the use of a single database. Since this approach greatly improves the identification rate, and is not significantly more complicated or time consuming than other approaches, we recommend its routine use when working with non-model species. In this work we demonstrate how the construction of a custom database (DB) gathering all available RNA sequences and its use in combination with Viridiplantae public DBs (NCBI, UniProt) significantly improve protein identification when working with non-model species. Protein identification rate and quality is higher to those obtained in routine procedures based on using only one database (commonly Viridiplantae from NCBI), as we demonstrated analyzing Quercus seeds and Pine needles. The proposed approach based on the building of a custom database is not difficult or time consuming, so we recommend its routine use when working with non-model species. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

The fronds tonoplast quantitative proteomic analysis in arsenic hyperaccumulator Pteris vittata L.

Pteris vittata, the first known arsenic hyperaccumulating plant, can accumulate very high concentration arsenic in its aboveground tissues, while low in roots. Previous studies have suggested that arsenic vacuole compartmentalization may play an important role in the arsenic-hyperaccumulation in P. vittata, but the mechanism(s) of arsenic transport to vacuole are largely unknown. We obtained tonoplast isolated from fronds of P. vittata sporophyte grown under minus and 1mM arsenate for 3weeks by iodixanol step gradient centrifugation method, and then used TMPP protein labeling technology followed by liquid chromatography-a linear ion trap-Orbitrap hybrid mass spectrometer analysis for the quantitative detection of proteins. And we designed and used an "artificial" database for database searching. In total, 56 tonoplast proteins were identified; more than 70% of them were transport proteins. Under arsenate treatment, one TDT transporter protein, a member of the TerC family and a PDR-like protein were upregulated differentially. While V-ATPase subunits c, E, and G, and V-PPase, were downregulated. Additionally, the identified tonoplast proteins in our present study provide an informative basis for arsenic carriers or channels and help to clarify the regulation of tonoplast arsenic transport processes in P. vittata. Vacuole compartmentalization is crucial to As hyperaccumulator P. vittata, while there is limited known arsenic transport proteins involved in vacuole compartmentalization. In this paper, we obtained tonoplast of P. vittata fronds by iodixanol step gradient centrifugation method and then used TMPP protein labeling proteome technology for the quantitative detection of fronds tonoplast proteins. Our findings are the first challenge to the tonoplast proteins data mining of P. vittata which provide an informative basis for As carriers or channels. The proteomic approach in our study is suited for detecting alterations tonoplast protein and help to clarify the regulation of tonoplast transport processes. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Marine dinoflagellate proteomics: Current status and future perspectives

Dinoflagellates are not only the important primary producers and an essential component of the food chain in the marine ecosystem, but also the major causative species resulting in harmful algal blooms (HABs) and various shellfish poisonings. Although much work has been devoted to the dinoflagellates, our understanding of them is still extremely limited owing to their unusual features. Proteomics, a large-scale study of the structure and function of proteins in complex biological samples, has been introduced to the study of marine dinoflagellates and has shown its powerful potential with regard to revealing their physiological and metabolic characteristics. However, the application of proteomic approaches to unsequenced dinoflagellates is still in its infancy and faces considerable challenges. This review summarizes recent progress in marine dinoflagellate proteomics and discusses the limitations and prospects for this approach to their study. The dinoflagellates are the major causative agent responsible for harmful algal blooms and paralytic shellfish poisoning around the world. However, our understanding of them is still extremely limited owing to their unusual features, such as large genome size and permanently condensed chromosomes, which impedes the monitoring, mitigation and prevention of HABs. Proteomics, a large-scale study of the structure and function of proteins in complex biological samples, has been introduced to the study of marine dinoflagellates and has shown its powerful potential with regard to revealing their physiological and metabolic characteristics. This review summarizes recent progress in marine dinoflagellate proteomics with regard to methodology, cell growth, toxin biosynthesis, environmental stress, cell wall and surface, and symbiosis, and discusses the limitations and prospects for this approach to dinoflagellate study. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

ITRAQ-based quantitative proteomic analysis reveals acute hypo-osmotic responsive proteins in the gills of the Japanese eel (Anguilla japonica)

Osmoregulation in fish has been a classical research topic for several decades. Salmon and eels are the widely used model animals because of their wide distribution in different geographical locations and spawning migration between fresh- and salt-water habitats. Numerous fish osmoregulatory hormones and ion transporters were identified for their essential roles in acclimation and adaptation to waters of different salinities. Because of the lack of a genomic database, the scope of most studies, however, is very limited. Recently, our group reported the first high-throughput transcriptomic and proteomic studies to identify hyperosmotic-responsive genes/proteins in gills of Japanese eels. In this study, we aimed to decipher changes in hypo-osmotic-responsive proteins in fish acclimating from seawater (SW) to freshwater (FW) conditions. We collected gill samples from SW-adapted and SW-to-FW-acclimating fish. The respective gill proteins were extracted and labeled using isobaric tags for relative and absolute quantitation (iTRAQ) and analyzed using a high-resolution mass spectrometer. In the short-term transfer from SW to FW, 51 hypo-responsive proteins were detected, and 24 unique hypo-osmotic-responsive proteins were identified (15 up-regulated and nine down-regulated proteins). Our data support the use of an omics approach to facilitate the application of functional genomics in non-model organisms. By combining transcriptomic and proteomic approaches, the study has provided the most comprehensive, targeted investigation of eel gill hypo-osmotic responsive proteins that provides molecular insights of osmoregulation mechanisms in a non-model organism, eel. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Proteomic and biochemical analyses of short-tailed pit viper (Gloydius brevicaudus) venom: Age-related variation and composition–activity correlation

We conducted an in-depth analysis of the proteomic and biochemical profiles of the venom of neonate and adult short-tailed pit vipers (Gloydius brevicaudus). Identified proteins were assigned to a few main toxin families. Disintegrin, phospholipase A2 (PLA2), serine proteinase, cysteine-rich secretory protein, C-type lectin-like protein, l-amino acid oxidase and snake venom metalloproteinase (SVMP) were detected in both venoms, while 5'-nucleotidase was detected only in the adult venom. SVMP was the predominant protein family in both venoms (neonate: 65.7%; adult: 64.4%), followed by PLA2 (neonate: 13.4%; adult: 25.0%). Antivenomic analysis revealed that commercial G. brevicaudus antivenom almost neutralized the chromatographic peaks with medium and high molecular masses in both venoms, but did not completely recognize peaks with low molecular mass. Toxicological and enzymatic activities show remarkable age-related variation in G. brevicaudus venom, probably resulting from variation in venom composition. Our data demonstrate age-related variation across venomics, antivenomics and biochemical profiles of G. brevicaudus venom, and have implications for the management of G. brevicaudus bites, including improving antivenom preparation by combining both venoms. This study investigates the composition and biochemical activity of neonate and adult Gloydius brevicaudus venoms. We found remarkable age-related variation in venom biological activity, likely the result of variation in venom composition. Antivenomics analysis was used to explore difference in neonate and adult G. brevicaudus venoms. Our findings have implications for the diagnosis and clinical management of G. brevicaudus bites, and the design of venom mixtures that will increase the efficacy of commercial antivenom. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Elucidation of the unexplored biodiversity of ant venom peptidomes via MALDI–TOF mass spectrometry and its application for chemotaxonomy

The rise of integrative taxonomy, a multi-criteria approach used in characterizing species, fosters the development of new tools facilitating species delimitation. Mass spectrometric (MS) analysis of venom peptides from venomous animals has previously been demonstrated to be a valid method for identifying species. Here we aimed to develop a rapid chemotaxonomic tool for identifying ants based on venom peptide mass fingerprinting. The study focused on the biodiversity of ponerine ants (Hymenoptera: Formicidae: Ponerinae) in French Guiana. Initial experiments optimized the use of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to determine variations in the mass profiles of ant venoms using several MALDI matrices and additives. Data were then analyzed via a hierarchical cluster analysis to classify the venoms of 17 ant species. In addition, phylogenetic relationships were assessed and were highly correlated with methods using DNA sequencing of the mitochondrial gene cytochrome c oxidase subunit 1. By combining a molecular genetics approach with this chemotaxonomic approach, we were able to improve the accuracy of the taxonomic findings to reveal cryptic ant species within species complexes. This chemotaxonomic tool can therefore contribute to more rapid species identification and more accurate taxonomies. This is the first extensive study concerning the peptide analysis of the venom of both Pachycondyla and Odontomachus ants. We studied the venoms of 17 ant species from French Guiana that permitted us to fine-tune the venom analysis of ponerine ants via MALDI-TOF mass spectrometry. We explored the peptidomes of crude ant venom and demonstrated that venom peptides can be used in the identification of ant species. In addition, the application of this novel chemotaxonomic method combined with a parallel genetic approach using COI sequencing permitted us to reveal the presence of cryptic ants within both the Pachycondyla apicalis and Pachycondyla stigma species complexes. This adds a new dimension to the search for means of exploiting the enormous biodiversity of venomous ants as a source for novel therapeutic drugs or biopesticides. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Non-model organisms, a species endangered by proteogenomics

Previously, large-scale proteomics was possible only for organisms whose genomes were sequenced, meaning the most common model organisms. The use of next-generation sequencers is now changing the deal. With "proteogenomics", the use of experimental proteomics data to refine genome annotations, a higher integration of omics data is gaining ground. By extension, combining genomic and proteomic data is becoming routine in many research projects. "Proteogenomic"-flavored approaches are currently expanding, enabling the molecular studies of non-model organisms at an unprecedented depth. Today draft genomes can be obtained using next-generation sequencers in a rather straightforward way and at a reasonable cost for any organism. Unfinished genome sequences can be used to interpret tandem mass spectrometry proteomics data without the need for time-consuming genome annotation, and the use of RNA-seq to establish nucleotide sequences that are directly translated into protein sequences appears promising. There are, however, certain drawbacks that deserve further attention for RNA-seq to become more efficient. Here, we discuss the opportunities of working with non-model organisms, the proteomic methods that have been used until now, and the dramatic improvements proffered by proteogenomics. These put the distinction between model and non-model organisms in great danger, at least in terms of proteomics! Model organisms have been crucial for in-depth analysis of cellular and molecular processes of life. Focusing the efforts of thousands of researchers on the Escherichia coli bacterium, Saccharomyces cerevisiae yeast, Arabidopsis thaliana plant, Danio rerio fish and other models for which genetic manipulation was possible was certainly worthwhile in terms of fundamental and invaluable biological insights. Until recently, proteomics of non-model organisms was limited to tedious, homology-based techniques, but today draft genomes or RNA-seq data can be straightforwardly obtained using next-generation sequencers, allowing the establishment of a draft protein database for any organism. Thus, proteogenomics opens new perspectives for molecular studies of non-model organisms, although they are still difficult experimental organisms. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Critical roles of mitochondria in brain activities of torpid Myotis ricketti bats revealed by a proteomic approach

Bats are the only mammals that fly and hibernate. Little is known about their overall metabolism in the brain during hibernation. In this study, brain proteins of torpid and active Myotis ricketti bats were fractionated and compared using a proteomic approach. Results showed that 21% (23 proteins) of identified proteins with significant expression changes were associated with amino acid metabolism and proteostasis. The expression levels of proteins involved in energy metabolism (15 proteins), cytoskeletal structure (18 proteins), and stress response (13 proteins) were also significantly altered in torpid bats. Over 30% (34 proteins) of differentially expressed proteins were associated with mitochondrial functions. Various post-translational modifications (PTMs) on PDHB, DLD, and ARG1 were detected, suggesting that bats use PTMs to regulate protein functions during torpor. Antioxidation and stress responses in torpid bats were similar to those of hibernated squirrels, suggesting a common strategy adopted by small hibernators against brain dysfunction. Since many amino acids that metabolize in mitochondria modulate neuronal transmissions, results of this study reveal pivotal roles of mitochondria in neural communication, metabolic regulation, and brain cell survival during bat hibernation. This article is part of a Special Issue entitled: Proteomics of non-model organisms. This study reveals the mechanisms used by bats to regulate brain activities during torpor. These mechanisms include post-translational modifications and differential expression of proteins involved in mitochondrial electron transport, anaerobic glycolysis, TCA cycle efflux, cytoskeletal plasticity, amino acid metabolism, vesicle structure, antioxidation defense, stress response, and proteostasis. Our study provides insights in metabolic regulation of flying mammals during torpor and common strategies used by small hibernators in response to hibernation. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Vintage venoms: Proteomic and pharmacological stability of snake venoms stored for up to eight decades

For over a century, venom samples from wild snakes have been collected and stored around the world. However, the quality of storage conditions for "vintage" venoms has rarely been assessed. The goal of this study was to determine whether such historical venom samples are still biochemically and pharmacologically viable for research purposes, or if new sample efforts are needed. In total, 52 samples spanning 5 genera and 13 species with regional variants of some species (e.g., 14 different populations of Notechis scutatus) were analysed by a combined proteomic and pharmacological approach to determine protein structural stability and bioactivity. When venoms were not exposed to air during storage, the proteomic results were virtually indistinguishable from that of fresh venom and bioactivity was equivalent or only slightly reduced. By contrast, a sample of Acanthophis antarcticus venom that was exposed to air (due to a loss of integrity of the rubber stopper) suffered significant degradation as evidenced by the proteomics profile. Interestingly, the neurotoxicity of this sample was nearly the same as fresh venom, indicating that degradation may have occurred in the free N- or C-terminus chains of the proteins, rather than at the tips of loops where the functional residues are located. These results suggest that these and other vintage venom collections may be of continuing value in toxin research. This is particularly important as many snake species worldwide are declining due to habitat destruction or modification. For some venoms (such as N. scutatus from Babel Island, Flinders Island, King Island and St. Francis Island) these were the first analyses ever conducted and these vintage samples may represent the only venom ever collected from these unique island forms of tiger snakes. Such vintage venoms may therefore represent the last remaining stocks of some local populations and thus are precious resources. These venoms also have significant historical value as the Oxyuranus venoms analysed include samples from the first coastal taipan (Oxyuranus scutellatus) collected for antivenom production (the snake that killed the collector Kevin Budden), as well as samples from the first Oxyuranus microlepidotus specimen collected after the species' rediscovery in 1976. These results demonstrate that with proper storage techniques, venom samples can retain structural and pharmacological stability. This article is part of a Special Issue entitled: Proteomics of non-model organisms.